Pediatric Gender Assignment - A Critical Reappraisal PDF

PEDIATRIC GENDER
ASSIGNMENT
A Critical Reappraisal
ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
Editorial Board:
NATHAN BACK, State University of New York at Buffalo
IRUN R. COHEN. The Weizmann Institute of Science
DAVID KRITCHEVSKY, Wistar Institute
ABEL LAJTHA, N. S. Kline Institute for Psychiatric Research
RODOLFO PAOLETTI, University of Milan
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PEDIATRIC GENDER
ASSIGNMENT
A Critical Reappraisal
Edited by
Stephen A. Zderic
Douglas A. Canning
Michael C. Carr and
Howard McC. Snyder, III.
Children s Hospital of Philadelphia
Philadelphia, Pennsylvania
Springer-Science+Business Media, LLC

Library of Congress Cataloging-in-Publication Data
Pediatric gender assignment: a critical reappraisal/edited by Stephen A. Zderic ... [et al.].
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-4613-5162-7 ISBN 978-1-4615-0621-8 (eBook)
DOI 10.1007/978-1-4615-0621-8
I. Gender identity disorders in children-Congresses. 2. Hermaphroditism-Congresses.
3. Generative organs-Abnormalities-Congresses. 4. Sex orientation-Congresses. I.
Zderic, Stephen A.
[DNLM: l. Sex Differentiation Disorders-therapy-Child-Congresses. 2. Sex
Differentiation Disorders-therapy-Infant-Congresses. 3. Genitalia,
Female-abnormalities-Child-Congresses. 4. Genitalia,
Female-abnormalities-Infant-Congresses. 5. Genitalia,
Male-abnormalities-Child-Congresses. 6. Genitalia,
Male-abnormalities-Infant-Congresses. 7. Hermaphroditism-Congresses. 8. Sex
Determination (Analysis)-Congresses. WS 320 P3687 2002]
RJ506.G35 P436 2002
618.92'65-dc21
2002032120
ISBN 978-1-4613-5162-7
©2002 Springer Science+Business Media New York
Originally published by Kluwer Academic I Plenum Publishers, New York in 2002
Softcover reprint ofthe hardcover 1st edition 2002
All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written
permission from the Publisher, with the exception of any material supplied specifically for the purpose of being
entered and executed on a computer system, for exclusive use by the purchaser of the work
CONTRIBUTORS
Anthony Atala
Children's Hospital and Harvard Medical School
Boston, Massachusetts
Laurence S. Baskin
Univeristy of California San Francisco
San Francisco, California
Jonathon Bingham
Columbia University
College of Physicians
Babies and Children's Hospital of New York
New York, New York
Douglas A. Canning
University of Pennsylvania School of Medicine
The Children's Hospital of Philadelphia
Michael C. Carr
Wilson C.l. Chung

Netherlands Institute for Brain Research
Amsterdam, The Netherlands and
University of Massachusetts
Amherst, Massachusetts
Timothy M. Crombleholme
Patricia K. Donahoe
Pediatric Surgical Research Laboratories
Massachusetts General Hospital
Harvard Medical School
v
vi CONTRIBUTORS
Roger A. Gorski
University of California Los Angeles
School of Medicine
Los Angeles, California
Faruk Badziselimovic
The Basel Children's Hospital
Basel, Switzerland
Terry W. Hensle
Columbia University
College of Physicians
Babies and Children's Hospital of New York
New York, New York
Michael A. Hofman
Graduate School Neurosciences Amsterdam
Amsterdam, The Netherlands
Dale Huff
Douglas A. Husmann
Mayo Clinic
Rochester, Minnesota
J ulianna Imperato-McGinley
Weill Medical College of Cornell University
New York, New York
Tatjana A. Ishunina
Amsterdam, The Netherlands and
Kursk State Medical University
Kursk, Russia
Brian K. Jordan
CONTRIBUTORS vii
Gerald H. Jordan
Eastern Virginia School of Medicine
Norfolk, Virginia
Christine Kodman-Jones
Frank P.M. Kruijver

David T. MacLaughlin
Pediatric Surgical Research Laboratories
Massachusetts General Hospital
Harvard Medical School
Laurence B. McCullough
Baylor College of Medicine
Houston, Texas
J. William McRoberts
U ni versity of Kentucky School of Medicine
Lexington, Kentucky
Heino F.L. Meyer-Bahlburg

Columbia University
New York, New York
Michael E. Mitchell
University of Washington School of Medicine
Children's Hospital and Regional Medical Center
Seattle, Washington
J. Chadwick Plaire
Children's Hospital and Regional Medical Center
Seattle, W ashi ngton
William G. Reiner
Johns Hopkins Medical Institutions
Baltimore, Maryland
viii CONTRIBUTORS
Natalie E. Rintoul
Richard C. Sadove
University of Kentucky School of Medicine
Lexington. Kentucky
Howard McC. Snyder, III

Dick F. Swaab
Eric Vilain
Perrin C. White
University of Texas Southwestern Medical Center
Dallas. Texas
Stephen A. Zderic
PREFACE
This book represents the proceedings from a conference that took place in Dallas
in the spring of 1999 which was entitled "Pediatric Gender Assignment - A Critical
Reappraisal". Some participants rightfully argued that the conference really focused
on the issue of pediatric gender assignment, and that reassignment was not applied in
most cases. Their comments were reflected in the title of this monograph. This
multidisciplinary meeting was sponsored by a conference grant from the National
Institutes of Health, and a broad inquiry into this complex topic took place from
many points of view. Basic scientists offered insight into mechanisms of sexual
differentiation of the gonads, physical phenotype and imprinting of the central
nervous system. Endocrinologists reviewed their experience in diagnosis and
management, surgeons described traditional as well as innovative approaches, and
there was strong representation from the ethical and behavioral sciences. In putting
together such a panel, it was essential that we identify a cast of speakers who could
address their viewpoints with strong convictions, and yet not let their passions render
the meeting counter productive. We were not disappointed. While many differing
points of view were firmly expressed by the panelists and audience, all viewpoints
were accorded the respect they deserved.
The concept behind the meeting and this book really originated in 1997 shortly
after Diamond and Sigmundson published their long term follow up study of the
John/Joan case. Our first thoughts centered around the concept of the numerator
versus the denominator. This high publicized case report represented a disturbing
example of gender reassignment for a high unusual indication, but it represents a
single case report. Even more disturbing was John Colapinto's personal account of
his experience which appeared that spring in Rolling Stone magazine. We began to
ask ourselves if there were any successful cases of neonatal gender reassignment.
We do not know about the denominator. Are there patients out there who are doing
well or are they all doing poorly? What becomes clear within this monograph, is that
more long term follow up information about these patients is needed.
Several themes emerged from this conference. First and foremost was the need
that all these patients and families have for long term counseling. No matter which
direction is taken with these incredibly difficult issues, counseling must be made a
cornerstone of their management. Psychiatrists, psychologists and social workers are
critical to a multidisciplinary team in order to produce the best possible long term
outcomes for the patient and the family. Logical as this might seem, individuals with
these skills and interests are hard to find. A second theme that emerged was that
patients have more medical knowledge than ever before. We must become
comfortable with life in the internet age where much more information is readily
available, some of which may not always be accurate. Patients will always
appreciate getting the information from their physicians, as opposed to finding it on a
web page. Recently after a long initial discussion with two very computer literate
ix
x PREFACE
parents, I referred them to the Intersex Society of North America (lSNAorg) web
site, and asked them to look through it. At the subsequent multidisciplinary team
meeting, they had additional information at their disposal. By empowering parents
and patients, it is possible to take what is initially threatening in the traditional
medical model, and have it benefit everyone. A third major theme that emerged was
that the human brain is a sexual organ. Since the human brain is sexually dimorphic,
it is not always possible to predict whether the adult will be happy with their gender
20 and 30 years after such a critical decision has been made in the first days of life.
This is underscored by the divergence of opinions expressed at the conference and
within this monograph about the issue of early neonatal corrective surgery versus
delaying any corrective surgery until the child can voice their gender identity.
As organizers, we never expected that one conference could resolve so complex
an issue and arrive at some simple management algorithm. Rather, our goals were
simply to initiate a dialogue amongst individuals with varying points of view. In
fact, the exchange of ideas after the presentations were so informative, that we have
included in this monograph the transcripts (with minimal editing) of the discussions
following each major presentation. We are especially hopeful that major centers will
set up a central registry so that long term outcomes can be obtained. The American
Academy of Pediatrics Section on Urology, and the Society for Fetal Urology have
formed committees that are working together to develop such a registry. Many of the
answers to the questions posed at this meeting already exist, but can be obtained only
if we can get as many patients as possible to return for long term evaluation. It is our
hope that at a future meeting, the answers to some of these questions will have
become apparent.
Stephen A Zderic, M.D.

For the Editors
ACKNOWLEDGEMENTS
Editors should never fail to acknowledge the tremendous team that it takes to
undertake publishing a book such as this. To our contributing authors, we extend our
thanks for producing manuscripts of such quality. Today more than ever, scholarly
writing takes place in an environment of busy clinical schedules, grant deadlines,
administrative burdens and countless other distractions. We also thank the reviewers
(whoever they might be) for recommending that our National Institutes of Health
conference grant be funded.
Within our department, we wish to thank collectively our entire office staff for
making things run as smoothly as they do. Without their dedication and efforts this
conference and book could not have become a reality. We especially appreciate the
assistance of Elaine Bacon, Karen Jackson and our division manager, Nancy Harriz
for the organizational efforts at the conference. Our thanks to Karen Jackson for her
transcription of the dialogue tapes, and assistance with the manuscripts. But without
a doubt, this mansucript would have never come to press without the persistant
efforts of Venita Joynes who wrestled with unfamiliar templates and countless jpg
files to produce the final camera ready mansucript. We also thank Andrea Macaluso
and Jennifer Stevens in the Kluwer Plenum editorial department for their assistance,
and hospitality.
Finally in closing, we wish to acknowledge the ongoing contributions of our wives
and children. They have endured our long hours, piles of reading, and an endless
stream of deadlines in pursuit of our scholarly interests. Without their love and
support, our work would not be possible.
The Editors
xi
CONTENTS
SRY AND THE GENETICS OF SEX DETERMINATION ................. .

Brian K. Jordan and Eric Vilain
GONADAL DIFFERENTIATION - NORMAL AND

ABNORMAL TESTICULAR DEVELOPMENT.................. 15
Faruk Hadziselimovic and Dale Huff
MULLERIAN INHIBITING SUBSTANCE:

AN UPDATE ............................................................... 25
David T. MacLaughlin and Patricia Donahoe
MICROPENIS: AN ANIMAL MODEL AND ITS

HUMAN CORRELATES ................................................ 41
Douglas A. Husmann
HYPOTHALAMIC IMPRINTING BY GONADAL

STEROID HORMONES................................................. 57
Roger A. Gorski
SEXUAL DIFFERENTIA TION OF THE

HUMAN HyPOTHALAMUS........................................... 75
Dick F. Swabb, Wilson c.J. Chung, Frank P.M. Kruijver,
Michael A. Hofman and Tatjana A. Ishunina
THE ENDOCRINOLOGIST'S APPROACH TO THE

INTERSEX PA TIENT ................................................... 107
Perrin C. White
5a-REDUCTASE-2 DEFICIENCY AND COMPLETE

ANDROGEN INSENSITIFITY: LESSONS FROM
NATURE ....................................................................... 121
J. Imperato-McGinley
THE CHOP EXPERIENCE WITH CLOACAL

EXSTROPHY AND GENDER REASSIGNMENT..................... 135
Stephen A. Zderic, Douglas A. Canning, Michael C. Carr,
Christine Kodman-Jones and Howard McC. Snyder
xiii
xiv CONTENTS
A FRAMEWORK FOR THE ETHICALLY JUSTIFIED

CLINICAL MANAGEMENT OF INTERSEX
CONDITIONS................................................................ 149
Laurence B. McCullough
GENDER IDENTITY AND SEX ASSIGNMENT:

A REAPPRAISAL FOR THE 21 ST CENTURy....................... 175
William G. Reiner
GENDER ASSIGNMENT AND REASSIGNMENT IN

INTERSEXUALITY: CONTROVERSIES, DATA
AND GUIDELINES FOR RESEARCH................................. 199
Heino F.L. Meyer-Bahlburg
PRENATAL DIAGNOSIS AND TREATMENT OF

INTERSEX STATES.. .. . . . . . . . .. . ... . . . . .. . .. . . . .. . . .. . . . .. . . .. .. . . . . . . .... 225
Natalie E. Rintoul and Timothy M. Crombleholme
ANA TOMICAL STUDIES OF THE FETAL

GENITALIA: SURGICAL RECONSTRUCTIVE
IMPLICA TIONS ............................................................. 239
Laurence S. Baskin
FEMINIZING GENITOPLASTY ................................................ 251

Terry W. Hensle and Jonathon Bingham
MANAGEMENT OF CLOACAL EXSTROPHy............................. 267

Michael E. Mitchell and Chadwick Plaire
TOTAL PHALLIC CONSTRUCTION, OPTION

TO GENDER REASSIGNMENT......................................... 275
Gerald H. Jordan
PENILE RECONSTRUCTION WITH A FREE

SENSATE OSTEOCUTANEOUS FIBULA
FLAP IN THE SURGICAL MANAGEMENT
OF THE INTERSEX PATIENT ........ '" . .. . . . .. . . .. . . . .. . . . . . . . . . . . . . .. 283
J. William McRoberts and Richard C. Sadove
TISSUE ENGINEERING APPROACHES FOR

GENITAL RECONSTRUCTION ......................................... 289
Anthony Atala
INDEX ................................................................................. 305

SRY AND THE GENETICS OF SEX
DETERMINATION
Brian K. Jordan and Eric Vilain

Department of Human Genetics UCLA School of Medicine, Los Angeles, California.
INTRODUCTION
Many consider the distinction between the two sexes, male and female, to be absolute.
Genetic studies of the mechanisms of sexual development have shown the complexity of
sex determination and have started to unveil, at the molecular level, how a number of
individuals can develop as intersex.
Genotypic and Phenotypic Sex
There are many ways to define sex. The genotypic sex of an individual is defined by
the genetic information leading to the development of one sex or the other. Typically, the
genotypic sex is determined by the chromosomal makeup of that individual, i.e. XY for
males, XX for females. However, mutations of genes important for sex determination
represent other genotypic mechanisms leading to more complicated situations such as XX
males or XY females. The gonadal sex of an individual corresponds to the type of gonad
present in that individual, either testis or ovary. A small number of patients, true
hermaphrodites, are born with presence of both tissues. The phenotypic sex of an
individual is defined by the primary and secondary sexual characteristics of that
individual. These characteristics, usually male or female, may be ambiguous. Sex of
rearing denotes the cultural characteristics that we ascribe to an individual. These
characteristics include not only the physical appearance of the individual's body, but also
cultural issues such as name and manner of dress. Finally, the legal sex offers no more
than two choices in our society: male or female.
Each of these differenf definitions corresponds to a different perspective on the
mechanisms of sexual development. They all contribute, to varying degrees, to each
individual's gender identity. We will focus essentially on the genetic factors responsible
for the development of an undifferentiated embryo into either a male or a female. Sex
determination refers to the developmental decision that directs the orientation of the non-
differentiated embryo into a sexually dimorphic individual. In mammals, this decision
occurs during the development of the gonads. For instance, once the testes are formed in
males, sex is determined. Following this sex determining decision, sexual differentiation
takes place, and testes will produce the male hormones responsible for male sexual
characteristics.
Pediatric Gender Assignment: A Critical Reappraisal

Edited by Zderic et al., Kluwer Academic/Plenum Publishers, 2002
2 B. K. JORDAN, ET AL.
In mammals, particularly in humans, embryos are initially not only sexually

undifferentiated, but also bipotential at all levels of sexual differentiation, including the
formation of the gonads and the differentiation of internal and external genitalia. Until
approximately the end of the seventh week of gestation, the gonads of males and females
remain undifferentiated and indistinguishable. At this time, the primordial germ cells
migrate from the posterior endoderm of the yolk sac to the developing gonad. Testicular
differentiation occurs during the eighth and the beginning of the ninth week of gestation,
and the male external genitalia differentiate during the tenth and eleventh weeks of
gestation. The ovary does not begin to differentiate until approximately the third month
(Grumbach & Conte, 1998). A number of genes, most of them still unknown, are
involved in this initial sex determination step. During the seventh week of gestation, the
genital ducts remain bipotential with both Mullerian and W olffian ducts present. In a
female embryo, the Mullerian ducts will develop into the uterus, Fallopian tubes and
superior third of the vagina, while the W olffian ducts will regress in the absence of a
sufficient amount of testosterone. In males, the Wolffian ducts will differentiate into the
epididymis, vas deferens and seminal vesicles under the influence of testosterone secreted
by the Leydig cells, while the Mullerian ducts will undergo a regression caused by the
secretion of Mullerian Inhibiting Substance (MIS) by the Sertoli cells. Development of
internal genitalia occurs during the third month of gestation. External
genitalia remain undifferentiated until the eighth week of gestation. A urogenic slit is
present, with urethral folds on either side of it and more laterally, labioscrotal swellings.
Anterior to the slit is the genital tubercule. By the twelth to the fourteenth week of
gestation, the urethral folds fuse completely in the midline in the male. The
differentiation of the male external genitalia requires the action of testosterone and its s-
a-reduced metabolite, dihydrotestosterone (DHT). While testosterone is mainly
responsible for the differentiation of W olffian ducts, DHT directs the development of the
penis and scrotum and also the appearance of secondary sexual characteristics.
Modern Concepts of Sexual Development
In 1947, Alfred lost performed castrations in fetal rabbits, showing that if

gonadectomy were performed early enough, the rabbit embryos would all become female
(Jost, 1947). These key experiments demonstrated the equivalence of sex determination
and testis determination. This concept, verified in almost all mammalian species, led to
the search for a sex-determining gene that had to be a Testis Determining Factor (TDF).
The second major concept of sex determination was established when the karyotype of
patients with Klinefelter syndrome (male 47, XXY) (Jacobs and Strong, 1959) and with
Turner syndrome (female 45, X) (Ford et aI, 1959), were discovered. The presence of the
Y chromosome was necessary for a male phenotype, and TDF had to be localized on the
Y chromosome. The molecular era of sex determination had begun.
SRY, the Primary Trigger

SRY A:\fD THE GENETICS OF SEX 3
The vast majority of known sex determining genes, including SRY, were identified by
the molecular study of abnormalities present in rare sex-reversed patients. These
individuals have a discordance between their phenotypic and their genotypic sex. Their
gonads are usually abnormally developed and are called dysgenetic. They are XX males,
XX true hermaphrodites or XY females with gonadal dysgenesis. XX males have normal
genitalia, small azoospermic testes and
no Mullerian structures. They may also present at birth with severe hypospadias or
sexual ambiguity. XX true hermaphrodites present with ambiguous genitalia, persistance
of some Mullerian structures, generally on the side of the ovary, and are definied
pathologically by the presence of both ovarian and testicular tissue in their gonads. XY
females with pure gonadal dysgenesis have normal female genitalia, including a normal
uterus, and fibrous streak gonads in place of the ovaries. When the gonadal dysgenesis is
partial, these patients may present with sexual ambiguity. These pathologies, occurring
with a frequency of 1130,000, have allowed the mapping of sex determining genes, and
TDF in particular. It was hypothesized that, as a result of an abnormal crossing-over
between the X and the Y chromosome, XX males and XX true hermaphrodites could be
explained by the presence of a fragment of a Y chromosome, containing TDF,
translocated on one of the X chromosomes (Ferguson-Smith, 1966). Conversely,
abnormal exchanges of genetic material could also result in the deletion of a portion of
the Y chromosome containing TDF and, therefore, produce an XY female phenotype.
Molecular analysis of XX males revealed the presence of Y chromosome markers in a
number of these patients, validating the initial hypothesis (Ohno, 1979). The goal was
then to define the smallest fragment of the Y chromosome necessary to produce a male
phenotype in an XX individuals. This positional cloning approach led
to the identification of a 35 kb Y chromosome region, translocated on the X chromosome
of 4 XX males and true hermaphrodites (Palmer et ai, 1989). This region contained a
gene, named SR Y (Sinclair et ai, 1990), encoding a 204 amino acid protein with the
ability to bind and bend DNA through an HMG (High Mobility Group) conserved motif
(Ferrari et ai, 1992; Giese et ai, 1992; Harley et ai, 1992; Giese et aI, 1994). Several
convergent arguments proved that SRY was TDF. SRY protein has biochemical
properties of a transcription factor (Harley et aI, 1992); it is localized in the expected
portion of the Y chromosome (Sinclair et ai, 1990) and its temporal profile of expression
is appropriate, since murine SRY is expressed between ElO.5 and El2.5,just prior to the
appearance of seminiferous tubules (Koopman et aI, 1990). More importantly, an XX
mouse transgenic for 14 kb of a genomic Y chromosome fragment containing SR Y
developed as a male (Koopman et at, 1991). Point mutations in SRY were also shown to
divert the fate of the bipotential gonad of an XY fetus from testicular to ovarian tissue
(Berta et aI, 1990; Jager et ai, 1990; Hawkins et ai, 1992a; Hawkins et ai, 1992b;
McElreavey et ai, 1992a; McElreavey et ai, 1992b; Muller et ai, 1992; Vilain et ai,
1992a; Affara et ai, 1993; Zeng et ai, 1993; Poulat et aI, 1994; Tajima et ai, 1994; Brown
et ai, 1998; Domenice et ai, 1998). These mutations were found in XY females with pure
gonadal dysgenesis.
Since the identification of SRY as TDF in 1990, a number of questions remain
unsolved. Does SR Y act as a transcriptional activator or repressor? What controls SRY
expression? What genes are controlled by SRY and what is its target? Does SRY bind to
other proteins? Does SRY explain all the human pathologies of sex detennination? Very
few of these questions have a satisfactory answer yet.
We have suggested that SRY may antagonize a repressor of male-detennining genes,
based on the autosomal recessive pattern of inheritance of SRY-negative XX males
(Vilain et aI, 1992b; McElreavey et aI, 1993). This suggested a loss-of-function mutation
in a gene normally repressed by SRY. This model is supported by in vitro evidence that
SRY can act as a repressor of transcription (Desclozeaux et aI, 1998), as well as by
observation of dosage sensitive sex reversal in XY individuals (Am et aI, 1994; Bardoni
et aI, 1994). However, one report suggests that SRY may also act an an activator of
transcription (Dubin & Ostrer, 1994).
SRY expression is spatially and temporally tightly controlled. It is expressed
specifically in the mouse genital ridges from days 10.5 to 12.5 (Koopman et aI, 1990). In
humans, expression of SRY in a variety of non-genital tissues has been noted, but its
physiological significance remains elusive (Clepet et aI, 1993). It is interesting to note,
however, that SRI expression in the brain has been shown (Lahr et aI, 1995; Jordan and
Vilain, unpublished data). Whether it is specific to a brain region, and whether it has a
physiological relevance in terms of sexual behavior is still unknown. Control of SRY
expression is also still unknown. Promoter studies have shown the existence of a GC-
rich, TATA-Iess promoter, containing two Spl sites (Vilain et aI, 1992c), but did not
demonstrate the binding of a specific transcription factor that could explain the precise
regulation of SRY expression.
The target of SRY has also remained unclear so far. SRY contains an HMG box of 79
amino acids, similar to a group of non-histone proteins that associate with DNA (Jantzen
et aI, 1990). The HMG motif confers the ability to bind specifically to the sequences
AACAAAG and AACAAT (Waterman et aI, 1991; Harley et aI, 1992; van de Wetering
& Clevers, 1992; Harley et aI, 1994). SRY can also bind to DNA four-way junctions
with no specific sequence requirement (Harley et aI, 1994). SRY is also able to bend
DNA by angles of 60 to 85 degrees, suggesting a role for SRY in the modification of
chromatin structure, and/or in the assembly of transcriptional regulators (Ferrari et aI,
1992; Giese et aI, 1992; Giese et aI, 1994). However, no physiologically relevant target
gene has been successfully identified so far.
One of the main unsolved questions regarding SRY is the mechanism by which it
interacts specifically with its target, especially in the context of the existence of a large
family of HMG proteins binding to similar sequences. This may be achieved via
interaction with specific cofactors. One of these SRY co factors may be SIP1, which
contains a PDZ protein interaction domain, and was shown to interact with the last seven
amino acids of SRY (Poulat et aI, 1997). SRY's HMG box was also found to be a
calmodulin binding domain, suggesting a possible interaction of SRY with calmodulin
(Harley et aI, 1996). Finally, a variable stretch of glutamine is present in mouse SRY, but
absent from its human homologue. Transgenic experiments revealed that this domain is
necessary for murine SRY function, suggesting a potential interaction between the
glutamine stretch and another protein in the murine system (Bowles et aI, 1999).
Genetic studies of sex-reversed patients have shown that while SRY is present in 90%
of XX males without ambiguities, it is detected in only 10% of XX true hermaphrodites
and in only 10% of XX ambiguous males (McElreavey et ai, 1995). Conversely, SRY
mutations are found in only 25% of XY females with gonadal ***dysgenesis
SRY AND THE GENETICS OF SEX 5
(McElreavey et aI, 1992a). The variability of the phenotype observed in XX sex-reversed

patients carrying SRY, from normal male to ambiguous, could be explained by
differences in the pattern of X-inactivation between individuals (Abbas et aI, 1993). SRY
analysis is inadequate to explain the phenotype of all the patients with pathologies of sex
determination. For instance, we have shown that a completely normal male phenotype
could occur in an XX patient without any Y chromosome sequences including SRY
(Vilain et aI, 1994). This suggests that genes other than SRY are needed for normal male
development.
Non-Y Chromosomal Genes and Sex Determination
New genes important for sex determination were identified essentially in XY female
patients with chromosomal abnormalities. These karyotypic anomalies allowed the
characterization of regions of the genome that were probably responsible for the sexual
phenotype observed in these patients.
DAXI
Duplications of a region of the short arm of the X chromosome (Xp21.3) were found
in several XY females with gonadal dysgenesis (Am et aI, 1994; Bardoni et ai, 1994).
The shortest duplicated region of the X responsible for sex reversal was found to be long
of 160 kb, and was named DSS (Dosage Sensitive Sex-reversal) (Bardoni et aI, 1994).
Within DSS, DAX1, a gene also responsible, when mutated, for Adrenal Hypoplasia
Congenita, was cloned (Zanaria et aI, 1994; Guo et aI, 1995). DAX 1 encodes an unusual
member of the nuclear hormone receptor superfamily, with a typical ligand-binding
domain, but a novel putative DNA-binding domain containing 3.5 repeats of 65-67 amino
acids that may represent zinc finger structures (Zanaria et aI, 1994). Although its
physiological target is still unknown, DAX1 was shown to bind to single-strand hairpin
DNA motifs and to act as a repressor of transcription (Zazopoulos et aI, 1997). DAX1
pattern of murine expression is consistent with a role in sex determination. It is expressed
at E11.5 in gonads of both sexes (Swain et aI, 1996), which corresponds in males to the
peak of expression of SRY and to the first signs of testis differentiation. At E 12.5, DAX1
is turned off in the testis, but remains on in the ovary (Swain et aI, 1996). This suggests a
possible role of DAX1 in ovarian formation. DAX1 may also be part of genetic cascade
of successive inhibitions leading to testis formation (Figure 1). In addition, transgenic XY
mice carrying additional copies of DAXI develop as females, suggesting that DAX1 is
involved in sex determination by antagonizing the action of SRY (Swain et aI, 1998).
However, this was observed only with a specific strain of mice (Mus musculus
domesticus Poschiavinus), in which SRY is expressed at a relatively low level. No sex
reversal is observed with the more common mouse strain Mus musculus musculus,
leaving open the hypothesis that there are additional sex determining genes within the
DSS locus.
SOX9
Chromosomal rearrangements of chromosome 17 were observed in patients with

campomelic dysplasia (Tommerup et ai, 1993), a severe skeletal dysplasia in which a
majority of XY patients are phenotypic females. This allowed the cloning of SOX9, a
member of a family of transcription factors, the SOX genes, related by the presence of an
HMG box (Foster et ai, 1994; Wagner et ai, 1994). Point mutations in SOX9 associated
with campomelic XY females showed that it was a sex-determining gene (Foster et ai,
1994; Wagner et ai, 1994). It also binds to the same DNA targets as SRY in vitro
(Bardoni et ai, 1994; Bell et ai, 1997). Although the physiological target of SOX9
remains unknown, there is some evidence that it can regulate the transcription of MIS in
association with SFI (de Santa Barbara et ai, 1998). However, several indirect arguments
challenge the hypothesis that SOX9 regulates MIS expression directly. The fact that XY
patients with mutations in SOX9 are sex reversed while XY patients with mutations in
MIS are male (Behringer et ai, 1994) suggests the existence of a number of genetic
intermediates between these two genes. In addition, it was shown in chickens that MIS is
expressed prior to SOX9 (Oreal et ai, 1998), suggesting alternative regulation in this
species. Recently, an XX male patient was shown to carry a large duplication of
chromosome 17 including SOX9 (Huang et ai, 1999). This is the first example of XX
sex-reversal not caused by SRY in humans. It suggests that SOX9 may be antagonized by
SRY. We propose here a model in which SOX9 would in tum inhibit the action of
DAX 1, a repressor of testis development. This hypothetical cascade of negative
regulations is illustrated in Figure 1.
D --.J 0 --.J~-' 0 -. -.
o - - - - - - --- - - - - - - - - - - - - - - - - - - - - - - - -
XY Genotype: Testis
Development
SRY DAXI MIS
---+-
WT-l & SF!
xx Genotype:
O~D-.O
DAXI SOX9 MIS
Absence of
- . -'Testis
Development
Figure 1. Simplified model of gene interactions in male sexual development. In XY individuals, SRY
would antagonize DAXl, which would in tum inhibit SOX9, resulting in male phenotype. In XX individuals,
DAX! would inhibit the action of SOX9, resulting in the absence of male development. This model is
compatible with all molecular defects observed so far in pathologies of human sex determination.
SFl and WT-l:
Steroidogenic Factor 1 (SF1) and Wilms Tumor 1 (WT-l) encode two transcription
factors expressed as early as E9 during murine gonad development, which play major
roles in gonad morphogenesis (Pelletier et aI, 1991 a; Ikeda et ai, 1994). When disrupted
in a mouse knock-out model, the observed phenotype is the absence of gonad

development, associated with renal agenesis for WT-l (Kreidberg et aI, 1993), and
adrenal agenesis for SF 1 (Luo et aI, 1994). In humans, mutations in WT-1 are observed in
Denys-Drash and Frasier syndromes, consisting of severe renal disease and XY gonadal
dysgenesis (Pelletier et aI, 1991b; Bardeesy et aI, 1994; Barbaux et aI, 1997). Wilms
tumors are present only in Denys-Drash syndrome. In addition, a mutation of SFI was
identified in a patient with adrenal hypoplasia and XY gonadal dysgenesis (Acherman et
aI, 1999).
Wnt-4:
Wnt-4 is the only signalling molecule known to have a role in sex determination. It
was shown that a targeted disruption of this gene in an XX mouse leads to a male
phenotype (Vainio et ai, 1999). The authors suggest that Wnt-4 may have a role in the
development ofMiillerian structures as well as in ovarian differentiation.
Sex Determination, a Complex Network of Genes
All the sex-determining genes presented above are summarized in Table 1. They are
probably part of a complex genetic pathway leading to gonad differentiation. These genes
interact with each other via protein-DNA and protein-protein interactions. For instance,
an SF1-response element was found in the DAX] promoter (Burris et aI, 1995; Guo et aI,
1996), and it was shown that SF] upregulates the expression of DAXI in an
adrenocortical carcinoma cell line (Vilain et ai, 1997). DAXI and SFI also interact at the
protein level as part of a multi-protein complex (Ito et ai, 1997). In fact, it was
demonstrated that SFI acts synergistically with WT-l to upregulate MIS expression, and
that this activation could be blocked by DAXI (Nachtigal et aI, 1998).
Genes Involved in Sexual Differentiation
In contrast to sex determination, most genetic players of sexual differentiation are

known. They consist of all the genes encoding the enzymes for testosterone biosynthesis,
the gene encoding the androgen receptor, and the genes encoding MIS and its receptor.
Mutations in all these genes have been identified and are known to result in
pseudohermaphroditism, with normally developed gonads and abnormal differentiation
of internal and/or external genitalia.
The two most frequently encountered situations are androgen resistance and
congenital adrenal hyperplasia (CAR). Mutations in the androgen receptor can be
relatively easily identified by sequencing, although the large size of the gene has
hampered a systematic screening for mutations in XY females (Quigley et aI, 1995). In
the case of CAR, the diagnosis is suspected when the levels of 17-hydroxyprogesterone
are high in an XX intersex patient and is easily confirmed by genetic screening for a few
well characterized mutations (White et aI, 1987).
Table 1. Genes Involved in Sex Detennination
Gene Localization Gene family Putative Phenotype of mutations

function
SF-I 9q33 Nuclear receptor Transcription Gonadal dysgenesis and adrenal
factor insufficiency
WT-I IIpl3 Zinc finger Transcription Denys-Drash and Frasier
protein factor syndromes
SRY Ypll HMGprotein Transcription XY gonadal dysgenesis
factor
DAX Xp21,3 Nuclear receptor Transcription Duplication: XY gonadal
1 factor dysgenesis
Mutation: Adrenal Hypoplasia
Congenita
SOX l7q24 HMG protein Transcription Duplication: XX sex reversal
9 factor Mutation: Campomelic dysplasia
with XY~onadal dysgenesis
MIS 19q13 TGF- Growth factor Persistent Mullerian Duct
Syndrome
Wnt- ? Wnt Growth factor XX sex reversal in mouse
4
Conclusion: A Molecular Approach to the Diagnosis of Intersex

Patients
Despite many ffilssmg pieces of the genetic puzzle of sexual development, our
knowledge of the mechanisms of sex determination
and sexual differentiation has recently improved considerably. This molecular
understanding allows a new diagnostic approach to the patient with sexual ambiguity.
Molecular tools provide a more rapid and more precise method to establish a diagnosis.
We propose here a simplified decision tree for the diagnosis of intersex patients including
the possibilities given by molecular analysis (Figure 2).
xx (nonCAH)
A
SRYpresent SRYabsent
xx male
/
xx true hennaphrodite
Maternal exposure to
XX true
progesterone or
hermaphrodite
androgens
xx male
Xy
B
Uterus present
Uterus absent
Normal renal andlor

adrenal function 1 Adrenal disease
j
Low testosterune Nonna! or high
Renal dlsease
testosterone
1 Mutation in WT-J
1 /
Mutation in SF-I
j
1
Mutation in SR r or SOX9 5·(X·reductase
Duplication of DAXI deficiency Mutation in
androgen receptor
Testosterone synthesis defect
Denys·Drash syndrome Leydig cell hypoplasia
Frasier syndrome
XV gonadal dysgenesis Androgen

resistance
Ftgure 2. Simplified decision tree for molecular diagnosis of sexual ambiguities. A: XX karyotype, B: XY
karyotype
Figure 2. Simplified decision free for molecular diagnosis of sexual ambiguities. A: XX karyotype, B: xy
karyotype.
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Questions for Dr. Vilain
Question - I am curious about the expression of SRY in the brain? At what stage of
development was that? It does not coincide with the development of the gonad.
Answer - So far we have done it at 11.5 days worth, and it also, expressed in the gonad.
Weare doing the full profile, but so far it is expressing the brain at the same time as it is
expressed in the gonad which is a short window of development between 10.5 and 12
days in the mouse. We are doing a full study of SRY ontogeny in the mouse brain.
Question - I would like to take an issue with you about the fact that the brain is a little
gonad from the point of view that the SRY and DAX1 are actually determining the
gonads and subsequently the gonads pattern the brain.
Answer - It is very true. At this time it is just an interesting observation that there is SRY
gene expression in the brain. We have not shown you everything today, but it is
expressed in specific structures within the brain and this observation has to be validated
by more complex behavior analyses and experiments in the mouse. For example,
producing a transgenic mouse that will have expression of SRY only in the brain without
any steroid interference. I would agree entirely. This is just an observation at this point
in which I thought was both relevant and interesting.
Question - At a recent seminar there seemed to be two positions about SRY. One is that
SRY is a mammalian gene, that is either on the way out, in terms of evolution, or still
very important for sex determination. What is your position?
Answer - I kept saying that SRY was conserved throughout evolution and it is actually
evolving. If you look at SRY among primates, and we are also doing that in the
laboratory, it is evolving extremely rapidly and it is rare for a gene, which is important in
development, to evolve rapidly and most of the developmental genes are very highly
conserved. We do not know why SRY and DAXI evolve rapidly, but this is what seems
to be happening. We do not know if it is actually because they are very important to stay
on top of the hierarchy, that they have to adapt. They have to run fast to stay at the same
place. In evolution there is this so called red queen hypothesis, or like in Alice in
Wonderland, the red queen has to run very fast to stay at the same place. You can
imagine that SRY could be in this position to stay on the top of the hierarchy, it has to
evolve rapidly. Another point of view would be to say, "well, it is just going to be on its
way out". But, I do not think so. For reasons that we do not understand, there is some
sort of selective evolution. We do not know what the forces are that push us away to
evolve rapidly, but I think it is a very important gene.
Question - Ninety percent of 46XY true hermaphrodites did not show up as having SRY,
SOX-9, Wnt-I, or DAXI expression. How do you propose that they develop testicular
tissue?
Answer - It just means that we still have a lot to learn about other sex determining genes
and there have to be mutations in other genes. We actually have familial cases of XX
true hermaphrodites who do not have SRY and do not have any mutations in the known
sex determining genes and these familial cases segregate in a recessive way which means
that there must be a more genes involved that we do not yet know about. So the answer
is we need to analyze more patients to understand what is going on in this sex experiment
to a hermaphrodite. But there must be mutations in other sex determining genes, for sure.
Question - How rapidly can you make a molecular diagnosis?
Answer - It depends. If it is peR based it is a matter of days. If it is like looking for a

presence or absence of SRY or looking for sequence mutations in SRY, I would say
realistically it takes about a week. If it's based on Southern techniques such as looking
for a duplication index one, it takes about 2 to 3 weeks.
GONADAL DIFFERENTIATION - NORMAL AND
ABNORMAL TESTICULAR DEVELOPMENT
Faruk Hadziselirnovic, Dale Huff

The Basel Children's Hospital, and the Department of Pathology, Children's H05pital of
Philadelphia
INTRODUCTION
This chapter concerning normal and abnormal testicular development represents 28

years of our experience based on semi-thin histologic sections of over 13,000 testicular
biopsies mostly from patients with cryptorchidism and male infertility. The series also
includes testicular biopsies from 30 testes of complete Androgen Insensitivity Syndrome
(ArS), 20 of mixed gonadal dysgenesis, 24 of XXY Klinefelter syndrome, 6 of male
Turner syndrome (Noonan syndrome), 10 of XX-male syndrome, and 10 of hernia uteri
inguinalis (persistent Mullerian duct syndrome). Special effort was taken to compare
abnormal testicular development from intersex patients with that of normal testicular
development from birth to adulthood.
Normal Testicular Development
The development of the normal human testicle is a continuous process from birth until
puberty (Hadziselimovic, 1977). The diameter of seminiferous tubules increases
constantly until the 1t h year. But the dramatic increase in its diameter occurs only after
the development of the lumen.
Testicular Development During the First Year of Life

Gonocytes, spermatogonia and Sertoli cells are evident in the seminiferous tubule
immediately after birth. The gonocytes, the primitive reproductive cells, are located
primarily in the center of the tubule and tend to migrate towards the basement membrane.
There are, therefore, two distinct types of cells: those that eventually come in contact
with the basement membrane evolve into spermatogonia, the largest cells in the infant
seminiferous tubule. The most common cell in the seminiferous tubule in the first year of
life is the Sertoli cell, an oval or polarized cell which, by definition, is always in contact
with the basement membrane. These are small cells (4911J.3) that perform many functions,
including phagocytizing, hormone-producing and a nutritive supporting role.
Pediatric Gender Assignment: A Critical Reappraisal 15

16 F. HADZISELIMOVIC, ET AL
The peritubular connective tissue forms the wall of the seminiferous tubule; it is
composed of the basement membrane consisting of one layer of collagen fiber and
fibroblasts. These cells form concentric rings around the tubule. The interstitium contains
mainly well-developed fetal Leydig cells that remain until the second year.
Development During the Fourth Year of Life
The ultrastructural appearance of the seminiferous tubule at this age is quite different
from that of a one-year old boy. Gonocytes are no longer evident. In addition to the A-
type spermatogonia already present in a one-year old child, B-type spermatogonia are
encountered for the first time. At the same time, primary spermatocytes are found in the
seminiferous tubule. The Sertoli cells have completed their transformation from fetal
cells into Sa- and Sb-type cells. The Sa-type cell is the most common of the Sertoli cells
in the seminiferous tubule in children this age. B-spermatogonia and primary
spermatocytes, and Sb-type Sertoli cells are found in an increasing abundance during the
fourth year.
Apart from the distinctive widening of the tubule, there are no qualitative changes in
the peritubular connective tissue. The basement membrane still consists of one layer, and
the knob formation is not discernible. The collagen fiber layer is wider and its cellular
stratum is composed of fibroblasts. The interstitium contains mainly precursors of Leydig
cells; occasionally, particularly between the ages of four and eight years of age, juvenile
Leydig cells are clustered around the vessels.
Changes Occurring During Puberty
During puberty the final development of all four testicular components is completed.
The Sertoli cells increase significantly in size, as much as five times their previous state;
and complete the final transition to the Sc-type cell. The number of Sertoli cells has
steadily decreased from birth to puberty. The lumen of the seminiferous tubule is
pronounced. Degenerating cells are seldom apparent. Germ cells are now comprised of
spermatogonia, primary and secondary spermatocytes, and spermatids. Late spermatids
were not evident in the testes before the age of fourteen; and they occur only when the
Sertoli cells are fully developed.
Gonadotropins influence changes in the peritubular connective tissue. The basement
membrane becomes multilayered; collagen fibers form a matrix in which fibroblasts
transform into myofibroblasts. The seminiferous tubule acquires its characteristic
contractility, and the blood testis barrier is established subsequently.The Leydig cells at
this time are well differentiated, having a particularly noticeable increase in the amount
of smooth endoplasmic reticulum. However, as yet, no Reinecke's crystalloids are
evident.
GONADAL DIFFERENTIATION 17
The Significance of Postnatal Gonadotropin Surge for Testicular

Development in Normal Testes
A sharp increase in serum testosterone has been observed in the normal male infant at
about 2 to 3 months of age (Forest et aI, 1974); an event that appears to be blunted
significantly in the child with cryptorchidism (Gendrel et aI, 1978). The elevation of
testosterone occurs as a consequence of an increase in plasma gonadotropin levels
(Winter et aI, 1976) but the physiological reason for the phenomenon remains an enigma,
although some believe that it is important for virilization of the hypothalamus (Grumbach
et aI, 1974). As the development of the testes is followed chronologically, it becomes
apparent that whenever there is an increase in testosterone, transformation of the
gonocytes into spermatogonia occurs
(Hadziselimovic, 1977; 1983). Impairment of this transformation adversely affects later
fertility. Those postnatal changes occurred in the normal testes at the same time that there
was an increase in both the luteinizing and follicle-stimulating hormones during the first
few months of life; these changes are believed to be a consequence of this hormonal
activity. Therefore, neonatal activation of the hypothalamic-pituitary-gonadal axis is
essential for adequate transformation of gonocytes to spermatogonia (Hadziselimovic et
aI, 1986).
ABNORMAL TESTICULAR DEVELOPMENT
Complete Androgen Insensitivity Syndrome
Although complete AIS is rare, found in from 1 in 60,000 to 1 in 100,000 male births,
it is the most common form of male pseUdohermaphroditism (Brown et ai, 1993). The
phenotypic expression of AIS is extremely variable and ranges from patients with normal
female external genitalia to patients having male external genitalia with hypospadias
(Brown et aI, 1993). Rare patients may even be otherwise completely normal males with
azoospermia (Aiman et aI, 1982).
We have evaluated semi-thin section of testicular biopsies from 15 patients with
complete AIS. It is a common belief that prepubertal patients with complete AIS have
normal testicular development (Muller, 1984; Rutgers et aI, 1987). In contrast, in 10 of
the 12 cases in our biopsy series, abnormal testicular development is obvious as early as
the first month of age. At this age, the transformation of gonocytes into adult dark (Ad)
spermatogonia is absent even though the total germ cell count is within normal limits,
Severe Leydig cell hyperplasia is characteristically present at this age, but Sertoli cell
development is unaffected. After the age of three, no sperrnatocytes were observed in our
prepubertal patients. As prepubertal development advances, 80% of patients progress to
absence of germ cells or Sertoli cell-only syndrome (SeQ). Leydig cell hyperplasia
becomes more severe. By late prepubertal life, Sertoli cell development becomes
abnormal. While in adulthood, the remainder of this group loose all germ cells, and the
entire group has severe Leydig cell hyperplasia.
The remaining 3 of the 15 patients with AIS had phenotypic female external genitalia
but varying degrees of masculinization of secondary sex characteristics and less severe
testicular histopathology representing those incomplete variables of complete AIS. The
transformation of gonocytes into Ad spermatogonia occurred but was impaired in
infancy. In the prepubertal period, spermatogenesis was impaired, and maturation arrest
at the primary spermatocyte level was present in adults. These may subsequently become
the 30% of patients with AIS over 50 years of age who have carcinoma-in-situ and
seminoma (Rutgers et ai, 1987).
All 15 AIS-patients had intraabdominal or high canalicular located testes. Despite the
similar testicular positions, the two distinct patterns described above emerged. In 80%,
the transformation of gonocytes into Ad spermatogonia failed completely while in 20%
the transformation occurred but was incomplete. This supports the idea that this critical
transformation of gonocytes into Ad spermatogonia is testosterone dependent and
temperature independent. Cryptorchidism, a forme fruste of hypogonadotropic
hypogonadism, gonadotropin deficiency causes Leydig cell hypoplasia and reduced
testosterone production that causes defective germ cell transformation. In AIS, defective
testosterone receptor function blocks the action of testosterone which causes defective
gonocyte transformation, and furthermore, blocks the negative feedback on hypothalamic
and pituitary level, inducing increased LH release, and finally, Leydig cell hyperplasia.
XXY Klinefelter Syndrome
Klinefelter syndrome is a relatively common chromosomal abnormality found in 1 m

700 to 800 males (Rutgers et aI, 1987). The karyotype is XXV in 80%; the rest have other
complications of multiple X- and one or more Y chromosomes, or mosaic karyotypes
with one of the cell lines being XXV (Zang, 19XX). The adult testis is atrophic and
histologically exhibits severe tubular atrophy, peritubular hyalinosis, and nearly complete
loss of germ cells and Sertoli cells. Severe Leydig cell hyperplasia is always present.
Spermatogenesis can be found in histologic sections of testicular biopsies of only
occasional young adults, and in these the spermatogenesis is abortive. Siebeman found
evidence of spermatogenesis in only 4 of 20 patients with Klinefelter syndrome
(Siebman, 1958). It was incomplete in all four. A few late spermatids were found in only
two (Siebman, 1958). In the testes of a 21 week fetus with Klinefelter syndrome, Leydig
cell hyperplasia was already present. There were two types of germ cells present: two
disparate gonocytes, and the absence of fetal spermatogonia. The total germ cell count,
the number Sertoli cells, tubular diameter, and peritubular connective tissue were all
within the normal range. During prepuberty, tubular atrophy, loss of germ cells, Leydig
cell hyperplasia, and peritubular hyalinoses all increased with increasing age. The
indication for surgery was usually undescended testis.
The most obvious difference in the testicular histopathology of Klinefelter syndrome
and cryptorchidism is in Leydig cells. Leydig cells are severely hyperplastic in
Klinefelter syndrome and hypoplastic in cryptorchidism. A histopathological pattern
similar to that seen in Klinefelter syndrome was observed in 6 testes from three XX-male
patients and in 4 testes from two patients with Noonan syndrome ("male Turner
syndrome") indicating common pathogenetic mechanisms and histopathology in X

chromosomal anomalies.
The Persistent Mullerian Duct Syndrome
Anti-Mullerian hormone is secreted by Sertoli cells in fetal and prepubertal testes. It is a

member of transforming growth factor ~ superfamily of growth and differentiation
factors (Josso et aI, 1993), and is processed intracellularly and secreted in its mature bio-
active form (Cate et ai, 1986; MacLauglin et ai, 1991), It induces regression of the
Mullerian duct. Persistent Mullerian duct syndrome (PMDS) is a heterogenous condition,
an inherited autosomal recessive (sex-limited). Two anatomic forms of PMDS are
described. In the more prevalent form, there is a hernia containing a scrotal or an
incomplete descended testis. The ipsilateral Fallopian tube and occasionally a uterus are
in the hernia. All of the testes in our study belonged to this first form. In the second form
ofPMDS, the uterus, tubes, and testes are in the pelvis (Nachtigal et aI, 1996).
Testicular differentiation and function are normal (Grumbach et ai, 1998), but
increased prevalence of testicular degeneration has been described (Imbeaud et ai, 1995).
Early orchiopexy, proximal salpingectomy, and complete hysterectomy are recommended
as a useful surgical approach (Guerrier et ai, 1989:. Loeff et ai, 1994) During puberty,
germ cell histology ranged from normal number and morphology of late spermatids to
severe oligospermia.
All 5 patients studied were less than one year of age; their surgery was performed
because of inguinal testes or scrotal hernia. During the surgical exploration, tube and
subsequently uterus was found. All testes had a normal number of germ cells, and in 8
out of 10 testes, a few Ad spermatogonia were observed. Two patients were followed
throughout puberty and re-biopsied during andrological work-up because of infertility.
The testes had Leydig cell hyperplasia and Sertoli cells loaded with lipid indicating a
metabolic dysfunction of these cells. The fact that these patients had Leydig cell
hyperplasia and severe germ cell arrest despite early orchidopexy (in their first month of
life) indicates that defective Sertoli cell metabolism adversely affects Leydig cell-Sertoli
cell ultra-short hormonal loop and Leydig cell hyperplasia with oligospermia develops.
Therefore, not all patients with PMDS will benefit from an early surgical procedure.
Mixed Gonadal Dysgenesis
Mixed gonadal dysgenesis is characterized by gonads composed histologically of a

mixture of streak gonad and testis combined with a variety of genital malformations. The
most common karyotype is XO/XY mosaicism. Cryptorchidism is present in 90%. The
external genitalia are ambiguous. Testicular neoplasms,
gonadoblastomas, and seminoma occur in 9 - 25% and are observed as early as a few
months of age (Hedinger et aI, 1991). The typical histopathology in prepubertal gonads
are: (a) developmental arrest of the tubules which resemble those of embryos; (b)
frequent atypical germ cells; (c) severe interstitial fibrosis; (d) Leydig cell hypoplasia of
juvenile forms; (e) Sertoli cell maturation arrest at the Sa stage; and (j), an ovo-testis or
streak-testis constellation in 28%. Because of the high incidence of gonadal neoplasms,
the testes are generally removed in early pre-pubertal life. Therefore, we have no
experience with the histopathology in later pre-puberal life, puberty, or adulthood.
REFERENCES
Aiman .I, Griffin .IE. The frequency of androgen receptor deficiency in infertile men. J Clin Endocrinol Metab
54: 725-732, 1982.
Brown TR, Scherer PA, Chang Ying-Tai, Migeon CI, Ghirri P, Murano K, Zhou F. Molecular genetics of
human androgen insensitivity. Eur J Pediatr 152:(Suppl 2) S62-69, 1993.
Cate RL, Mattaliano RJ, Hession C, et al. Isolation of the bovine and the human genes for Mullerian inhibiting
substance and expression of the human gene in animal cells. Cell 45:685-689, 1986.
Forest MG, Sizonenko PC, Cathiard AM, and Bertrand .I. Hypophyso-gonadal function in humans during the
first year of life. 1. Evidence for testicular activity in early infancy. J C/in Invest 53: 819, 1974.
Gendrel 0, Job JC, and Roger M. Reduced post-natal rise of testosterone in plasma of cryptorchid infants. Acta
Endocr 89:372, 1978.
Grumbach MM, Roth JC, Kaplan SL, and Kelch RP. Hypothalamic-pituitary regulation of puberty: evidence
and concepts derived from clinical research, in: The Control of the Onset of Puberty. MM Grumbach, GO
Grave, FE Mayer., eds., John Wiley and Sons, New York, Chapter 6, pp. 115-181,1974.
Grumbach M, Conte F. Disorders of sex differentiation, in: Williams Textbook of Endocrinology, J Wilson, D
Foster, eds., Saunders, 1998.
Guerrier 0, Tran 0, Vanderwinden JM et al. The persistent Mullerian duct syndrome: a molecular approach. J
Clin Endocrinol Metab 68:46-52, 1989.
Hadziselimovic F. Funktionelle Morphologie des kind lichen Hodens im ersten Lebensjahr. Acta Anat 99: 342
abstract 378, 1977.
Hadziselimovic F. Cryptorchidism: Ultrastructure of Normal and Cryptorchid Testis Development. New York.
Springer- Verlag, 1977.
Hadziselimovic F. Cryptorchidism: Management and Implications. New York: Springer- Verlag, 1983.
Hadziselimovic F, Thommen L, Girard J, and Herzog B. The significance of postnatal gonadotropin surge for
testicular development in normal and cryptorchid testes. J Urol136: 274-276, 1986.
Hedinger C, Dhom G. Pathologie des manlichen Genitale. Springer Verlag Berlin, Heidelberg pp. 70-100, 1991.
Imbeaud S, Rey R, Berta P, et al. Testicular degeneration in three patients with the persistent Mullerian duct
syndrome. Eur J Pediatr 154: 187 -190, 1995.
Josso N, Picard .lY, Imbeaud S, Carre-Eusebe D, Zeller J, Adamsbaum C. The persistent Mullerian duct
syndromne: a rare cause of cryptorchidism. Eur J Pediatr 152:(Suppl 2) S76-S78, 1993.
Loeff D, Imbeaud S, Reyes HM, et al. Surgical and genetic aspects of persistent MUllerian duct syndrome. J
Pediatr Surg 29:61-65, 1994.
Maclaughlin DT, Epstein J, Donahoe PK. Bioassay, purification, cloning, and expression of MUllerian
inhibiting substance. Methods in Enzymology 198:358-369, 1991.
Moller .I. Morphometery and Histology of gonads from twelve children and adolscents with the androgen
insensitivity (testicular feminization) syndrome. J c/in Endocr Metab 59:785-789,1984.
Nachtigal MW, Ingraham HA. Bioactivation of Mullerian inhibiting substance during gonadal develpment by a
kex2/subtilisin-like endoprotease. Proc Am A cad Sci USA 93:7711-7716, 1996.
Rutgers .lL, Scully RE. Pathology of the testis in intersex syndromes. Semin Diegn Pathol4: 275-291, 1987.
Siebeman R. Die Gonadendysgenesien: Klinefeltersyndrom, Tumersyndrom und echter Hermaphroditismus.

VivchowsArch [A] 331:417-440, 1958.
Winter lSD, Hughes I, Reyes FI, and Faiman C. Pituitary-gonadal relations in infancy: 2. Pattems of serum
gonadal steroid concentrations in man from birth to two years of age. J Clin Endocr Metab 42: 679, 1976.
Zang KD. Genetics and cytogenetics of Klinefelter's syndrome:, in: Klinefelter Syndrome, Hl Baudmann, R
Brut, E Perwein, eds., Springer Verlag, New York, pp 12-23, 1991.
Questions for Dr. Hadziselimovic
Question - With regard to your patients with the persistent Mullerian duct syndrome, you
mentioned he had two patients that you followed through puberty. Were both those
unilateral undescended testes?
Answer - Yes.
Question - Did not have an occasion to biopsy the good testicle?

Answer - We biopsied both testes at the time when they were adults, because of
andrological work-ups and they were the same.
Question - What was done to the persistent Mullerian ducts? Were they removed and if
they were do you think there was damage, perhaps distally downstream to the vas that
sometime is very adherent? If there was damage to the distal vas as it got close to the
ejaculatory ducts, perhaps you were seeing some back pressure affects.
Answer - They were removed. That was a common policy at the time. I do not think that
there is damage downstream although I cannot be sure of that. However, the contralateral
testis appeared almost identical to the cryptorchid testis which was taken down. These
findings are definitely different from those 150 biopsies taken from vasectomized males
at the time of vasecetomy reversal. They show completely different testicular
histopathology.
Question - Were those two patients azosperrnic then?
Answer - They were not completely azosperrnic. They were Oligo-terato-sperrnic, but
they did have some sperm.
Question - It has always puzzled me Faruk, how it is that the high undescended testicles
which have no germ cells form germ cell tumors so often. Where do the germ cells come
from, that do that?
Answer - Yes, they have germ cells. The high undescended testes are completely normal
in the first three months of age and they do not have any transformation, they have a
gonocyte. They have a normal number of the genn cells in the first three months and
then they rapidly go down. And this non transformation of the gonocyte into the
spermatogonia. This is the main reason, I think, why they develop neoplasia later on.
Question - When you say that there are no germ cells present, they are present?
Answer - They are very present. The picture was taken at four years of age, four
different groups of boys, four years of age. If they were to be three months of age or two
months of age, this picture would be completely different.
Question - So they are there somewhere?
Answer - Yes, the germ cells are there, but their gonocytes, in the first three months you
have in the intra-abdominal testes predominately gonocytes and they do not transform.
This lack of transformation is crucial.
Question - In your complete androgen insensitivity patients, were the epididymis and the
vasa normal?
Answer - We have fifty patients of thirty testes biopsied what I get from all around the
world. I do not have all clinical pictures of those patients. That what I got, they were not
normal. The epididymis was not completely normal, but it was there. We also had some
cases in which the clinicians told us it is called complete androgen insensitivity yet they
had some remnants. They had a little bit uterus they found. It is not completely clear.
Question - If we stay with the Wolffian structures for a moment, I think that to most
clinicians in the CAIS patients the epididymis appears pretty normal. And 10hn Hodack
has asked the question as to whether this is the case. I would not like to diminish the
contribution of the French to sex; but if lost's experiments are correct, how do we explain
the W olffian development in the CArS patients.
Answer - The only explanation that I can offer you now, just of the question without
thinking to much would be that we do not understand completely the so-called complete
androgen insensitivity syndrome that must be receptor defect at different level and a
different time. And, this timing of when this receptor defect occurred and how much of a
defect we have give us a final clinical picture.
Question - I think one of the hypotheses proposed to explain these histories that there is
some type of hybridization between cells that do have normal androgen receptor and
some cells that have the complete androgen insensitivity, or the complete defective
receptor. I also believe that this hypothesis has gained more and more ground. Also, I
would like to ask you about a brief question about the incidents of malignant tumors on
patients who have complete androgen insensitivity. From my clinical experience we
have seen a lot of patients that we take the testes out later and later and so far I do not
know of many cases in which we do find clear malignancies. Can you comment on the
incidence of malignancy?
Answer - r have had almost 20% from those 30 testes what we have analyzed (it is not
too much) we did not find carcinomas inside of those testes. No atypical spermatogonia,
however, it has been said in literature that the patient has to go into his fifties to get a
tumor and all of our patients had been in surgery around the age of thirty. The oldest one
was forty.
Question - About the W olffian ducts differentiation into vas deferens and epidydimis. It
has been shown that it is not dependent on DHT receptor. It is directly dependent on
testosterone secretion and again like for MIS action it is dependent on testosterone action
inside the pelvis and not in the peripheral blood. So that accounts for why there are
normal vas deferens and epidydirnis in complete sensitivity to androgen. I have a
question about complete androgen insensitivity testes. We are discovering, more and
more, that these testes are accompanied by benign tumors such as hamartoma or
leiomyoma. Now we look for them and we find them quite in every case. Have you the
same experience?
Answer - I must admit I received only the biopsies, which represent a limited sampling.
I did not have the complete testes so I cannot answer that question.
MULLERIAN INHIBITING SUBSTANCE:
AN UPDATE
David T. MacLaughlin, Patricia K. Donahoe

Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts
General Hospital, Harvard Medical School, Boston, MA
INTRODUCTION
Among the many problems to be addressed when considering gender assignment, or

re-assignment in some cases, is determining gonadal status. Anatomical sexual
ambiguity can arise from abnormal fetal reproductive tract development secondary to a
failure of gonadal organogenesis. A major determinant in establishing phenotypic sex is
the expression of genetic sex early in fetal life, thereby leading to the differentiation of
indifferent gonads into testes or ovaries. Subsequently, and depending upon the type of
gonad that develops, male or female internal and external genitalia grow and differentiate
form the Wolffian and Mullerian ducts, respectively. Following the expression of the
testis determining factor gene locus, SRY on the Y chromosome (Goodfellow and
Lovell-Badge, 1993) the newly formed testes secrete Mullerian Inhibiting Substance,
MIS, also known as anti-Mullerian Hormone, AMH, from the Sertoli cells and the Leydig
cells produce testosterone. MIS interacts with its cell surface receptors to cause the
regression of the female reproductive tract precursor, the Mullerian duct, and testosterone
stimulates the growth of the androgen-dependent male reproductive tract including the
external genitalia. In the absence the Y chromosome, ovaries will develop and the
male genital tract atrophies due to the lack of testosterone. The female tract completes
differentiation into the uterus, Fallopian tubes, cervix and upper third of the vagina in
utero autonomously without sex-hormonal stimulation (for a recent review see
MacLaughlin et. al. 2001). Loss of function mutations of either the MIS gene or its cell
surface receptor gene leads to the persistence of Mullerian structures in males (for a
review, see Belville et. al 1999).
Of particular interest to a discussion of MIS and intersex anomalies is the fact that
MIS exhibits a sexually dimorphic expression pattern and that it is produced after birth at
a time period when testosterone is not. Although MIS gene transcription begins in tat the
end of the third trimester in fetal ovaries (Rajpert-De Meyts et. al. 2001), MIS protein is
not usually detectable in female serum until five or six years of age where it is always at
levels many fold below those of males until puberty.
Once human specific assays were established to measure MIS in biological fluids
including serum (Hudson et. al. 1990, Baker et. all. 1990 and Josso et. al. 1990), the
power of these measurements in certain clinical settings became immediately apparent.
For example, MIS is a very useful marker of granulosa cell (Lane et. al. Rey et. a1.1996)

Edited by Zderic et at., Kluwer Academic/Plenum Publishers, 2002
26 D.T. MCLAUGHLIN, ET AL.
or sex cord (Gustafson et. aI.1994) tumor burden in women because most of these lesions
over express the protein. In other cases such as non-palpable gonads, serum MIS is a
more accurate indicator of the presence of testes than measuring testosterone, particularly
in pre-pubertal children (Lee et. aI. 1997). Furthermore, MIS and testosterone levels
assess different testicular compartments. Testosterone is an indicator of Leydig cell
function particularly in response to gonadotropin stimulation and MIS is a measure of
Sertoli cell function. As is discussed below, however, serum MIS can be a more precise
predictor of normal testicular histology and, therefore, testicular function (Lee et. aI,
1997). Therefore, serum MIS, measurements provide useful clinical information to aid in
the assessment of gonadal status of the intersex patient. This chapter reviews MIS
biology in normal individuals and the usefulness of measuring serum MIS in certain
intersex and other gonadal is discussed.
The MIS Protein and Its Receptors
Regression of the Mullerian ducts, a critical event during normal male fetal sexual
development, is mediated by the gonadal hormone Mullerian Inhibiting Substance, MIS,
also known as anti-mullerian hormone, AMH. The existence of MIS was first predicted
by Professor Alfred Jost who, in a series of in vivo rabbit experiments, ruled out
testosterone as the testicular product responsible for Mullerian duct regression in males
(Jost 1947, 1953). This finding explains why the female pseudohermaphrodite in
congenital adrenal hyperplasia retains Mullerian structures and the male
psuedohermaphrodites due to androgen resistance do not. Although sufficient androgens
are secreted to virilize the female patient, there are no testes, therefore, MIS is not
produced. In males completely resistant to testosterone the Mullerian ducts regress in
utero because the testes still secrete bioactive MIS.
MIS is a homodimeric glycoprotein of 140 kilodaltons, is a member of the
transforming growth factor -beta (TGF-~) family of peptide hormones that regulate
growth and differentiation (figure 1) (Lane et. aI. 1998; Josso et. aI., 1993;; Lee et. aI.
1993; for a review see Teixeira et. al. 2001). The human MIS gene is located on
chromosome 19 (Cohen- Haguenauer et. al.) and has 5 exons and 4 introns. The protein
is a homodimer of two identical subunits held together by disulfide bonds (Cate et. al.
1986). In the human, each subunit is 535 amino acids in length and each is glycosylated
at N-linked sites in the amino terminal domain of the protein (Cate et. aI. 1986).
Approximately fifteen percent of the total protein is carbohydrate.
The source of MIS in males was first identified as Sertoli cells in calf testes (Josso et.
a1.1973) and ovarian granulosa cells were shown to synthesize MIS in rodents (Takahashi
et. al. 1986) and in human (Seifer et. al. 1993; Rajpert-De Meyts et. al. 200). MIS is
synthesized as a pro-hormone which is proteolytic ally cleaved by biosynthetic proteases
(Nachtigal and Ingraham et. al. 1996) to yield the 25 kilodalton bioactive carboxy-
terminus. The carboxy-terminus is then secreted along with the amino terminus in non-
covalent association. A mutated form of MIS that lacks this cleavage site has been
created in the laboratory (Kurian et. al. 1995) and, as expected, it is not biologically
active. Among mammals, the carboxyterminal domain of MIS is very highly conserved
MULLERIAN INHIBITING SUBSTANCE 27
(for a review see Teixeira et. al. 2001) varying only in one or two residues in the108
amino acid fragment. Therefore, species specific immunoassays employ antibodies that
interact with the amino terminal 110 kilo dalton region of MIS. Therefore, serum MIS
immunoassays detect the species specific aminoterminal domains and not necessarily
biologically active species of the protein, In fact patients with persistent Mullerian duct
syndrome and measurable serum MIS have been reported (Imbeaud et. al. 1994). Among
these false positives were cases in which MIS gene was truncated to completely delete
the carboxyterminal domain.
Although the specific mechanisms of human MIS gene regulation are not fully
understood, a number of transcription factors have been shown to up- or down-regulate
MSI gene expression. Among these are TFII-I which interacts with initiator elements
close to the transcription start site (Morikawa et. aI. 2000), the steroidogenic factor-I, SF-
1, which recognizes the proximal promoter (Wong et. al 1997; Parker et. aI. 1999), and
Sox 9, an Sry related homeobox gene (Wagner et. al 1994) all of which are required for
MIS gene transcription. The GATA-4 DNA binding protein acts in synergy with SF-l
(Viger et. aI. 1998; Tremblay and Viger 1999, 2001), WT-l (Nachtigal et al. 1998)
enhances MIS expression while Dax-l, a gene associated with adrenal hypoplasia and sex
reversal (Bardoni et. al. 1994; Muscatelli et. al., 11994), inhibits MIS gene expression
(N achtigal et. al. 1998). The specific factors that control the sexually dimorphic
expression of MIS, however, remain to be completely elucidated.
Human MIS Gene
Transcription 1 Translation
:~
i
53S aa
S3S iIiI
Cleavage Site
Blosynt.etlc Protease 1
Bioactive MIS Protein
~= AminOrermmus t;arboxyterminus
Figure 1. Drawing of the MIS gene and protein product (partial mutation map).
Signaling by MIS and other TGF-B family members is mediated by two types of
serine/threonine kinase receptors, called type I (Visser et. at 2001; Clarke et. al., 2001)
and type II (Baarends et. al., 1996; di Clemente et. al. 1994; Teixeira et. al. 1996) (figure
2). These two receptors are single membrane spanning molecules similar chemically and
differ in size. The type II receptor has 11 exons and 10 introns. Receptor activation
occurs when MIS binds first to the type II receptor, which then recruits the smaller 55-60
kilodalton type I receptor to form a heterodimeric complex. Phosphorylation of the type
I receptor by the type II receptor causes receptor-specific downstream molecules such as
the sma homolog of mothers-against-decapentaplegic (SMAD) molecules to associate
with the type I receptor and initiate a signaling cascade. The Smads 1,5 and 8 are
signaling molecules for the MIS type I receptor, also know as ALK2 (activin-like kinase
receptor 2) (Clarke et. al. 2001; Visser eLl. 2001).
Human MIS Type II Receptor Gene
IExtracellular
I~
Transmembrane
Domain
Intracellular Domain
/
Domain
Figure 2. DraWing of the MIS type II receptor gene (partial mutation map).
Another type I receptor candidate for the MIS type II receptor is ALK 6 which co-
precipitates with the type II receptor in certain transfected cells (Gouedard et. al. 2000).
However, it is not expressed in fetal Mullerian duct mesenchyme and animals in which
this gene was knocked out do not have retained Mullerian ducts (Yi et. al. 2000).
Therefore, ALK-6 may playa role in MIS action in other tissues.
As expected, the MIS type II receptor and the type I receptors are co-expressed in the
cells surrounding the fetal Mullerian duct, in the fetal gonads, and in the pubertal and
adult rat gonad as is the (Clarke et al. 2001).
Non Mullerian Activities of MIS
A non-Mullerian fetal activity of MIS may be to slow the pace of male fetal lung
development (Catlin et. al. 1997). Gestational prematurity more often affects the lungs of
male infants than females and a possible reason why this may be true is the influence of
MIS on the lungs. As gestation approaches term, lung development is completed in both
sexes, possibly due to the loss of MIS responsiveness in the males, with females reaching
maturity sooner than the males.
Postnatal, non-Mullerian roles for MIS in both males and females are areas of active
investigation. Normal fertility found in male and female MIS deficient "knockout" mice
suggests that MIS is not required for normal gonadal junction in rodents (Behringer et. ai,
1994) but the possibility has not been excluded for other animals. For example, MIS
inhibits estradiol production by human granulosa-luteal cells (Kim et. al 1992) and that
follicular MIS concentrations are inversely related to the mitotic index of granulosa cells
(Seifer et. al. 1993) indicating a role for MIS in the functioning of normal ovarian
follicles. Furthermore, Fallat and colleagues showed that human sperm binds MIS, and
that sperm from men with sperm abnormalities had decreased binding, suggesting a
potential role for MIS in human spermatogenesis (Fallat et. aI, 1998). Recently,
inhibition of basal and gonadotropin stimulated testosterone synthesis by MIS in vivo and
in vitro was demonstrated (Teixeira et. al. 1999; Trbovich et. al 2001) indicating a
possible paracrine role for MIS in postnatal testes, as well. It is clear that there are
numerous, possible non-Mullerian target tissues for MIS and the future directions of MIS
research will include more extensive characterizations of such tissues.
Developmental Changes in Serum MIS Concentration
The expression of MIS is sexually dimorphic. In human males MIS is first secreted
during the seventh to eighth week of gestation, when it causes dissolution of the
mesenchyme surrounding the Mullerian ducts (figure 3). The period of Mullerian duct
responsiveness to MIS is brief- limited to 6 to 9 weeks, and complete regression occurs
by 9 112 weeks gestation (Taguchi et. aI, 1984). Because the granulosa cells of the ovary
do not secrete measurable amounts MIS prenatally, MIS only affects the sexual
development of the male fetus.
Several assays have been developed for the measurement of serum MIS concentrations
in humans (Baker et. aI, 1990; Hudson et. aI, 1990; Josso et. al., 1990). The enzyme
linked immunosorbent assays (ELISA) use mouse monoclonal antibodies raised against
recombinant full-length human MIS. The assays have lower limits of detection in the 0.5
nglml range and they do not recognize inhibin, activin, LH, or FSH. It is important to
note that in comparing clinical MIS studies the various established assays have not been
uniformly standardized. Each of the assays uses different preparations of recombinant
human MIS for standards, and unique antibodies for the detection of MIS. Thus, the
normal reference ranges are specific for each assay. In spite of these differences,
however, results obtained by the various assays are qualitatively comparable.
MIS Expression is Sexually Dimorphic

Fetus Neonate Puberty Adult
MIS, male
\.
Testosterone
\,........
. /. . . . . ii'! ""
...... 1
' . ./.'' ' I
--- ......... --
MIS, female MIS, tumor
Figure 3. Graph showing the relationship of serum MIS vs testosterone vs age and sex.
Using an ELISA, our laboratory has established the mean and normal ranges for serum
MIS concentrations from infancy to adulthood in both sexes (Lee et. aI, 1996). Six
hundred serum samples obtained cross-sectionally and longitudinally from 288 normal
males and from 179 females were collected from subjects at the Massachusetts General
Hospital and several other institutions for MIS determination. MIS concentrations in
male infants are low at birth with a mean of 6.8 ng/rnl at day 1 to 3 of life. Serum levels
rise four fold to a mean of 26 ng/rnl by 3-30 days of age, they continue rising during the
first six months of life to a mean peak of 115.5 ng/rnl, and then decline from late infancy
until puberty and adulthood when the mean value is 4.0 ng/m!.
MIS values in female infants are even lower or undetectable at birth and remain low
throughout the first year of life, with a mean of 0.66 ng/m!. MIS concentrations rise
slowly in childhood and puberty to a peak of 4.7 ng/ml at 14 - 16 years of age, and then
decline. After menopause, levels become undetectable. There is no overlap between the
lower limits of normal in boys and the upper limits of normal in girls before the age of
three. In fact, median MIS values in males are 10·20 fold higher than in females until the
age of 12 years old. However, with declining male levels, extensive overlap of the male
and female normal ranges exists by the time of puberty.
The dimorphic pattern of MIS expression in serum, particularly the inverse
relationship of serum MIS and testosterone concentrations, can be exploited in the study
of intersex anomalies and in the valuation of patients with certain gonadal tumors.
CLINICAL UTILITY OF SERUM MIS l\lEASUREMENTS
Serum MIS in Children with Intersex Conditions
MIS determination has been very useful in the evaluation of children with non-
palpable testes and ambiguous external genitalia (Gustafson et. al. 1994; Rey et. al 1999).
Chronically elevated MIS levels are often definitive Jin patients with ambiguous genitalia
abnormalities. MIS is absent in patients with congenital adrenal hyperplasia, but very
high in patients with Male Pseudohermaphroditism (Gustafson et. al 1994) despite
indistinguishable appearance of the external genitalia, which are discordant with the
chromosomal sex. Anorchic subjects, on the other hand, have low or undetectable serum
MIS, as predicted by the absence of Sertoli cells. Intermediate serum MIS levels were
detected in patients with Mixed Gonadal Dysgenesis, depending upon how much
destruction of the gonad had occurred (Donahoe and Schnitzer, 1996). The assay has
been very helpful in patients with true hermaphroditism after partial gonadectomy to
confirm that all testicular tissue has been removed in patients to be raised as females
(Gustafson et. al. 1994).
Prior to the development of the MIS assay, only the functional capacity of the Leydig
cell component of the testis could be assessed, by measuring testosterone. Obtaining a
basal testosterone concentration may not be helpful diagnostically, because unstimulated
testosterone levels in prepubertal males and females are usually indistinguishable (figure
3). Thus, the more involved approach of sampling testosterone following stimulation
before and after gonadotropin treatment is often used in the evaluation of ambiguous
genitalia and non-palpable gonads. Serum MIS measurements in such patients assesses
the Sertoli cell activity and facilitate the diagnostic process. Rey and colleagues have
also reported low testosterone coupled with low or undetectable MIS in ambiguous
genitalia cases is consistent with the diagnosis of testicular dysgenesis (Rey et. aI., 1999).
Lee et. al (1997) and Rey et. al. (1999) agree that normal MIS for age indicates the
presence of normal Sertoli cell function and that ambiguity in these males may relate to
defects in androgen synthesis or action.
Therefore, MIS measurements should be used in conjunction with those for
testosterone in patients with ambiguous genitalia. This approach allows for assessment
of both the Sertoli cell functional capacity and the Leydig cell steroidogenic capacity, and
is specific for detecting histo-pathologically abnormal testes. Undetectable MIS
associated with non-palpable testes and a normal phallus, for example, is consistent with
the absence of testes as is true in a male with vanishing testes. The failure of testosterone
to increase following hCG stimulation in a patient with undetectable serum MIS confirms
anorchia or fully virilized congenital adrenal hyperplasia, and a rise in testosterone would
suggest the presence of testes and the diagnosis of persistent Mullerian duct syndrome,
PMDS, indicating the need for surgical exploration.
Patients with PMDS have 46, XY karyotype, a normal phallus and retained Miillerian-
derived structures, often with unilateral or bilateral cryptorchidism. Loss of function
mutations in the MIS gene (Imbeaud et. al. 1994) or in the MIS type II receptor gene
(Belville et al. 1999) are the most frequent cause of PMDS and the serum MIS
concentration may be used to predict the molecular basis of the disorder. Patients with
mutations in the MIS gene have low or low-normal MIS concentrations, while those with
mutations in the MIS type II receptor have high-normal or elevated concentrations. No
mutations in either the gene or receptor have been identified in a third group (20 % of
cases) of patients (Josso et. a1. 1997).
Serum MIS can also be useful in evaluating cases of partial androgen resistance. The
phenotype of these patients is often ambiguous and a specific diagnosis is often difficult
to confirm. Rey and coworkers measured serum MIS in subjects with impaired
androgen secretion or androgen resistance (Reyet. a1.,1994;1999). These patients had
normal or elevated MIS values, while subjects with gonadal dysgenesis had low or
undetectable MIS. Therefore, elevated MIS concentrations are indicative of impaired
androgen action, but additional tests are required to make the specific diagnosis of
androgen insensitivity.
MIS Measurements in Patients with Sex Cord and Granulosa Cell

Tumors
Stromal tumors ovary, such as granulosa cell tumors (GCT) and sex-cord tumors with
annular tubules (SCTAT) are often hormonally active and secrete the peptide hormones
inhibin and MIS, as well as large amounts of estrogens, and sometimes androgens.
Because MIS values in adult females are low, MIS determination is useful to identify the
presence of tumor to evaluate the extent of surgical removal of the lesion, and to assist in
monitoring the recurrence of disease (Gustafson et. aI., 1992; Gustafson et. aI., 1993;
Lane et. aI., 1999; Rey et. a1. 1996).
Granulosa cell tumors are categorized into adult GCTs, occurring in women in the
fourth decade and older, and Juvenile GCTs (JGCT), typically occurring in children and
young women. Adults can present with signs of abnormal estrogen exposure including
irregular or postmenarchal bleeding, breast swelling and tenderness, and abdominal
symptoms such as distention or cramps including non-centrally mediated precocious
puberty with or without abdominal symptoms in young girls. At diagnosis, 80 - 90% of
patients have stage I disease, with tumor confined to the ovarian capsule and an excellent
prognosis with 96% long-term survival. The long-term survival of those with higher
stage tumors decreases to 30%. Recurrent adult GCTs are typically slowly growing,
with a poor prognosis, while recurrences in JGCT are more rapid and fulminant with a
mean survival of just over a year.
In our stUdy, serum MIS concentration, measured preoperatively was elevated in 75%
of patients with JGCT, and in 78% of subjects with adult GCT. Rey and colleagues also
reported elevated MIS concentrations in 8 of 9 patients with GCT pre-operatively (Rey
et.al, 1996). Serum MIS, therefore is elevated in the majority of patients with GCT, and
would be of benefit as a tumor marker. In cases where postoperative samples only were
measured, nearly one third had at least one elevated post-operative value. Recurrent or
incompletely resected tumor was observed in 40%, and 25% had sustained, significant
rises suggesting the presence of small, slowly growing tumors not yet detectable
clinically. One third had with no evidence for tumor but had a transient rise in MIS
concentration with a return to undetectable levels, suggesting the need for serial sampling
to confirm elevated values. Finally, there was no evidence for clinical recurrence in
subjects who had normal postoperative MIS concentrations.
MIS determination, therefore, is helpful in the follow-up of women with GCT that
secrete MIS. A pre-operative MIS value provides a baseline for comparison with
postoperative values, which is especially important when a normal contralateral ovary is
not removed. The only known source for circulating MIS is either granulosa cells or
gonadal-derived tumor cells, and therefore, any sustained, measurable values following
bilateral oophorectomy indicates either incompletely resected tumor, or recurrent tumor.
Elevations in serum MIS concentration due to the presence of recurrent tumor may
precede any clinical or radiographic evidence of the tumor, and such a rise would merit
more frequent examinations and/or imaging to search for the source. We recommend
obtaining MIS values at semi-annual intervals for 4 to 5 years in all patients with adult
GCT's and those with stage I JGCT's. More frequent testing is recommended for any
JGCTs with extension outside the ovarian, as these tumors have a rapidly progressive
course.
Our group has measured serum MIS in a small number of SCTA T patients and the
findings very similar to those with GCT. We recommend obtaining a pre-operative value
and obtaining semi-annual MIS measurements in post-operative patients with this type of
sex cord stromal tumor (Gustafson et al. 1992).
Use of MIS in the Treatment of Ovarian Epithelial Carcinoma
Ovarian tumors derived from the coelomic epithelium are the most frequent cause of
gynecological cancer deaths, and despite advances in the treatment of these tumors the
overall survival has not changed significantly over the past twenty years. Because
ovarian epithelial tumors originate from the same cells that into the Mullerian ducts, these
tumors may retain responsiveness to MIS. Thus, the possibility of using MIS as a
chemotherapeutic agent is being explored. Expression of the MIS type II receptor mRNA
and protein in human ovarian cancer cell lines was demonstrated by reverse transcriptase
PCR (RT-PCR), and Northern and Western blot analysis. The presence of the MIS type
II receptor on the cell surface was then confirmed by the binding of biotinylated MIS in
flow cytometry experiments Masiakos et. al. 1999). These ovarian cancer cell lines
were cultured in semisolid agar and the ability of MIS to inhibit colonies growing under
these conditions was greater than fifty percent in 75% of the experiments.
Primary human ovarian cystoadenocarcinoma cells from ascitic fluid collected from
patients surgically also express the MIS type II receptor immunohistochemistry
(Masiakos et. al. 1997, 1999). These experiments demonstrate that ovarian cancer cell
lines and primary tumor cells from many patients express a MIS type II receptor that
binds MIS, and that colony formation is inhibited in the presence of MIS. Furthermore,
these experiments suggest that pre-screening ovarian tumor patients for active expression
of the type II receptor may allow the selection of those most likely to have a beneficial
response to treatment with MIS. Currently, large-scale production of purified rhMIS is
being planned for anticipated human trials in patients with ovarian epithelial cancer.
Currently, serum MIS measurement has diagnostic utility in the evaluation of children
with non-palpable gonads with or without ambiguous genitals, in the pre- and post-
operative management of women with ovarian sex-cord tumors, and in the
immunohistochemical evaluation of these tumors. Ongoing studies on the therapeutic
uses of MIS offer promise for the treatment of ovarian epithelial tumors. As our
knowledge of both the prenatal and postnatal biological roles for MIS continue to be
discovered and defined, the clinical applications of MIS may expand from strictly
diagnostic uses to exciting new therapeutic areas, particularly for chemotherapy.
SUMMARY
The decades long study of Mullerian Inhibiting Substance by numerous laboratories

around the world has been driven, in large part, by pediatric surgeons and pediatric
endocrinologists who have a keen interest in the molecular pathophysiology of genital
tract defects that are visited upon their patients. A better understanding of the genes
involved in the development of the normal reproductive tract in males and females should
lead to a more rational analysis of the diseases caused by their abnormal function.
Furthermore, a translation of this knowledge from the bench to the bedside may lead to
clinically useful advances in the diagnosis and management of intersex patients. The
molecular analyses of MIS and MIS receptor gene mutations and persistent Mullerian
duct syndrome and the development of MIS ELISAs to evaluate testicular function as
well as to follow the progress of gonadal tumors are several clear examples of successes
over the years. It will be interesting to see what lies ahead.
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Questions for Dr. MacLaughlin
Question - What happens when you have a testis on one side and an ovo-testis or an
ovary on the-other side in terms of how this will affect the child's internal anatomy?
Answer - The cases with testis on one side, ovo-testis on the other results in complete
Mullerian regression. With an ovo-testis on one side and an ovary on the other there is
also complete regression. A very small amount of MIS in a very small window of time
(maybe a week in the human, days in a mouse) causes complete Mullerian regression. If
you wanted to repeat the Jost experiment that was referred to earlier very, very small
amounts of MIS do the job, so I do not think that there is a dose response. It seems much
more qualitative at the early going. Its gene is turned on, and the rest happens very
quickly.
Question - I do not agree with your last answer because we have shown that MIS is
active inside the pelvis and not with elevated peripheral blood concentrations. We have
quite a huge series of true hermaphrodites, and when there is one testis on one side and
one ovary on the other side, there is a hemi-uterus on the side of the ovary and the MIS
concentration is not normal but not very low. I would like to add two comments. When
dealing with sex assignment in an intersex newborn we somehow rely on the level of
MIS to predict whether there will be a good uterus or any uterus or not. And for us it is a
great argument to raise these children as a girls when there is a hemiuterus or total uterus
so MIS may be useful. The other comment is that we have come with Natalie Joso to use
MIS level in fetal serum as an argument to predict what will be the choice of sex of
rearing after birth. So if we come to a prenatal diagnosis of ambiguous genitalia, we
always screen cord blood to know what is MIS level because before birth testosterone
level is not very discriminating between girls and boys; therefore, we see a great
advantage of measuring MIS levels before birth.
Answer - I am very interested in your last comment. I think that is a terrific idea to look
at the fetal cord blood. We tried to look at the amniotic fluid to look at this as an
indicator of sex of the embryo early, and it was not helpful at all. We often thought of
doing what you have done, and that is great. As to the effects of the siting of oval tests
versus testis and hemi-uterus, we have not got any more evidence than we have
developed in vitro which was with implants and we always had a complete response.
Since we are using exogenus MIS we may be dealing with a completely different story
than what was seen normally in the fetus.
Participant - In vitro, there is a lateral effect. If you put the MIS crystal on the lateral part
of the pelvis, it will only act on that side. If you put it in the middle, it will act on both
sides.
Question - Is the MIS receptor linked to androgen transport?
Answer - I do not know the answer to that. The most recent work on the receptor has
been to do two different types of investigations. One is the pathophysiology of intersex,
where you have a nice clinical example and go back and clone the gene and sequence it.
The other is try to do experiments that a number of labs are doing, including ours, which
is to transfect the gene and look at specific down stream regulation. But the transport has
not been one of them yet.
Question - Because you have the elevation of MIS in the androgen insensitivity, patients
now wondered if that was cause and effect?
Answer - Yes, I think if you look at the sort of add back experiments this certainly is the
endpoint. Where that happens is not clear. For example, the MIS gene does not have an
androgen receptor element that anybody can find. downstream. There was an estrogen
receptor element discovered a long time ago, but it is a thousand bases away from the
start site of the gene. So there is definitely the effect, but the mechanism is not
completely worked out.
40 D.T. MCLAUGHLIN. ET AL.
Question - I noticed a couple of your testicles at low MIS levels and I was just wondering
if you had looked within an individual on an hourly or daily basis, do you get sort of a
circadian variation of MIS?
Answer - We have done that in two setting. The short answer was no. We have tried it in
females after puberty when they have measurable levels of MIS to see if there was
anything to do with the ovarian cycle, not seen. The only data we have the serial data we
have is when gonadectomies were performed or surgery was done to remove granulosa
cell tumors and we have looked at the decay and then looking only daily though. We
have not looked for very, very short time periods for pulses.
Question - How do you explain the persistence of the prostatic utricle in phenotypically
normal males? Is there a sequential regression of the Mullerian system?
Answer - We only have sort of theoretical answers for that. At least that is all I have.
The very important time and the temporal relationships between expression of MIS, MIS
receptor and their subsequent activities seems to be very, very tightly controlled and there
must be examples where that is slightly aberrant and activity is not completely seen.
Other than that, I do not have an answer. Maybe with these newer tools for identifying
receptor and downstream signaling molecules, we can look at that at a molecular level.
MICROPENIS: AN ANIMAL MODEL AND ITS HUMAN
CORRELATES
Douglas A. Husmann, M.D.

Division of Pediatric Urology, Mayo Clinic, Rochester Minnesota
DEFINITION AND GENERAL COMMENTS
Micropenis is defined as a perfectly formed abnormally small penis, with a stretched

penile length that is < 2.5 standard deviations below the established mean value for race.
The psychological concern and anxiety of the parents and patient regarding the adequacy
of penile size makes treatment of this entity challenging. Since the early 1970's androgen
therapy for micropenis has been the mainstay of tre:atrnent. Although testosterone will
usually enhance penile growth in the prepubertal microphallic patient, not all of the
infants or children that experience a response to androgens maintain their enhanced
phallic size into adulthood. Indeed several of these patients will redevelop micropenis as
they mature. Although most of the adults with micropenis adjust well to the male sexual
role and develop stable heterosexual relationships, some refuse to become sexually
active. Indeed occasional patients will prefer to maintain a life of sexual abstinence,
rather than face their fears of rejection. The social and psychological consequences seen
within this latter select group of patients is what prompted our interest into research on
penile development and the treatment of micropenis. (Schonfeld et ai, 1942; Walsh, et aI,
1978; Allen, 1986; Lee, et ai, 1980; Money, 1984; Reilly et ai, 1989; Woodhouse, 1998).
Current Controversies Regarding Micropenis
There are currently two major controversies that exist in the field of micropenis.
These are: 1) Does intermittent infantile and childhood androgen treatment substantially
increase phallic size in adulthood or does it only bring about premature penile growth?
2) Do patients with persistent micropenis in adulthood have significant gender
dysmorphia and/or psychosexual abnormalities? If so, should infants with androgen
insensitive micropenis be sexually reassigned? {Allen, 1986;Guthrie et aI,
1973;Bumstein et ai, 1979; Money, 1984,1985; Reilly et aI, 1989; Reiner, 1997,
Diamond et ai, 1997; Husmann et ai, 1998; Woodhouse, 1998).

42 D.A. HUSSMANN
Physiology of Penile Growth
The growth of the penis is classically defined into two different categories, androgen
dependent and androgen independent penile growth.
Androgen Dependent Penile Growth
Androgen dependent penile growth is believed to be responsible for 70-75% of the

adult penile length. Three important periods of penile formation and/or growth are known
to be under androgenic control: 1) virilization of the penile shaft and early fetal penile
growth 2) neonatal penile growth and 3) pubescent penile growth.
Virilization of the phallic shaft, that is, formation of the male urethra and early fetal
penile growth occurs from gestational weeks 8-18. During this formative time interval,
the penis increases little in length, an average of only 2-3 mm. Although it was
previously believed that virilization of the phallic shaft was predominantly controlled by
placental gonadotropin (heG) current experimental evidence suggests that initiation and
early function of the Leydig cell is autonomous and not dependent upon the presence of a
gonadotropin during gestational weeks 8-12. The concept that autonomous Leydig cell
activity is responsible for the initial production of testosterone and virilization of the
phallic shaft is based upon three important facts: One, fetal luteinizing hormone (LH) is
not detected until the 12th week of gestation. Two, the receptors for heG or LH
(necessary prerequisites) for gonadotropin stimulation of the Leydig cells to secrete
testosterone, are absent from the fetal testis during this time span. Three, patients with
gonadotropin receptor defects may have histologically normal Leydig cells that fail to
respond to gonadotropin stimulation. Physical evaluation of these patients reveal a
perfectly formed but microphallic penis. After the 12th week of gestation the fetal
hypothalamic and pituitary gland initiate their function reaching peak fetal LH values in
the 22nd week. It is believed that the growth of the penis, from gestational weeks 12-18 is
probably under gonadotrophic control. It is unclear if the maternal heG or the fetal
gonadotropins are chiefly controlling the fetal surge of testosterone during this time span
since both are elevated within the fetal serum. Ironically fetal testosterone levels decline
between gestational weeks 14-18 reaching sexually indifferent values after the 18th week.
This decline in fetal testosterone occurs despite high levels of fetal (LH) and placental
(heG) gonadotropins and is due to direct suppression of the fetus's testicular
steroidogenesis by material estradiol and prolactin.
Neonatal penile growth is stimulated by two different androgen surges and occurs
between birth to 6 months of age. An extremely rapid and dynamic androgen surge
occurs within 24 hours after birth. This initial surge of androgens is believed to be do to
the loss of direct testicular suppression by high levels of circulating maternal estrogens
and prolactin. As the maternal estrogens and prolactin continue to decrease in the fetal
serum, loss of the inhibitory control of the neonatal hypothalamus occurs, this results in a
rise in fetal LH and eventually translates into a second surge in neonatal testosterone. The
second increase in fetal testosterone begins between 5-7 days postpartum and reaches its
peak in the 2nd to 3rd month of life, by the 6th month of life serum testosterone levels
fall to sexually indifferent levels, where it will remain until puberty. During this initial 6-
months of life the neonatal penile length will increase by 5-8 mm.
MICROPENIS: AN ANIMAL MODEL 43
Pubescent penile growth occurs between 12 to 18 years of age. Average penile length
gained during this time period is approximately 10 em. It is noteworthy that the growth of
the penis at puberty may in part be detenruned by the fetal and neonatal surges of
testosterone. In specific, experimental data has demonstrated that either an excess of
and/or the lack of androgens during key points of embryogenesis can result in a
diminished growth response of an organ to testosterone, i.e., androgen imprinting. It is
therefor theoretically possible that inappropriate androgen secretion during fetal or
neonatal life may permanently impair the ability of the penis to grow in response to
androgens at puberty. (Schonfeld, 1942;Feldman et ai, 1975;Danish et ai, 1980; Husmann
et ai, 1998; Reyes et aI, 1974; Wilson et aI, 1973; Catt et aI, 1975; Hostetter et ai, 1984;
Naslund et ai, 1986; Rajfer et aI, 1979; Even et ai, 1991; George et aI, 1979).
Androgen Independent Penile Growth
Penile growth is not always correlated to elevated levels of testosterone. Indeed,

approximately 25-30% of penile length is obtained when androgen levels are sexually
indifferent. There are two major penile growth intervals that appear to occur in the
absence of androgens. The first happens between 20-40 weeks of gestation and ends with
the neonatal testosterone surge. During this time period penile growth reaches its greatest
velocity, with penile length increasing by approximately 2.5-3 cm. The second interval
for androgen independent penile growth begins after the neonatal androgen surge is
complete. This period extends from 6 months of age until the onset of puberty. During
this time the average penis grows approximately 2..5 em in length. In total, 5-5.5 cm of
penile growth (average penile length 16.7 cm) occurs at a time that androgen levels are
sexually indifferent. See figure 1.
The growth of the penis during the apparent absence of androgens suggest one of two
possibilities, first, is the biological phenomena of two step steroid amplification. In the
first step testosterone or dihydrotestosterone directs the activation of a few specific genes,
which in tum will produce first step proteins. The first step proteins induce a multitude
of additional genes to produce second step proteins. In this hypothesis the second step
proteins are responsible for the physiologic effect of the hormone, i.e. penile growth. If
this hypothesis is true, androgen independent penile growth is actually a misnomer, with
penile growth occurring in the absence of androgens due to a delay in the production of
the second step proteins. An alternative hypothesis is that an androgen independent
growth factor or factors may playa minor role in penile growth. Androgen independent
growth factors may act directly on the penis or may indirectly enhance penile growth by
modulating androgen physiology. The most likely non-androgen hormone to alter penile
growth is growth hormone. The hypothesis that growth hormone or the growth hormone
activated growth factor, Insulin Like Growth Factor - 1 (ILGF-l) maybe partially
responsible for penile growth is based upon the following findings: 1) Patients with
congenital growth hormone deficiency or Laron dwarfism (dwarfism due to a growth
hormone receptor defect) may have micropenis despite normal serum levels of
androgens. 2) Both growth hormone and ILGF-l aid in gonadal growth and enhance
androgen production. 3) Growth hormone deficiencies result in impaired 5 a. reductase
activity and a quantitative decrease in the penile androgen receptor. 4) ILGF -1 has been
44 D.A. HUSSMANN
documented to result in proliferation of penile fibroblasts in the absence of androgens.

These accumulative findings suggest that growth hormone secretion and ILGF-l
production may either directly or indirectly affect penile growth. (Schonfeld, 1942;
Feldman et ai, 1975; Danish et ai, 1980; Money et ai, 1984; Husmann et ai, 1998; Alberts
et ai, 1983; Isaksson et ai, 1988; Levy et ai, 1996).
16 400
- Penile length en
E --- Testosterone Q)
.£. 12 --
.. Sexually indifferent 300 >
~
.E!2
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t:
t: ,,'...... ....
0
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I I ,
.E!2 I ' I Ci.i
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I '
I
,
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4 I:
I '
'
,
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ro \
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0 0 en
8 12 1822 30 1 2 3 6 1 2 3
_ Gestation ~ Growth 1 Growth 1
1
(weeks) -~ (months) - - (years) -
Birth
Figure 1. Serum Testosterone Levels Correlated to Penile Growth: Human (Fetus to 3 years of Age).
Hormonal and stretched penile length values for fetal and neonatal ages have been derived from a variety of
sources. (Schonfeld et al. 1942; Feldman et ai, 1975; Allen, 1986; Money et al. 1984; Husmann et ai, 1998).
The Use of Animal Models to Study Human Diseases
Animal models allow us the ability to select, control and isolate experimental factors
that would be technically impossible or impractical to alternate within the human
population. This is especially true for a disease process, like micropenis, where the
population affected is limited in number. In a condition with a restricted number of
affected individuals, clinical treatment trials require a multi-institutional study with
decades of patient accumulation and follow-up necessary to provide conclusive data. The
use of animal models allows us to acquire useful insight into the problem in a short time
interval. If the experimental design and animal model are perfect the physician should
gain tremendous insight into the pathophysiology of the disease, allow the development
of new and/or alternative treatment plans and substantially circumvent the length of time
required to develop an effective cure. The ideal animal model to study a human disease
process should exactly mimic the human condition and occur naturally within the species.
In determining the applicability of the findings of the animal model to the human
condition, vanatIOns between the two species regarding their anatomical and
physiological differences must be taken into account.
Etiologies of Micropenis (Human)
Micropenis within the human is due to a variety of etiologies. Combined data from
several large case reports reveal that approximately 50% of all patients with micropenis
will have hypo gonadotrophic hypogonadism, 25% will have hypergonadotrophic
hypogonadism, 15% will have end organ hormonal insensitivity, predominantly due to
androgen insensitivity from growth hormone defects, and 10% are idiopathic. Due to the
multiple etiologies of micropenis it is impossible to have a single representative animal
model to investigate this disease process. To limit the scope of our experimental
endeavors we elected to investigate micropenis using an animal model for the most
common etiology of micropenis, hypogonadotrophic hypogonadism. (Husmann et aI,
1998).
DEVELOPMENT OF AN ANIMAL MODEL FOR MICROPENIS
Micropenis Secondary to Hypogonadotrophic Hypogonadism (Murine

Model)
Although we have developed multiple animal models for the investigation of

micropenis the most clinically applicable is a naturally occurring strain of mice with
hypogonadotrophic hypogonadism. This murine strain has a defect in the gonadotrophic
releasing hormone region of chromosome 14. Animals that are homozygotic for this
defect have an isolated hypothalamic gonadotropin releasing hormone defect resulting in
hypogonadotrophic hypogonadism with micropenis. (Husmann et aI, 1994; Levy et aI,
1996; Tietjen et aI, 1998a; Tietjen et aI, 1998b).
Animal Model for Micropenis: Anatomical Differences of the Penis

(Rodent versus Human)
The chief anatomical difference in the penile structure between rodents and humans is
the presence of the os penis. The os penis is an androgen sensitive phallic bony structure
that along with the penile corporal bodies make up the bulk of the penis. High
concentrations of androgen receptors are found within the os penis during puberty with
down regulation of the receptors and subsequently loss of androgen responsiveness found
at adulthood. No analogous anatomical structure can be found within the human phallus.
(Takane et aI, 1991; Origuchi et aI, 1998).
46 D.A. HlJSSMANN
Animal Model for Micropenis: Cessation of Penile Growth (Rodent

versus Human)
During maturation the penis is stimulated to begin its pubescent growth spurt by
androgens. Interestingly, at adulthood growth of the penis stops despite high levels of
circulating testosterone. The cessation of phallic growth is associated with an 80%
decline in penile androgen receptor levels. The decline in the penile androgen receptor at
maturation is particular to that organ, with high levels of androgen receptor activity
persisting in other androgen sensitive structures after puberty. It is controversial whether
the cessation of phallic growth is due to the down regulation of the penile androgen
receptors or if the receptor's down regulation is a direct consequence of the cessation of
penile growth.
Penile androgen receptor down regulation and cessation of phallic growth is
discordant between the rodent and the human. In the rodent androgen receptor down
regulation is dependent upon both the cellular component of the penis along with the age
of the animal. Significant down regulation of penile androgen receptor is found to occur
with maturation in the corpora cavemosa and as penis. With androgen receptor down
regulation occurring to a much lesser extent in the urethra and penile skin. In the rodent
down regulation of the penile androgen receptor precedes the cessation of phallic growth.
See Figure 2.
Human data regarding the relationship of the penile androgen receptor to cessation of
penile growth is somewhat scientifically flawed. Specifically, unlike the rodent where
we have measurements of all of the penile components (penile shaft skin, urethra, corpora
cavemosa, os penis), we have only the analysis of human penile shaft skin for evaluation.
Nevertheless, human studies reveal that down regulation of the penile androgen receptor
occurs at 21-22 years of age. Interestingly, cessation of penile growth in the human
occurs at 17-18 years of age, or 3-4 years prior to penile androgen receptor down
regulation, see figure 3. These findings suggest that cessation of penile growth occurs in
the human at a time when penile androgen receptor expression is high, with down
regulation of the receptor resulting as a consequence of penile growth cessation. See
figure 3.
The discordant results between the between the human and the rodent regarding the
down regulation of the penile androgen receptor and the cessation of penile growth may
be due to either an interspecies variation or due to a variation in the expression of penile
androgen receptor in the human penile skin versus the rodent's penile body, i.e., corporal
tissues and os penis. It is however, absolutely important to note that the contradictory
findings between rodents and humans may have a significant impact on the applicability
of using the rodent penis as a model for human phallic development. Although the
timing of the down regulation of the penile androgen receptor is different between
humans and rodents, what is similar is that once down regulation of the penile androgen
receptor has taken place, additional androgenic stimulation of penile growth does not
occur. (Husmann et aI, 1998; Takane et aI, 1991; Rajfer et ai, 1980; Roehrbom et aI,
1987; Takane et aI, 1990; Gonzales-Cadivid et aI, 1991; Takane et aI, 1991b; Baskin et
aI, 1997).
400
--
-0-
Penile weight
Androgen receptor
'1.2
'1.0
Oi
:::1-
::::
0
Oi 300
.s 0.8
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~
.E 0
0) E..
'CD 200 0.6 (I)
:= 0
~
.~ 0.4 c::
c:: (I)
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a.. 100 0 0~
"" 0.2 "0
" ""'0.-- ___ «
c::
0 0
4 8 12 16
Age (weeks)
C8J
CA·t'4414B-Q1.
Figure 2. Penile Androgen Receptor Levels Correlated to Cessation of Penile Growth: Rodent. Penile
androgen receptor content was detennined by western blot analysis of homogenized penile tissue.
Quantification of the androgen receptor protein was accomplished by using a Scatchard analysis of a standard
preparation of androgen receptor from the cytosol of a Dunning rat prostate tumor sample. See reference 31 for
methodology. The mean androgen receptor content is given in fmol!j.lg of protein. This value is plotted against
the mean wet penile weight. Ten animals were evaluated at the various ages listed (Husmann, unpublished
data).
18 240
-e- Penile length
,
15 -0- Androgen receptor 200 al al
/°-0 c :J
E a Mean o (/)
~ 12 \ 160 (5
c-
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0 5 10 15 20 25
Age (years)
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Figure 3. Penile Androgen Receptor Levels Correlated to Cessation of Penile Growth: Human. The penile
androgen receptor content is expressed as fmol of [JH] methyltrienolone per gram of tissue. Analysis used the
direct hydroxyapatite assay. The sum of the cytosol and nuclear extract (total binding) is plotted against the
mean value for stretched penile length for age. The androgen receptor value plotted is the mean value for 5
different patient assays obtained from foreskins removed at the time of circumcision at the various ages listed.
See references Feldman et aI, 1975;Walsh et aI, I 978;Allen, I 986;Roehrbom et aI, 1987 (Husmann unpublished
data).
48 D.A. HUSSMANN
Animal Model for Micropenis: 5 Alpha Reductase (Rodent versus

Human)
In addition to the down regulation of the human penile androgen receptor that
transpires with age, 5 alpha reductase has also been documented to decrease within
human penile skin with maturation. In essence, it appears that the ability of the human
penis to respond to androgens becomes blunted with maturation due to the down
regulation of the penile androgen receptor and the progressive loss of the 5 alpha
reductase enzyme. At this time it is unknown if the rodents penile 5 alpha reductase
enzyme down regulates with age. (Husmann et aI, 1998; Wilson et aI, 1969).
Extrapolation of Animal Model Data to Humans. Prepubertal

Androgen Therapy for Micropenis Due to Hypogonadotrophic
Hypogonadism: How Effective Is It?
There are some authors who believe that the infant with micropenis at birth is destined
to have micropenis at adulthood. Treatment with testosterone doing nothing more than
causing premature growth of the penis. To evaluate this hypothesis we decided to
perform two separate investigations. One involves our murine animal model with
micropenis, the other involving a retrospective review of our patient population.
In our murine animal model we divided newborn mice into four different study
populations: hypo gonadotrophic hypo gonadal mice with and without testosterone
treatment, and normal wild type animals with and without testosterone treatment.
Testosterone therapy 2.5 mg 1100 gm body weight was initiated during the prepubertal
time span, i.e., day15 of life and continued at weekly intervals until 90 days of life. The
dose of testosterone chosen resulted in a serum testosterone level that was 1.5 to 2 folds
higher than normal 1 week after injection. This investigation revealed that
supraphysiologic levels of testosterone did not have a harmful effect on penile growth.
Specifically in the normal animals the mean wet penile weight (mean value of 10
animals) was 38.5 +/- 3.5 mgs (plus or minus one standard deviation) compared to a
mean wet penile weight of 41 +/- 4.5 mgs in normals treated with testosterone. The
hypogonadotrophic hypogonadal untreated micropenile animals were significantly
smaller than normal animals, mean wet penile weight of 8.5 +/-4.5 mgs, p < 0.001.
Treatment of the hypo gonadotrophic hypogonadal animals with testosterone significantly
increased the mean weight of the penis to 26.5 +/- 2.0 mgs. The testosterone treated
micropenis was significantly larger compared to the untreated micropenis, p < 0.001.
Unfortunately, the testosterone treated hypogonadotrophic hypo gonadal micropenis
remained significantly smaller than the normal penis, p < 0.001. In essence this study
revealed that testosterone significantly enhanced but did not normalize growth of
hypogonadotrophic hypo gonadal micropenis.
To see if our clinical data would match the animal studies we reviewed our patient
population with micropenis secondary to hypo gonadotrophic hypogonadism due to an
isolated gonadotrophic defect. A total of twenty patients were identified. Only nine of
these patients had received intermittent prepubeltal androgen therapy for micropenis.
Androgen therapy consisted of 25 mg of testosterone given 1M every 3 weeks for 4
injections. Median age at which the testosterone was first administered was one year of
age, range, 7 days of age to 6 years. If the penis did not respond by normalization of the
stretched penile length or if the penis grew adequately but later fell off of the standard
penile length growth curves, additional testosterone injections were given. The median
number of hormonal treatments was 4, the range was 2-8. Routine monthly testosterone
replacement was initiated at 12 years of age. Median standard deviation for the stretched
penile length at the time of initial treatment was -3.7, range -5.8 to -2.5. At a median
follow-up age of 18 years, range 16-22 yrs, only 22% (2/9) had maintained a stretched
penile length within normal limits. The median stretched penile length was-3.4, range -
5.9 to -2.3. It is however, noteworthy that our clinical data may be affected by a
significant referral bias. Specifically, 55% (5/9) of the patients involved in this study
were referred to us due to an inadequate response of the micropenis to testosterone.
The combination of animal and human studies suggests that prepubertal testosterone
therapy for micropenis due to hypogonadotrophic hypogonadism does not establish
normal penile growth. (Walsh et aI, 1978; Guthrie et aI, 1973; Burnstein et aI, 1979; Lee
et aI, 1980; Danish et aI, 1980; Money et aI, 1981; Reilly et aI, 1989; Husmann et aI,
1998; Tietjen et aI, 1998a, 1998b; Allen, 1978).
Penile Androgen Receptor Expression in Micropenis Due to

Hypogonadotrophic Hypogonadism
The recommendation to treat micropenis with intermittent prepubertal hormonal

therapy is based upon the fact that the normal penile androgen receptor is elevated during
the prepubertal and pubertal time phase with down regulation of the receptor occurring at
adulthood. Theoretically administration of androgens prior to the maturation induced
down regulation of the penile androgen receptor would result in optimal penile growth.
We hypothesized that failure of the micropenis to fully respond to androgens could be
due to 3 possibilities: An abnormal expression of the micropenis androgen receptor
during maturation. An inadequate response of the micropenis androgen receptor to
androgens, i.e., a inadequate peak concentration of the androgen receptor or a inadequate
length of time that the penile androgen receptor is up regulated following the
administration of androgens. Alternatively, inadequate penile growth could be due to a
still undefined abnormality in penile growth physiology not directly related to androgen
receptor expression.
To determine if there is a difference in the expression of the penile androgen receptor
between the normal and microphallic animal we characterized penile androgen receptor
expression within the micropenis during maturation. This study revealed a significant
difference in the penile androgen receptor expression between the normal and
microphallic mouse. Specifically, mice with hypo gonadotrophic hypogonadism exhibit
extremely low levels of penile androgen receptor expression during the prepubertal time
50 D.A. HUSSMANN
span; with the penile androgen receptor being 2.5 fold lower than normal during the
prepubertal time span and 4 fold lower than normal during puberty. A slow but steady
increase in receptor expression is seen with maturation with equivalent levels of penile
androgen receptor found in the adult microphallic and normal animals. In essence,
hypo gonadotrophic hypogonadism significantly alters the temporal expression of the
penile androgen receptor, with the highest levels of penile androgen receptor expression
occurring in adulthood. This study characterized the penile androgen receptor in
micropenis and resulted in a number of questions. Would androgen therapy up regulate
the micropenis androgen receptor? If so would the androgen receptor up regulate to
normal values? Would androgens up regulate the receptor for the maximal time span
needed for full phallic growth? If we can normalize the penile androgen receptor
expression in micropenis would adequate phallic growth result? (Walsh et aI, 1978; Lee
et aI, 1980;Danish et aI, 1980;Money et aI, 1981; Reilly et aI, 1989; Tietjen et aI, 1998a,
1998b; Allen, 1978).
Response of the Micropenis Androgen Receptor to Testosterone

Treatment
To answer these questions we evaluated androgen receptor expression in

hypo gonadotrophic hypogonadal mice with and without testosterone treatment.
Testosterone therapy 2.5 mg 1100 gm body weight was initiated during the prepubertal
time span, i.e., day15 of life and continued at weekly intervals until 90 days of life.
Animals were killed at various time points during maturation and androgen receptor
content evaluated. This study demonstrated that supraphysiologic levels of testosterone
resulted in a significant up regulation of the micropenis androgen receptor during
maturation. The peak concentration of the penile androgen receptors in the treated
microphallic animal eventually reached values that were 1.4 fold higher than that of
normal animals. In addition to the up regulation in concentration, the duration of up
regulation was significantly enhanced over normal from 60 to 75 days, a 25% increase.
In spite of androgen administration enhancing both the peak concentration and duration
of the penile androgen receptor expression, phallic growth did not normalize. (A
significant difference in the mean wet penile weight was found between all three study
groups: normals 38.5 +/- 3.5 mgs, micropenis untreated animals 8.5 +/-4.5 mgs and in
micropenis animals treated with testosterone 26.4 +/-2.0 mgs, p < 0.001.) Two
possibilities exist to explain our findings. First, it is theoretically possible that the
absence of fetal androgens resulted in the loss of androgen imprinting. Failure of the
androgen sensitive organ to be imprinted hindered the growth response of the micropenis
to testosterone replacement therapy. The second possible explanation is that
normalization and or cessation of penile growth is not solely dependent upon androgen
receptor expression. The finding that micropenis persisted despite enhancing the peak
concentration and lengthening the duration of the penile androgen receptor up regulation
strongly suggests that cessation of penile growth and penile size is predominantly
regulated by non androgen dependent mechanisms. In view of these findings we
recommend that further experimental work regarding normalization of phallic growth in
micropenis should concentrate on stromal epithelial interactions and not androgen
receptor expression. (Hostetter et ai, 1984;Naslund et ai, 1986; Rajfer et ai, 1979;Even et
ai, 1991;Tietjen et aI, 1998a, 1998b; Allen, 1978).
Does Persistence of an Androgen Unresponsive Micropenis Affect

Psychosexual Function?
Since our experimental and clinical investigations revealed that a substantial number
of animals and or patients had a persistent micropenis in adulthood we believed it was
necessary to determine the psychological well being and/or social functioning of our
persistently microphallic patients. To evaluate their psychosexual function we identified
13 adult patients (18 years of age or older) with micropenis secondary to an isolated
hypogonadotrophic defect. All of these patients are currently on standard testosterone
replacement therapy as an adult. The individuals were interviewed to determine their
perception of their sexual gender, sexual orientation, sexual function, psychological
concerns regarding adequacy of penile size and the impact of their penile size on their
social functioning. Specific questions included: Is the appearance of the penis
satisfactory? Are there difficulties in relating to your male peers? What sexual interests
do you have? Have you had a sexual relationship? Are erections sufficient for vaginal
penetration? Have you or your partners ever brokl~n off a sexual relationship due to
feelings of penile inadequacy? Have you ever been through psychologic counseling?
Have you ever had counseling or treatment for depression or self-destructive behavior?
If so, were the depression and/or self-destructive behavior linked to your concerns
regarding the adequacy of penile size?
The median age at the time of their interview was 22 years, range 18-30 years. Median
stretched penile length of these patients in adulthood is -3.5 standard deviations, range -
5.9 to -2.8. All patients had been identified in childhood as having a micropenis that
failed to normalize or repeated fell off of standard penile growth curves following
androgen therapy. Median age at the time of diagnosis of an androgen unresponsive
micropenis was 6 years, range 3 to 16 years. None of these patients had been offered
sexual conversion do to their advanced age at the time of their diagnosis. All 13 (100%)
report a normal male gender identity in adulthood. Nine (69%) are or have been sexually
active, 4 (31%) are not active by choice or lack of opportunity. Two (15%) of the
sexually active patients have been rejected by partners do to phallic inadequacy. Both of
these patients have undergone psychiatric counseling regarding their sexual rejection and
their own feelings of penile inadequacy. One of the two patients required in hospital
stays on two separate occasions for suicidal ideation directly related to his feeling of
penile inadequacy.
In essence, all of our patients with persisting micropenis had a male gender identity
with the majority demonstrating normal psychosexual behavior. The finding that the
majority of men with micropenis function adequately as males is similar to that reported
previously by Drs Reilly and Woodhouse. The combination of these published reports
along with our own data suggest that the vast majority of men with androgen
unresponsive micropenis will function adequately as males in adulthood with excellent
psychosocial adaptation to their body habitus. However, on occasion phallic inadequacy
will lead to significant impairment of psychological and or social function. This data
52 D.A. HUSSMANN
suggests that sexual conversion of patients with an androgen insensitive micropenis

should not be recommended on a routine basis. (Money et al, 1981; Money, 1985;
Glassberg, 1999; Reilly et aI, 1989; Woodhouse, 1998).
Conclusions
1. Rodents have an androgen sensitive os penis. No analogous structure exists within

the human. It is unknown if the anatomical difference between the two species
affects the ability to correlate rodent data to the human.
2. Rodents predominately down regulate their penile androgen receptors in the corporal
bodies and os penis prior to cessation of phallic growth.
3. Humans down regulate phallic skin androgen receptors after the cessation of penile
growth. In humans it is unknown if or when the androgen receptors in the penile
corporal bodies down regulate.
4. Current experimental data in both the humans and rodents suggest that penile
androgen receptors down regulate do to the cessation of phallic growth. It is not the
penile androgen receptor down regulation that causes the cessation of penile growth.
5. In our experience, in both rodents and humans, prepubertal androgen therapy for
treatment of micropenis due to an isolated gonadotropin defect does not result in
normalization of phallic growth. Unfortunately our human data maybe biased do to
referral patterns. To accurately answer this question clinical investigations will
require a multi-institutional study.
6. Persistent micropenis in the human does not affect gender identity. The majority of
adult patients with micropenis will have normal psychosexual function. Only on
occasion will patients with micropenis require psychotherapy for feelings of sexual
inadequacy and suicidal ideation.
7. Sexual conversion of patients with an androgen insensitive micropenis should not be
recommended on a routine basis.
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54 D.A. HUSSMANN
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Tietjen DN, Uramoto GY, Tindall DJ, Husmann DA. Micropenis in hypogonadotrophic hypogonadism:
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Questions for Dr. Husmann
Question - It has been proposed to use testosterone for babies with micropenis as a
predictor for gender assignment and the responsiveness to testosterone early in life as
being one of the criteria to decide if this baby is going to have a functioning penis or not.
I would agree with you that most of the patients do not have actually sexual gender
dysphoria later on - the patients who had micropenis, but can you please comment on
this?
Answer - I am a strong believer in the use of neonatal testosterone to assess penile

growth. However, I want to mention that of the most recently of the individuals who we
have not had respond to androgens, they have all been raised as males. We have had an
extensive discussion with the families and right now they are all being raised as males.
What will happen with these individuals later on, I don't know. It is certain from our
preliminary data and from what Dr. Riley and Dr. Woodhouse have predicted, it looks
like the majority will do very well.
Question - Doug, I believe there is some data that suggest that DHT may be more
effective in increasing penile size and I wonder if you have any experience with, either
clinically or in the lab. What you might think the mechanism of that is as compared to
Testosterone?
Answer - I am going to speak from my laboratory experience. We have used DHT and
testosterone in the laboratory. Unfortunately, we used them in super-physiologic doses
and saw no difference in the growth response. So in our own experience, again using
super-physiologic doses, I have not seen a response. It would make some sense the DHT
would be better because it does have a 4-5 fold more affinity for the androgen receptor.
And if there is going to be a problem or partial in organ insensitivity to the androgen
receptor, it may be that the extra DHT may have an effect. We certainly have had a few
clinical cases where people have had a partial androgen insensitive penis. One of which
we wrote up that we actually almost quadrupled the dose of testosterone that we were
giving, not DHT, and saw substantial penile growth with high level of testosterone. In
that situation, I do think that if we would have used DHT they probably would have
responded at a lower dose.
Question - Have you used any HCG in your experiments? We know that Steve Koff
showed, last year, that you could alter a position of Ithe meatus in hypospadias, at least the
proximate portion. I was wondering ifHCG had any affect on micropenis?
Answer - We have not used any HCG in our experimental models because it would have
taken me one more step away from what I was looking for and I would have had to
attribute any sort of response to the testis then. We do use HCG on some intersex
patients to see how the testes are going to respond in increasing with testosterone. We
have some clinical use of it, but nothing experimentally. But if I am looking at penile
growth, although we may use HCG to see what is happening with the testes, I am going
to use testosterone because I want one less organ in the loop.
Question - Is there a dose response relationship between testosterone and increased

receptor activity? And, does it alter the pattern with which one should give testosterone
clinically? Are you giving a small priming dose fi)llowed by a growth dose or does it
make no difference at all?
Answer - What we have seen with testosterone is the occasional individual who has not
responded to what we would use as normal levels of testosterone as a neonatal trial, say
25 milligrams of testosterone 1M. If we had seen that they have responded, then we will
bump the testosterone level to give a trial with. We have certainly seen individuals where
their 25 milligrams of testosterone appears to be priming the pump, so to speak, and then
seen a significant response with the higher testosterone levels. I have not done that in
any of the animal experiments and I have not measured the androgen receptor in response
to that personally. Although my own conclusion would be is that in certain individuals
you will see a proper response in the androgen receptor with giving an initial small dose.
Question - Can you tell us if there is any influence of FSH on the androgen receptors?
56 D.A. HUSSMANN
Answer - I cannot answer that because I have not looked at that myself and I am unaware
of the literature.
Question - Have you looked at any IGF over expression in the transgenic lines? Are
there any available? Do you foresee the day when you will actually be able to target the
corpora specifically with certain markers that are corpora's specific?
Answer - Our experience with Insulin Like Growth Factor (IGF) 1 right now is in treating
the hypo gonadotropic animals with ILGF-l and with growth hormone. And what we
have seen is that we do cause penile growth with excess growth hormone. Not to the
level of testosterone. The next question to answer would be if we administer both
growth hormone and testosterone, will we see an increase difference? I cannot answer
this because we have not tried this to date. As for specific corporal markers that time
will be coming, but we have not had any experience with that.
HYPOTHALAMIC IMPRINTING BY GONADAL
STEROID HORMONES
Roger A. Gorski
Department of Neurobiology, UCLA School of Medicine, Los Angeles, CA 90095
INTRODUCTION
Over forty years ago Phoenix and his colleagues (Phoenix et al,1959) proposed an
heuristically valuable concept, namely that one can view gonadal hormone action on the
brain as an activational effect, i.e., a transient modification of neuronal function, or an
organizational effect, i.e., a permanent modification of neuronal function - at some level.
In the subsequent years much has been learned about the organizational effects of
gonadal hormones which appear to be responsible, at least in large part, for the normal
masculinization and defeminization of the brain of the male of a given mammalian
species. Enough has been learned, in fact, to challenge current clinical practice in cases
of ambiguous or underdeveloped genitalia in the newborn human infant. The goal of this
discussion is to bring clinicians up to date about our current understanding of
hypothalamic imprinting by gonadal hormones or, perhaps the more precise concept of
the sexual differentiation of the brain, focusing on the laboratory rat as a model system.
Laboratory rats are clearly not human beings, perhaps not even wild rats, but they are
mammals as are human beings and have been studied extensively and provide a wealth
of basic information with which to interpret the limited data from human subjects.
Sexual Differentiation of the Brain, The Concept
It is well documented in many species including human beings that the presence of
hormonally active testes during prenatal development leads to the suppression of
Mullerian duct development (due to the production of Mullerian Duct Inhibiting
Hormone) and the stimulation of W olffian duct derivatives and the masculinization of the
single primordia for the external genitalia (due to the production of androgens).
Although the determination of the gonad is rather complex genetically (see Vilain
chapter, this volume), once functional testes are formed and provided that the individual
can respond to testicular hormones, this individual, regardless of "his" chromosomal sex
(i.e., XY or XX [if the testis determining gene has crossed over to an X chromosome
during meiosis D, will be male in terms of his external and internal genitalia. Although
genetic factors are most certainly involved, hormone activity does not appear to be

Edited by Zderic et al .• Kluwer Academic/Plenum Publishers. 2002
58 D.A.GORSKI
particularly important for female development. Nature's blueprint for the internal and
external genitalia appears to be female!
Parts of the brain, particularly the hypothalamus, are an integral component of the
reproductive system. The hypothalamus and through it, higher brain centers, control the
release of gonadotropic hormones from the pituitary, including the surge of luteinizing
hormone (LH) necessary for ovulation. In addition, gonadal hormones acting on the
brain promote, or in human beings at least facilitate, reproductive behavior. If, as I
suggest, the brain is part of the reproductive system, this question arises: Does the brain
also undergo a process of hormone-dependent sexual differentiation analogous to that of
the genitalia? The results of literally thousands of studies over the last 40 years clearly
demonstrate that the process of hormone-dependent sexual differentiation does indeed
apply to the brain of non-human animals. Thus, for many mammals, the brain appears to
be inherently female or perhaps neuter. For the development of functional,
neurochemical and neuroanatomical characteristics that are typical of the male of a given
species, the developing brain must be exposed to testicular hormones (Goy et ai, 1980;
DeVries et aI, 1984; Gorski, 1996; Woodson and Gorski, 1999; Matsumoto, 1999).
If the brain is neuter at birth in rats, it is possible that exposure to low levels of
estrogen are indeed required for the normal development of the female brain. In this
case, or if the brain is inherently female, exposure to a higher level of gonadal hormones
makes the individual a male, not his genome per se. (See below for a discussion of the
fact that testosterone produced by the testes is a prohormone and the masculinizing
steroid for the brain in many animals is actually estrogen.) It must be emphasized that
what we have learned about the development of the rat brain must provide at least some
clues about the development of the human brain.
What have we learned about the rat brain? The experimental approach to proving the
sexual differentiation of the rat brain has been the administration of exogenous hormones
to the neonatal female or the removal of endogenous hormones by castration of the
newborn male or treatment with anti-hormones perinatally. Two fundamental functional
sex differences illustrate the value of this approach: ovulation and sexual behavior.
Ovulation is the result of estrogen-induced positive feedback action presumably in the
hypothalamus, possibly in the preoptic area and more specifically, the anteroventral
periventricular nucleus (AVPV) (Teresawa et aI, 1980), which is critical for the surge of
LH releasing hormone that is indirectly necessary for ovulation. The normal male rat
simply does not have the ability to respond to estrogen's positive feedback action and
cannot support ovulation in ovarian grafts or the necessary surge of LH. This functional
sexual dimorphism is essentially an all or none difference. In contrast, the sexual
dimorphism in sexual behavior is quantitative rather than qualitative. Female rats will
mount other females and normal male rats will exhibit the female characteristic lordosis
reflex under some circumstances (Beach, 1938; Soderston et aI, 1983). But even the
intact normal male rat will occasionally exhibit the lordosis reflex to the mounting
activity of a stud male. Nevertheless, the normal male and female clearly differ in their
rate of lordosis responding or of mounting activity.
These classical functional sex differences are not the result of sex differences inherent
in the brain. Rather, they are produced by the action of testicular hormones. The
injection of testosterone, usually in the propionated form, to the female rat within the first
week of postnatal life permanently de feminizes (estrogen-induced positive feedback and
HYPOTHALAMIC IMPRINTING 59
therefore ovulation does not occur, lordosis behavior is suppressed) and masculinizes
brain function (mounting, intromission and even ejaculatory behavior are enhanced). On
the other hand, castration of the newborn male rat feminizes the brain (estrogen-induced
positive feedback can occur and lead to ovulation in ovarian grafts and these males
exhibit female levels of lordosis responding upon appropriate activational effects of
ovarian hormones as well as reduced levels of masculine behavior, again under
appropriate activational actions of gonadal hormones). Following these general
observations it was shown that in the male rat, bUit not the female, there is a surge of
testosterone around embryonic day 18 (Weisz et aI, 1980;Sinha, 1997;Hsu et aI, 2000).
This prenatal surge of testosterone is important for the establishment of normal levels of
masculine sexual behavior and as will be discussed below, for the full development of at
least one structural sex difference in the rat brain.
This endocrinological approach (administering testosterone to females or castrating
newborn males) has established that the sexual differentiation of brain function applies as
well to aggressive, social and play behaviors, the regulation of food intake and body
weight (Goy et aI, 1980;DeVries et aI, 1984;Gorski, 1996;Matsumoto, 1999), and
learning strategies in rats (Williams et aI, 1991). Male and female rats appear to use
different strategies to learn a radial maze presentation of food pellets. Females rely on
landmarks as well as the geometry of the testing environment whereas males rely solely
on geometry. Thus, if only the geometry of the testing environment is altered, male rats
are negatively affected more so that females which still can utilize the unchanged
landmark clues. The important point is that these learning strategies are dependent on the
hormonal environment postnatally, exactly as predicted by the concept of the hormone-
dependent process of sexual differentiation of the brain (Williams et aI, 1991). It is likely
that if one looks closely, many functional processes differ between the male and female
of a given species. However, one must always keep in mind the different hormonal status
of intact adult males and females. True organizational sex differences have to be
demonstrated in adulthood under the same hormonal conditions in both sexes which
actually may not be that easy to accomplish because of sex differences in liver catabolic
activity.
Structural Sex Differences
One scientific development which has stimulated further acceptance of the concept of
the sexual differentiation of the brain has been the identification of structural sex
differences in the central nervous system which are determined, at least in part, by the
gonadal hormone environment during development (Gorski, 1996;Woodson et aI, 1999).
Remember that many of the sex differences in brain function are quantitative, not
qualitative, e.g., female rats do show some mounting behavior and male rats can exhibit
lordosis behavior. Thus, the male rat clearly has the neural circuitry to evoke the lordosis
reflex. This is further emphasized by experimental surgical procedures which appear to
disinhibit the lordosis reflex in normal male rats (Nance et aI, 1975;Yamanouchi et aI,
1975,1985). These facts led this author initially to conclude, albeit erroneously, that
structural sex differences in the central nervous system were unlikely. If the neural
circuitry for the lordosis reflex is present in the male, for example, the basis of the
60 D.A.GORSKI
functional sex difference in this behavior might more logically lie in the sensitivity of that
circuitry to the activational effects of gonadal hormones. However, as stated above it is
now clear that developmentally hormone-dependent structural sex differences, some quite
marked, do exist in the central nervous system of rats.
It may be interesting to recount the author's change of view from doubting the
existence of hormone-dependent structural sex differences to becoming a strong advocate
of the view that structural sex differences in the brain underlie many of the observed
functional sex differences. The first critical observation was the finding of Raisman and
Field (1971) that at the level of the electron microscope, sex differences did exist in the
synaptology of a specific region within the medial preoptic area. Moreover, these sex
differences were modified by postnatal hormonal manipulations in a way predicted from
the concept of sexual differentiation. Just a few years later, Nottebohm and Arnold
(1976) reported the existence of marked sex differences in the nuclear organization of the
neural system which controls song production is some species of birds. Then, a few
years later, this laboratory identified what is still one of the more marked sex differences
in the mammalian hypothalamus, the sexually dimorphic nucleus of the preoptic area
(SDN-POA) (Gorski et aI, 1978, 1980).
Figure 1. Representative coronal sections through the sexually dimorphic nucleus of the preoptic area (SDN-
POA) in adult rats. All at the same magnification. A. male. B. female. C. female treated perinatally with
testosterone propionate. D. female treated perinatally with the synthetic estrogen, diethylstilbestrol. AC =
anterior commissure; OC = optic chiasma. SCN = suprachiasmatic nucleus. Modified from Dahler et aI, and
reprinted with permission from Elsevier Science Publishers.
These three observations in the 1970s opened the floodgates for the demonstration of
other hormone-dependent structural sex differences in the rat brain and in other species
including that of human beings. It appears that a photograph showing the SON-POA of
the male and female rat (Figure 1), is more convincing proof of the process of the sexual
differentiation of the brain than any illustration showing lordosis quotients or other
functional parameters. Perhaps the apparent presence of structural sex differences in the
human brain (Swaab et aI, 1985; Allen et aI, 1989; Byne et aI, 2000 and also see Gorski,
1996; Woodson et aI, 1999), even though they have not been shown to be hormone
dependent, has had an equally significant effect on the belief that sexual differentiation
of the human brain does occur.
Of course, the existence of a structural sex difference in the hypothalamus, or many
such differences, does not establish the process of sexual differentiation of brain
structure. A structural sex difference could merely reflect the differing hormonal milieu
produced by the ovary and testis in the adult and/or be determined genetically. There is
evidence for morphological effects of the normal variation in hormone levels during the
rat's four day ovarian cycle (Woolley et al,1992), or in response to treating the adult with
gonadal hormones (Bloch et aI, 1988;Cooke et aI, 1999), but none of these even approach
the several fold sex difference in SON-POA volume. Moreover, the marked sex
difference in SON-POA volume persists after gonadectomy (Gorski et al,1978;Jacobsen
et aI, 1981). It must be pointed out that there could indeed be a genetic basis for this sex
difference. In fact, the results of early studies of the effect on the SON-POA of injecting
testosterone to females or castrating males could have been interpreted to mean that there
was indeed a genetic component. Female rats given a single but relatively massive
injection of testosterone propionate (1.25 mg) on postnatal day five did show a
statistically significant increase in SON-POA volume in adulthood. However, the
volume of the nucleus did not approach that of the male. Similarly, castration of the male
rat on the day of his birth reduced the volume of the SON-POA in the adult, but only by
about 50% (Jacobsen et aI, 1981). Even though this animal, which we have called the
fale, a neologism for feminine male (Gorski, 1967), can exhibit estrogen-induced
positive feedback and female levels of lordosis responding, SON-POA volume is still
considerably larger than that of a female.
However, before accepting a genetic component to 1the development of the SON-POA,
we argued that it may be difficult to reproduce a possibly prolonged developmental
process by a single injection of exogenous hormone. Moreover, perhaps the male
specific surge of testosterone prenatally sensitizes SON-POA neurons to hormone titers
postnatally (Weisz et aI, 1980), i.e., sexual differentiation of the rat brain actually begins
before birth. When we exposed rat pups to testosterone from embryonic day 16 to birth
via injections into the pregnant rat and then by direct subcutaneous injections for the first
10 days of postnatal life, there was a complete sex-reversal of SON-POA volume in
females (Oohler et aI, 1984). Interestingly, all that exogenous testosterone had no effect
of SON-POA volume in males. Thus, in the female rat the hormonal environment alone
can produce full masclinization of the SON-POA. However, since we are dealing with a
pharmacological exposure to hormone, in dose and/or temporally, these results cannot
rule out a possible role for genetic factors.
However, when male rats are treated for a similar period perinatally with the anti-
estrogen tamoxifen, the male's SON-POA is also completely sex-reversed in volume
62 D.A.GORSKI
(Dohler et aI, 1986). This result clearly supports the critical role of testicular hormones
in the sexual differentiation of the SDN-POA. However, it has been reported that sex
differences independent of gonadal hormones appear to exist (Beyer et aI, 1992;Vladivia
et aI, 1993;Pilgrim et aI, 1994;Arnold, 1996). Moreover, the testis determining gene,
SRY, and other factors involved in sex determination are actually expressed in the
developing brain (Zwingman et aI, 1993; Lahr et aI, 1995; Guo et aI, 1995). Although
gonadal hormones play a critically important role in the sexual differentiation of the
brain, these hormones may not be the only players.
Testosterone as a Prohormone
By the 1950s it had been shown that prolonged exposure to estrogen postnatally had
permanent deleterious effects on ovarian function (Takasugi et aI, 1953). This author
later demonstrated that a single injection of estradiol benzoate could masculinize brain
function (Gorski, 1963), but concluded that since estradiol is a more potent steroid, this
was most likely a pharmacological artefact. Wrong again. It is now well established that
for the sexual differentiation of the brain, testosterone secreted by the testes is a
prohormone and is converted to estrogen locally by the enzyme aromatase (McEwen et
aI, 1977; Toran-Allerand, 1984; Hutchison, 1999). This seemingly bizarre notion that
estrogen is the masculinizing hormone for the brain, posed a problem which to this day is
not resolved. When radioimmunoassays were developed for steroid hormones, it became
possible to measure plasma levels of estrogen in neonatal rats, and in both sexes plasma
estrogen levels were quite high neonatally, higher than adult proestrous levels (Weisz and
Ward, 1980)!
If estrogen is the masculinizing hormone in rats and both sexes are exposed to high
levels during the period of sexual differentiation of the brain, there would be no normal
female rats, all would be masculinized and sterile and the species would have become
extinct. This clearly has not happened. One potential solution to this dilemma involves
the fact that an estrogen binding protein made by the liver, alpha-fetoprotein (AFP),
which is found in high concentration in the blood of rats around the time of birth (Toran-
Allerand, 1984), but falls to low levels by postnatal day seven (Ali et aI, 1981). Thus, it
can be argued that AFP binds plasma estrogen and functionally sequesters it in both
sexes. However, the testes produce testosterone which is not bound by AFP so it enters
neurons where it is aromatized to estrogen. If we assume that through evolution estrogen
became the important hormone for masculine neuronal development, the AFP story
provides a clever mechanism to protect the brain of the developing female.
On the other had, there are data in the literature which suggest that exposure to some
level of estrogen is actually necessary for the normal development of the female rat's
brain (Toran-Allerand, 1984; Dohler, 1978; Dohler et aI, 1984). In this case, Nature's
blueprint for the rat brain would be neuter and the action of some level of estrogen,
presumably low, is necessary for the normal development of the female's brain while
higher local levels of, estrogen derived from testicular testosterone are necessary to
masculinize the brain. Too much of a good thing, from the female's perspective, makes
you male.
Although as will be discussed below, the process of hormone-dependent sexual

differentiation of the human brain probably does occur, the major focus of human studies
has been on androgen per se as in cases of congenital adrenal hyperplasia or the synthetic
estrogen, diethylstilbestrol which was administered to many pregnant women
therapeutically (Hines, 1999). The syndrome of complete androgen insensitivity, where
the individual does not have androgen receptors and has female genitalia and feminizes
morphologically at puberty, may call into question the role of estrogen in the sexual
differentiation of the human brain (Hines, 1999). However, estrogen levels during the
presumed prenatal period of sexual differentiation in such individuals are unknown.
To make the story complete, testosterone is also a prohormone in terms of the sexual
differentiation of the external genitalia in human beings. The conversion of testosterone
to dihydrotestosterone by the enzyme 5-alpha-reductase is required for the
masculinization of the male's external genitalia (Gmmbach et ai, 1998). Once again,
Nature has evolved a mechanism to convert a testicular product into a more active form
at the local level. Individuals with an absence of 5-alpha-reductase represent an
interesting "Experiment of Nature" in terms of human psychosexual differentiation (see
Imperato-McGinley chapter, this volume).
Steroid Action on the SDN-POA
Several facts about the SDN-POA are relatively unique: a significant percentage of its
neurons are born an outstanding several days later than neurons in the surrounding medial
preoptic area (Jacobsen and Gorski, 1981). The significance of this observation is
unknown, but it allowed us to specifically label with tritiated thymidine neurons destined
to form part of the SDN-POA and observe their migratory pathway during perinatal
development (Jacobsen et ai, 1985). However, no apparent sex differences were
observed. The mechanism of action of estrogen converted from testosterone secreted by
the testes appears, at least in part, to involve the prevl~ntion of apoptotic neuronal death.
The number of neurons within the SDN-POA decreases in females after postnatal day
four unless they are treated with testosterone (Dodson et aI, 1993). Moreover, the period
of apoptotic cell death postnatally within the SDN-POA is significantly more prolonged
in females than in males (Davis et aI, 1996). Finally, in/ales treatment with testosterone
significantly suppresses the incidence of apoptosis (Davis et aI, 1996). In the near future,
it should be possible to identify genes which are either turned on ("survival genes") or
turned off ("apoptosis genes") by exposure to testosterone-derived estrogen postnatally
within the SDN-POA.
Function of the SDN-POA
It is difficult to assign a specific function to the neurons of the SDN-POA. The

transplantation of punches containing the SDN-POA from newborn males directly into
the medial preoptic area of littermate females does result in enhanced masculine sexual
behavior in the recipient females when adult, but these females also exhibited increased
64 D.A.GORSKI
lordosis responding (Arendash and Gorski, 1982). Small electrolytic lesions of the SDN-
POA have been reported to have no effect in sexually experienced rats (Arendash and
Gorski, 1983), or to suppress masculine sexual behavior in sexually naIve rats (Dejonge
et aI, 1989). In studies currently published only as a doctoral dissertation, SDN-POA
volume in males from a strain of rats bred for aggressiveness is very large and small
lesions of the nucleus do reduce intermale aggressive behavior in the relatively non-
aggressive Sprague-Dawley strain (Hori, 1995).
Thus far, our most dramatic results have involved the electrical stimulation of the
SDN-POA in normal male rats. The rats were implanted with a chronic electrode aimed
at the SDN-POA unilaterally and adapted to a behavioral testing arena while connected
by a cable to a stimulator. A receptive female was introduced into the testing arena and
the animals allowed to interact for 30 seconds after which 30 second periods of electrical
stimulation were alternated with 30 second periods without electrical stimulation. When
the stimulating electrode was in or very close to the SDN-POA, the only sexual behavior
that occurred without electrical stimulation occurred during the first 30 seconds of the
test. After that, sexual behavior was confined to the 30 second periods of electrical
stimulation and was markedly enhanced (Hori, 1995). Electrical stimulation in the region
of the AVPV, which is just anterior to the SDN-POA, resulted in supression of masculine
behavior during the 30 second periods of stimulation (Hori, 1995). Electrical stimulation
of the SDN-POA also enhanced aggressive behavior but this was not studied
systematically. Thus far, the function of the SDN-POA of the male rat appears to be
related to masculine sexual behavior, particularly ejaculatory behavior, but it may be
involved in aggressive behavior as well.
The Anteroventral Periventricular Nucleus (AVPV)
Although numerous structural sex differences have been reported in rats and other
mammals, the present discussion has emphasized the SDN-POA. This is because the
development and function of this nucleus has been extensively studied. In addition, as
indicated above, possible homologous nuclei have been reported in the gerbil and ferret
and most recently in the rhesus monkey (Byne, 1998) and human beings (see Swaab
chapter, this volume). However, an exclusive focus on the SDN-POA fosters
unwarranted generalization. For example, the AVPV, initially called the medial preoptic
nucleus (Bleier et aI, 1982;lto et aI, 1986;Murakami et aI, 1989) is larger in the female
(Bleier et aI, 1982;Simerly et aI, 1985) and testosterone treatment of neonatal females
leads to a smaller nucleus in adulthood (Ito et aI, 1986). Testosterone treatment for the
first five postnatal days has been reported to increase the incidence of apoptosis within
the female rat's AVPV (Murakami et aI, 1989). Interestingly, the sexual dimorphism in
AVPV volume, in spite of the significant action of testicular hormones in early postnatal
life, currently inexplicably, does not develop until approximately postnatal day 40 and
may be related to puberty (Arai et aI, 1993 ;Davis et aI, 1996). The point that must be
emphasized with these data is that the action of testis-derived hormones on the
developing brain is complex and not limited to a single mechanism.
Structural Sex Differences in the Human Brain
If, as this author believes, sexual differentiation of the brain also occurs in human
beings, one might predict the existence of structural sex differences in the human brain.
At the outset of this consideration, the differences between the preparation of animal
brains for anatomical study and human brain tissue must be stressed. With laboratory
animals, the subject is anesthetized and while still alive perfused intracardiacally with
buffered saline followed by a fixative. Tissue preservation is usually very good. In the
case of a human being, the story is quite different. Death must come first followed by
unequal delays between subjects in terms of the issuance of valid death certificates and
brain removal. In addition, the brain is not perfused but it, or somewhat berter, fragments
of it, are immersed in fixative and preserved by diffusion, which is a slow process.
During all this time, the brain will be deteriorating and the quality of human tissue may
be poor.
In spite of these difficulties related to the deterioration of brain tissue before adequate
fixation, several statistically significant sex differences in the human brain have been
reported (Swaab et aI, 1985;Allen et aI, 1989;Byne et aI, 2000 and see Gorski,
1996; Woodson et ai, 1999). It is important to stress that these differences have to be
confirmed in independent samples and by different investigators. Thus far, only one
reported sex difference, the third interstitial nucleus of the anterior hypothalamus has
been confirmed twice (Byne et aI, 2000;LeVay, 1991) after its initial discovery (Allen et
ai, 1989). Although it is tempting to assume that hormones, presumably from the testes,
create or at least influence these sex differences, this has not been documented and may
never be. Since it is unethical to manipulate the gonadal hormone environment in the
developing human being, we are basically left with uncontrolled and usually retrospective
studies of the "Experiments of Nature," such as females with congenital adrenal
hyperplasia or males with androgen insensitivity or 5-alpha-reductase deficiency. The
results of the study of these individuals certainly may give us important clues, but may
not offer experimental proof. The observations that girls with congenital adrenal
hyperplasia prefer to play with toys preferentially played with by boys (Berenbaum et aI,
1992) and are considered tomboys (Ehrhart et aI, 1974), that genetic males with complete
androgen insensitivity are psychosexually female (Hines, 1999), and that many
individuals with 5-alpha-reductase deficiency and female-like external genitalia
ultimately consider themselves male (Imperato-McGinley chapter, this volume), lend
some support to a meaningful influence of gonadal hormones on the developing human
brain.
To date, individuals with these abnormalities have not be studied in terms of their
brain structure. However, Swaab and his colleagues (Zhou et aI, 1995) have reported
that the central component of the bed nucleus of the stria terminalis, which they find to be
larger in men than in women and which does not vary with sexual orientation in men,
was female-like in volume in six postoperative and hormone treated male to female
transsexuals. The authors argue against an effect of postsurgical estrogen treatment on
brain structure, but we must keep open the possibility that this structural difference may
reflect hormone therapy rather than an inherent structural difference which might have
led these individuals, or predisposed them, to transsexualism. There also have been three
different but unconfirmed reports of structural differences between the brains of
66 D.A.GORSKI
homosexual and apparently heterosexual men (LeVay, 1991;Swaab and

Hofman,1990;Allen and Gorski, 1992). In such cases, not only do we have no
information on possible hormonal effects on these structural differences, we do not know
which came first or if there is a causal relationship between the structural differences in
the brain and sexual orientation. In cases such as those briefly mentioned here, there is
little that medical science can do, except to offer potential explanations for what may
have transpired in an individual's life.
However, there are groups of individuals for whom the concept of the sexual
differentiation of the human brain is of immense potential significance in a very proactive
way, i.e., individuals with congenital morphological anamolies such as penile agenesis,
micropenis or cloacal extrophy in which hormonal influences on the developing brain
might be expected to have been normal during prenatal development, but the external
genitalia are not. Such individuals will be considered in several subsequent chapters in
this volume. In part because of the apparently erroneous conclusion that it is readily
possible to successfully assign a genetic male with such anomalies to the female sex
(Kipnis and Diamond, 1998), the possible effects of testicular hormones on the
developing human brain have, until recently, been ignored.
Summary and Conclusions
The results of more than four decades of research on different mammalian species
have established that the brain, like the rest of the reproductive system, is esentially
basically female. For the male to develop structural and functional characteristics typical
of his species, his brain must be exposed to testicular hormones during a critical period,
or critical periods, of development. As mammals, human beings are most likely subject
to this process of the hormone-dependent sexual differentiation of the brain, but proving
it will be difficult. Common sense ethics preclude experimental procedures such as
castration of neonatal infants or exposing the female fetus to testosterone perinatally.
Thus, scientists are restricted to the retrospective study of "Experiments of Nature." The
results of such studies support to a degree a meaningful role of hormones in the
development of the human brain. The concept of the sexual differentiation of brain
structure and function has a potentially profound influence on clinical decisions with
respect to sex assignment and clinical mana$ement of infants with ambiguous or poorly
developed external genitalia. Because of the importance of a baby's sex in our culture,
parents of such infants must be given consideration, but so should the infant whose
hormonal environment prenatally may well have produced permanent changes in the
structure and functional potential of his/her brain.
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70 D.A.GORSKI
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Questions for Dr. Gorski
Question - 1 would like to discuss one of your slides in which you showed the findings
from a 1974 study which reported some virilizing effects on the females who had
congenital adrenal hyperplasia and they found some tomboyish behaviors. The fact is
that in those the years, the patients with CAH were really identified much later in life,
and they were not effectively treated. So therefore, there are components here which
have to do with actually their developments of self image, somatic effects of androgen
excess on muscle development, which in tum might have had an effect on their brain and
their own perception. Specifically, for the patients of CAH that now we feel more and
more, that the female sex is the more appropriate sex, and this is actually one of the
diseases that most of the people agree that actually these girls have much more feminine
potential. Many people feel that these effects which have been reported in the literature
are actually consequence of being exposed much longer time on androgens because of
misdiagnosis and or delayed medical treatment.
Answer - Yes, 1 think you raised some of the complex issues that we face. There is
another one - did not leave the slide on very long; but, one thing was that they preferred
to wear slacks rather than dresses. Culture has changed since 1974, and in our culture it
is easier for a woman to have a successful life, being a little bit more masculine (meaning
a masculine appearance) as opposed to an effeminate boy. I mean, as you can see many
in the audience, even the lady standing in front of me, have slacks on. Today in 1999, I
don't think anything of that.
Question - You mentioned experiments of nature, such as androgen insensitivity

syndrome to try to help define how real these findings are in humans, but in fact the
experiment of nature that is available is the TFM mouse and have you looked at all that
brain structures and sizes and so forth because that would be very nice way to compare
estrogen versus testosterone effects in the real world.
Answer - Yes, two interesting components about that: One is the mouse does not have an
SDM; but the rat does have the TFM model and in the TFM rat, (I should indicate that
there is a structural sex difference in the spinal cord of motor neurons that innervate
penile muscles [spinal nucleus of the bulbocavernosus muscles - 5MB]). In TFM rats,
the 5MB is feminized. The SDM is masculine. Again, consistent with the view that it is
estrogen i.e. estradiol which is the masculinizing hormone.
Question - We seem to know very little about the burst of androgens in first four months
of life in the male infant. And that leaves us in the dark in terms of how we treat patients
and maybe you can help us on your experiments with rats and mammals. For example,
we have the patients with ambiguous genitalia that we are going to raise as a female; but
yet it may be a true hermaphrodite or it may be a mixed gonadal dysgenesis. We have
testes in there so it would seem logical to get those testes out before the burst, so that is
the first week of life. That's number one and I don't know if anybody else wants to
comment on that. But, we don't know enough about the burst and what it does to later
identity and orientation. Number two, we have the individual with ambiguous genitalia
that we want to test and see how this patient might respond later on, so we give the
patient HCG but we give the patient testosterone and end up going the female route and
now we've given this individual a burst of testosterone to make the phallus grow just to
test it. So it confuses our issue on what to do, how to test these patients, when to remove
gonads and it may confuse the individual's self identity when older, because we have
done something contrary to what we actually want to raise that child as or what we
perceive as the ideal gender assignment.
Answer - Remember I said at the beginning of my talk that we are approaching your
problem from a great distance; but we are suggesting that there may in fact be important
hormone dependent differences in the brain that clinicians have to consider. Now you
have to consider the many factors that impact on the quality of the individuals life, and I
envy people that attempt to help those families and individuals with these very serious
problems. Dick may comment on that because he has shown, in the human sex
differences, more of a developmental time course: I think beginning after you are 5, or
something like that. In the case of the rat, my feeling is this process is over by about day
10 of life because neurons (and I am using the SDM as a model) are committed to die by
about day 6 unless they have seen testosterone. But again, just thinking back at ones own
puberty, I think that is another areas where people have not studied enough and in the
human, is it possible that hormonal effects on the brain at puberty are far more important
than the perinatal hormone, which I think would be a very pleasing thought to you
clinicians, that is some of the things that you are doing on these kids might pale in
comparison to what hormones might do at the time of puberty.
Question - There is out there a patient who has no estradiol receptor alpha. It was
discovered, I think, by Philip Smith in Cincinnati. He identifies as a male and I think is
reproductively competent, I am not sure. But, I wonder if you can comment on how that
speaks to the early developmental model you have proposed.
Answer - Yes, it is very interesting. The prediction of course would be that he should be
female in his psycho-sexuality. A lot of knockouts do not follow the prediction because
as we have heard this morning, there are multiple genes. And, you pointed out that it is
estrogen alpha, there could be estrogen beta. There is growing evidence of a membrane
receptor mediated short-timed action of estrogen. So I think that the question is open.
We heard not too long ago, the SRY was the determining factor and we have learned
today that it is just part of a much greater cascade. I also think the same may be true for
the estrogen receptor. And, some of my clinical friends deny that estrogen could be the
masculinizing hormone in humans.
72 D.A.GORSKI
Question - We have found that in cryptorchid males, what we examined the percent of
cryptorchid males, that there is a significantly high level of alpha feto-protein content of
placenta than in the normal males and we have just recently be found that the
synciciotrophoblasts in the placenta of these cryptorchid males are expressing much
significantly high level of estradiol beta. Now my question to you is, could this be a
placental disease, now we suspect that cryptorchidism may be a placental disease at some
point. Could be a placental disease responsible for such behavior? It is possible that the
mother directly because the placenta's metabolism is not functioning correctly influence
the fetus ultimate gender identity?
Answer - I really cannot comment. I think the placenta is playing a more major role in
fetal development and possibly in sexual differentiation. I don't know anything
specifically about cryptorchid testes. But I think it is possible and one of the things we
all have to remember is that we've got psycho sexual differentiation the is hormonally
driven as well as learned behaviors. These are probably independent processes and may
some may be very brief. And so I think an alteration, even a temporary alteration in
placental function or adrenal or gonadal function could lead to the myriad of things that
we see.
Question - Roger, beautiful job, as always. There is another quirk of fate and that is the
one patient that has been identified without aromatase so they have no estrogen at all and
as I remember seeing and hearing both those presented, they do present as males their
male identity. I wondered if you had any experience with the alpha and beta estrogen
knockout mice at NIEHS and how they look?
Answer - No, I haven't. Again, for the last 20 or so years we have been focusing on the
SDN and as I said, the mouse doesn't have one. But I was careful today to mention that
the action of testosterone on AVPV seems to be opposite to that because I do not want
people coming away thinking that there is only one mechanism of androgen action on the
developing brain. There is many, but again, focusing on this one nucleus has kept our lab
busy for quite a while.
Question - I just wanted to discuss briefly the issue of aromatase deficiency and there is
an animal model which is the hyena. Now there is a placenta aromatase deficiency and
the female hyenas are actually born with significant virilization then subsequently as their
virilization declines they go and assume their female lives. So I think this is another
issue which actually, despite the fact that there is this clear hormonal effect early in life, it
appears that later on this is reversed, and the they move on to have female reproductive
function. So I was wondering whether you can comment on that.
Answer - No. I'll just add another complicating factor and that is the literature gets really
confusing because people often confuse activational with organizational. They may be
the same mechanism but conceptually they are different; but you report a sex difference
in an adult. You do not know if that is because of the sex differences in hormonal mileau
at the time, or some effect very early in life. And we tend to confuse these. I try to
convince my students if you want to look for a sex difference than undergo sex
differentiation - study the phenomenon under the same hormonal conditions. And that is
not always done.
Question - Have you looked at maternal administration of Tamoxafin, in terms of

blocking the effects of estradiol? Would it work or would you actually have to deliver or
target it directly to the eNS?
Answer - We did use Tamoxifin, and it did depress SDM development in development in
both males and female. Just one final comment, I am not a clinician but I was moved by
Diamond's papers and his supposed recommendations and the point seems to be that
there are hundreds of patients out there who have experienced and lived through some of
this stuff and it seems to me a pity that you as a profession have not obtained the
appropriate data on the success or lack there of, of this approach. So I thoroughly support
the concept of a registry.
SEXUAL DIFFERENTIATION OF THE HUMAN
HYPOTHALAMUS
Dick F. Swaab l , Wilson C.J. Chun§I,2, Frank P.M. Kruijverl, Michael A.

Hofman l and Tatjana A. Ishunina 1,
} Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research,
Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands, tel. (+31)20 5665500,fax
(+ 31)20 6961006, e-mail: w.verweij@nih.knaw.nI2 Department of Psychology,
University of Massachusetts, Amherst, USA 3 Department of Histology and Embryology.
Kursk State Medical University. Kursk. Russia.
INTRODUCTION
In analogy with observations in many mammalian species the human brain might well
undergo sexual differentiation during its development due to organizing effects of sex
hormones. Such a structural organization might be the basis for functional sex differences
(Pilgrim and Reisert, 1992; Anderson et aI, 1986; Pilgrim and Hutchison, 1994) and sex
differences in neurological or psychiatric diseases (Table I). In fact, remarkably little
attention has been paid so far to the possible structural basis of the often pronounced sex
differences in the incidence of such diseases (Swaab and Hofman, 1995). The proportions
of cases range from more than 75% female in Rett syndrome, anorexia nervosa and
bulimia to more than 75% male in dyslexia, sleep apnoea, Gilles de la Tourette's
syndrome, and Kleine-Levin syndrome (Table I). Not only might the number of cases of
disorders show clear sex differences, but the signs and symptoms and the course of the
disease might differ as well. Males not only suffer from schizophrenia 2.7 times more
often than females, they are also prone to a more severe form of this disorder, experience
an earlier onset, and exhibit more structural brain abnormalities. In addition, relapses are
more severe, and their response to neuroleptic medication is less favorable.
Another example is that, following restricted posterior left-hemisphere lesions, 41 % of
the males and 11 % of the females developed aphasia, whereas manual apraxia was found
in 6% of the females and 42% of the males (for references see Swaab and Hofman,
1995).
An important recent finding with respect to sexually dimorphic alterations in
neurological diseases is the observation of Schultz et al. (Schultz et aI, 1996). They
showed a conspicuous neurofibrillary Alzheimer-like pathology in the infundibular (=
arcuate) nucleus and posterior median eminence in most of the males over the age of 60,
and only in a small percentage of the females. The vessel-associated dystrophic neurites
develop independently of Alzheimer changes in the neocortex. In the arcuate nucleus of
postmenopausal women, LHRH neurons are strongly activated. We propose to explain
the lack of neurofibrillary changes in the mediobasal hypothalamus of females as an

Edited by Zderic et ai., Kluwer Academic/Plenum Publishers, 2002
76 D. F. SWAAB, ET AL.
illustration of how activated neurons are protected against the development of Alzheimer
changes, a principle we paraphrased as "use it or lose it" (Swaab, 1991).
Table 1. Ratios for females over males suffeing from particular neurological and psychiatirc
diseases.
Disease %
Female
Rett syndrome 100 ojmale
Lymphocytic hypophysitis 90 1010
Anorexia nervosa 93 7
Hypnic headache syndrome 84 16 8
Bulimia 75 25
Senile dementia of the Alzheimer type 74 26 5
Posttraumatic stress disorders 70 30 4
Multiple sclerosis 67 33 11
Anxiety disorder 67 33
Dementia 64 36 5
Unipolar depression 63 37
Severe mental retardation 38 62
Substance abuse 34 66 6
Autism 29 71
Stuttering 29 71
Schizophren ia 27 73
REM sleep behavioral disorder 24 76 1
Dyslexia 23 77
Sleep apnoea 18 82
Kallmann syndrome 17 83 7
Rabies 13 87 9
Gilles de la Tourette 10 90
Kleine-Levin 0 100 2
I Schenk et aI, 1993

2 Critchley, 1962; chapter 28.1
3' Naidu, 1997; chapter 2.5
4 Breslau et aI, 1997
5 Bachman et aI, 1992
6 Kessler et aI, 1994
7 Rugarli and Ballabio, 1993
8 Dodick et aI, 1998
9 Gomez-Alonso and Rabies, 1998
10 Maghnie et aI, 1998
II Sadovnick and Ebers, 1993
For further references, see Swaab and Hofman, 1995
SEXUAL DIFFERENTIATION 77
FACTORS INFLUENCING GENDER AND SEXUAL

ORIENTATION
Gender
One of the conditions under which sexual dimorphism is found, is transsexualism.

There are approximately 3 times more male-to-female transsexuals than female-to-male
ones (Dessens et aI, 1999). There is only little information available about prenatal
hormonal factors that may influence gender and calise transsexuality in humans (Table
II). Congenital adrenal hyperplasia, characterized by high androgen levels during prenatal
development, constitutes a risk factor for the development of gender identity problems.
Although it should be emphasized that the large majority of women with this disorder do
not experience marked gender identity conflicts. The risk that a genetic female with this
disease would be living, as an adult, in the male social role compared to genetic females
in the general population were found to be 608: 1 in the study of Zucker et al. (1996).
Table II: Factors that influence sexual differentiation of the human brain.
Gender identity (transsexualism)

prenatal hormones: (Diamond, 1997)
CAH girls with gender problems (Meyer-Bahlburg et aI, 1995; Zucker ct al. 1996).
5-a-reductase deficiency (Imperato-McGinley et ai, 1997; 1991).
phenobarbitalldiphantoin during pregnancy (Dessens et aI, 1999).
Intersex (Zucker et aI, 1987; Reiner et ai, 1996)
postnatal social factors? (Bradky et aI, 1998).
Sexual orientation
- genetic factors: twin studies (Kallman, 1952; Bailey and Bell, 1993)
molecular genetics (Hamer et aI, 1993; Hu et aI, 1995)
- prenatal hormones: CAH girls (Money et aI, 1984; Dittmann et aI, 1992; Zucker et
al,1996)
DES (Ehrhardt et aI, 1985; Meyer-Bahlburg et aI, 1995)
Male-to-female sex
reassignment (Bradley et aI, 1998)
Stress during pregnancy (Ellis et ai, 1988; Bailey et aI, 1991).
Postnatal social factors? Raising by transsexual
or homosexual parents does not affect sexual
orientation (Green, 1978; Golombok et aI, 1983).
CAH: congenital adrenal hyperplasia

DES: diethylstilboestrol
Recently Dessens et al. (1999) reported that some children born of a group of women
exposed to the anticonvulsants phenobarbital and diphantoin, which affect sex hormone
metabolism, were found to be transsexuals in adulthood. This happened remarkably
often in view of the rarity of this disorder. This exciting observation on compounds
known to alter steroid levels in animal experiments has to be examined further. Reiner
(1996) described a 46,XY child with mixed gonadal dysgenesis, one immature testis, one
ovary, and hypoplastic uterus clitoral hypertrophy, who was raised, unstigmatized, as a
girl, but who declared himself male at the age of 14. Following corrective surgery and
testosterone substitution he lived as a boy despite the social factors that were strongly in
favour of maintaining the assigned sex. Apparently the deficient testis had been able to
organize the brain during development even though the hormone levels were so
inadequate during early prenatal development of the genitalia that ambiguity of these
organs was induced.
Although postnatal psychosocial factors are generally presumed to play a role in
gender development (Zucker et aI, 1987; 1996; Bradley et aI, 1998), the evidence for
such an influence is not strong. Children raised by transsexuals do not develop gender
problems (Green, 1978). The famous John-Joan-John story is an example of a case story
pleading against the opinion that gender is determined by psychosexual factors. John's
penis was accidentally burned off in 1963, during his circumcision at the age of 8
months. When he was 17 months it was decided to tum him into a girl; his testicles were
removed, and at 21 months a vagina was constructed. He was also given female sex
hormones. However, despite having been raised as a girl and despite 2 years of estrogen
therapy, at age 14 this 'girl' declared that she had " ... suspected [she] was a boy since
second grade". He eventually underwent a mastectomy and received doses of male
hormones. At 25 he married a woman who already had children (Diamond, 1997). In a
second case of ablated penis where the decision was made to reassign the patient's sex
and tum a boy into a girl by removing the remainder of the penis and testes at an earlier
age (7 months) and raising the child as a girl, the girl developed a bisexual orientation.
She was mainly attracted to women, and her gender identity was female. The different
outcome of this case is explained by the authors as being due to the fact that the change
happened earlier (Bradley et aI, 1998). This explanation is, however, not supported by
another case history. A child with true hermaphroditism, 45x(l3%47XYY(87%) sex
chromosome mosaic pattern in blood, uterus, fallopian tubes, phallus, testicular tissue and
epididymis was assigned at birth to become a male. At 5 weeks the decision was made to
reassign him to the female sex. At l3 months the testicle was removed, and at 9 months
and 5 years operations were carried out to make the genitalia female. She was raised as a
girl, but had masculine interests and when she was around 8 years old she declared that
"God had made a mistake" and that she "should have been a boy." Apparently the male
sex hormones to which she had been exposed in utero had imprinted the male gender,
although the authors also presumed postnatal psychosocial factors to have played a role
(Zucker et aI, 1987).
Sexual Orientation
The determinants of human sexual orientation seem to be genetic as well as non-

genetic in nature, as appeared from studies in families, twins and through molecular
genetics (Kallman, 1952; Bailey and Bell, 1993; Hamer et aI, 1993; Turner, 1995; Hu et
aI, 1995; table II). The group of Hamer found linkage between DNA markers on the X-
chromosome and male sexual orientation. Linkage between the Xq28 markers and sexual
orientation was detected for the gay male families, but not for the lesbian families
(Hamer et aI, 1993; Hu et aI, 1995). Sex hormones during development also have an
influence on sexual orientation judging by the increased proportion of bi- and
homosexual girls in case of congenital adrenal hyperplasia syndrome (Money et aI, 1984;
Dittmann et aI, 1992; Zucker et aI, 1996). Then there is diethylstilboestrol (DES), a
synthetic oestrogen that increases the occurrence of bi- and homosexuality in girls
(Ehrhardt et aI, 1985; Meyer-Bahlburg et aI, 1995). Maternal stress is thought to lead to
increased occurrence of homosexuality in boys (Ellis et aI, 1988) and girls (Bailey et aI,
1991). As an interesting case history of this factor, Weyl (1987) mentioned that Marcel
Proust's mother was subjected to the overwhelming stress of the Paris commune during
the fifth month of her pregnancy in 1871 and that Mary, Queen of Scots, the mother of
the homosexual King of England, James I, toward the end of the fifth month of
pregnancy had the terrifying experience that her secretary and special friend Riccio was
killed. Although postnatal social factors are generally presumed to be involved in the
development of sexual orientation (Byne et ai, 1993; Zucker et aI, 1996), evidence in
support of such an effect has not yet been reported. In fact, the observation that children
raised by lesbian couples or by transsexuals generaHy have a heterosexual orientation
(Green, 1978; Golombok et aI, 1983; Kirkpatrick et aI, 1981) does not support the
possibility of the social environment being an important factor for determining sexual
orientation.
On the basis of animal experiments it is expected that all compounds that influence
neurotransmitter metabolism in development may affect sexual differentiation of the
brain as well (Pilgrim and Reisert, 1992). Prenatally alcohol-exposed young adult mice
have a decreased preference for females and an increased preference for males as a
partner (Watabe and Endo, 1994). Exposure during development to certain drugs (e.g.
barbiturates) cause deviations in testosteron levels that persist in adulthood. Exposure to
other drugs (e.g. opiates) led to behavioral changes despite apparently normal adult
gonadal hormone levels (Ward, 1992). Data-related humans are, however, lacking at
present. In connection with this observation, that points to an alternative mechanism of
sexual differentiation, it is of great interest that there is recent animal experimental evi-
dence for primary genetic control of sexual differentiation that does not involve sex hor-
mones. Results obtained from cultures of embryonic rat brain indicate that dopaminergic
neurons may develop morphological and functional sex differences in the absence of sex
steroids (Pilgrim and Reisert, 1992). Recent clinical observations also point to the
possibility that the interaction between sex hormones and brain development may not be
the only mechanism involved in the development of gender and sexual orientation. DNA
sequence variation in the androgen receptor gene was not found to be a common
determinant of sexual orientation (Macke et aI, 1993). Moreover, aromatase deficiency
due to a mutation was accompanied by psychosexual orientation of both brother and
sister, appropriate for their phenotypic sex (Morishima et aI, 1995) and a 28-year-old man
with estrogen resistance due to a mutation of the estrogen-receptor gene had no history of
gender-identity problems and had strong heterosexual interests (Smith et aI, 1994). We
have recently shown that two Y-chromosomal genes that are involved in sex
80 D. F. SWAAB, ETAL.
determination of the gonad, SRY and ZFY, are transcribed in the hypothalamus, the
frontal and temporal cortex of the adult human brain. These genes are candidates for
male-specific transcriptional regulators that could give human brain cells the potential for
hormone-independent realization and maintenance of genetic use (Mayer et aI, 1998).
Hormones and Sex Differences in Hypothalmic Development
The stages of development in which sex steroids determine sexual differentiation of

the human brain are most probably the three periods during which sexually dimorphic
peaks in gonadal hormone levels are found, viz. during the first half of gestation (when
the genitalia are formed) during the perinatal period, and during puberty (Swaab et aI,
1992). In human neonates of 34-41 weeks of gestation the testosterone level is 10-fold
higher in males than in females (De Zegher et ai, 1992). Few data are available on the
exact period in development when the human brain differentiates according to sex. Brain
weight is sexually dimorphic from 2 years postnatally onwards, taking differences in
body weight between boys and girls into account (Swaab and Hofman, 1984). The
supposition of Domer and Staudt (1972) that structural sexual differentiation of the
human hypothalamus would take place between 4 and 7 months of gestation was based
only on the observation that the matrix layer around the third ventricle, in which the
hypothalamic cells are presumed to have been formed, has disappeared by 7 months of
gestation. Indeed, exhaustion of the matrix layer of the third ventricle begins in the 14-
week-old embryo. A one-cell-Iayer ependyma appears from the 25 to the 28-week old
embryo. Although the exhaustion of the matrix layer near the arcuate nucleus is present at
23 weeks of gestation, a multilayer ependyma remains here. No sex differences were
observed in matrix exhaustion (Staudt and Stiiber, 1977). Yet about 80% of the cells of
the sexually dimorphic nucleus appeared to be formed postnatally (Swaab and Hofman,
1988) (Fig. 1). In mid-pregnancy the SDN-POA can already be distinguished in the
human fetal brain (Swaab and Hofman, 1988), yet the SDN-POA cell number and
volume at term birth are only 22% and 18%, respectively, of the values found between 2
and 4 years of postnatal age. During the first postnatal years, up to the age of 2-4, the
SDN-POA cell number rapidly increases at the same rate in both boys and girls, and only
after this age does the human SDN-POA differentiate according to sex, due to a decrease
in both SDN-POA volume and cell number in women. In men these parameters remain
unaltered up to the age of about 50 (Swaab and Hofman, 1988) (Figs. 1,4). The
surprisingly late postnatal sexual differentiation of the human SDN-POA may be a
general phenomenon in the human brain, as it seems as if the sex difference in the
volume of the BNST-dspm does not occur until adulthood (Allen and Gorski, 1990).
In addition, it also became clear that differences in cell death rather than in cell
division may be the most important mechanism in sexual differentiation of the nervous
system (Swaab and Hofman, 1988; Nordeen et ai, 1985). This mechanism seems to take
place in the human sexually dimorphic nucleus between 4 years and puberty (Swaab and
Hofman, 1988) (Fig. 1). After the age of 10 years a clear sex difference was found in the
vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus (Swaab et
ai, 1994). Although the testosterone peak during puberty is generally thought to be
involved in activation rather than organization, the neuron number of the female domestic
pig hypothalamus - to our surprise - showed a twofold increase in a sexually dimorphic

hypothalamic nucleus around puberty (Van Eerdenburg and Swaab, 1991), which means
that late organizational effects cannot be excluded. See Figure 1.
10~
.
•
.
L.
<IJ
.a
E
:;J
c:
Qi
u 10.
<i o
o0.. .
Z
o
VI
Birth Age - postconception (yrs)
Figure I. Development and sexual differentiation of the human sexually dimorphic nucleus (SDN-POA) of the
preoptic area of the hypothalamus. Log-log scale. Note that at the moment of birth the SDN-POA is equally
small in boys (A) and girls (0) and contains only about 20% of the cell number found at 2-4 years of age. Cell
numbers reach a peak value around 2-4 years postnatally, after which a sexual differentiation occurs in the
SDN-POA due to a decrease in cell number in the SDN-POA of women, whereas the cell number in men
remains approximately unchanged up to the age of 50. The SDN-POA cell number in homosexual men (-) does
not differ from that in the male reference group (for more data see Fig. 5). The curves are quintic polynomial
functions fitted to the original data for males (drawn line) and females (from Swaab and Hofman, \988, with
permission).
Recently we found that the sex difference in the central nucleus of the bed nucleus of
the stria terminalis (BSTc) in the human brain only occurred in adulthood (W.C.J. Chung
et aI, unpubl. observ.). Concluding one might say that the evidence that is currently
available suggests that sexual differentiation of the human hypothalamus becomes
apparent between 2 years of age and adulthood, although this may, of course, be based
upon processes that were programmed much earlier, i.e., in mid-pregnancy or during the
neonatal period.
The Hypothalamus and Sexual Behavior
Sex differences in the hypothalamus and strongly connected limbic structures are
thought to be the basis of sex differences in reproduction and sexual behavior (e.g., the
menstrual cycle in women, gender identity (i.e. the feeling one is either male or female),
gender identity problems (i.e. trans sexuality) and sexual orientation (homosexuality and
heterosexuality) (Gooren et aI, 1990; Swaab et aI, 1992; Zhou et aI, 1995c). There is an
extensive animal experimental literature showing that the hypothalamus is a key structure
for male and female copulatory behavior (Pilgrim and Reisert, 1992; Yahr et aI, 1994).
However, literature on hypothalamic structures involved in sexual orientation in experi-
mental animals is scarce (Paredes et aI, 1998; Kindan et aI, 1996; Paredes and Baum,
1995) and data on the hypothalamus in relation to gender identity in animals are, of
course, non-existent. There are a few studies in the medical literature implicating the
hypothalamus and adjoining structures in various aspects of sexual behavior. Direct
electrical or chemical stimulation of the septum may induce a sexually motivated state of
varying degrees up to penile erection in men and building up to an orgasm in both sexes
(Heath, 1964). Marked increased sexual behavior was observed following the placement
of the tip of a ventriculoperitoneal shunt into the septum in two cases (Gorman and
Cummings, 1992). Meyers (1961) described a loss of potency following lesion in the
septo-fornico-hypothalamic region. Precocious puberty and hypersexuality have been
reported following lesions in the posterior part of the hypothalamus and hypogonadism is
an early sign of pathology in the anterior part of the hypothalamus (Bauer, 1954; Bauer,
1959; Poeck and Pilleri, 1965).
Electrical stimulation of the mamillary body in monkeys induces penile erection
(McLean and Ploog, 1962; Poeck and Pilleri, 1965). In addition, in humans there are a
few case histories of changing sexual orientation - from heterosexual to pedophilic or
homosexual - on the basis of a lesion in the hypothalamus or in the temporal lobe, from
which the amygdala has strong connections to the hypothalamus (Miller et ai, 1986). A
German stereotactic psychosurgical study (Muller et ai, 1973) reported on 22 male
subjects, mainly pedo- or ephebophilic homosexuals (n=14), and 6 cases with
disturbances of heterosexual behavior (hypersexuality, exhibitionism or pedophilia). In
12 homosexual patients a lesion was made in the right ventromedial nucleus of the
hypothalamus. In 8 of these patients homosexual fantasies and impulses disappeared, and
6 out of these 8 showed a "vivid desire for full heterosexual contacts" after the operation.
In one pedophilic patient bilateral destruction of the ventromedial nucleus was performed
and he lost all interest in sexual activity after the operation. The heterosexual patients
reported a significant reduction of their sexual drive. Although this report suggests that
the human hypothalamus is involved in sexual orientation and sexual drive, the study is
highly controversial, both from an ethical and from a methodological point of view.
STRUCTURAL SEX DIFFERENCES IN THE HYPOTHALAMUS

AND ADJACENT STRUCTURES
Sexually Dimorphic Nucleus of the Preoptic Area (SDN-POA) in

Human
The sexually dimorphic nucleus of the preoptic area (SON-POA) of the hypothalamus
is located between the dorsolateral supraoptic nucleus (SON) and the mediodorsal pole of
the paraventricular nucleus (PVN) at the same rostrocaudal level as the suprachiasmatic
nucleus (fig. 2). It seems to be involved in aspects of male sexual behavior. The SON-
POA was first described in the rat by Gorski et al. (1978). The cytoarchitectonic sex
difference of this cell group, which is 3 to 8 times larger in male rats than in female rats,
is so evident that it can even be observed with the naked eye in Nissl-stained sections.
Lesions of the SON-POA affect masculine components of sexual behavior in rat
(Turkenburg et aI, 1988; Oe Jonge et aI, 1989) and. the positive correlation between the
volume of the SON-POA and both testosterone levels and male sexual activity in rat
studies suggest a similar relationship (Anderson et aI, 1986). On the other hand, the
extent of the changes in sexual behaviour following SON-POA lesions is so modest
(Turkenburg et ai, 1988; Oe Jonge et aI, 1989) that it is quite likely that the major
function of the SON-POA has not yet been established. We have found a sexually
dimorphic nucleus in the preoptic area of the human hypothalamus (Swaab et aI, 1985;
Swaab and Hofman, 1988; Hofman and Swaab, 1989) that we presume to be homologous
to the SON-POA in the rat as judged from its sex difference in young adults in size and
cell number, localization, cytoarchitecture and neurotransmitter/neuromodulator content
(Figure 2).
Immunocytochemical studies support such a homology between the SON-POA in rat
and human. Galanin- and galanin-mRNA containing neurons are present in the human
SON-POA (Gai et aI, 1990; Bonnefond et aI, 1990) and in the same area in rat (Bloch et
aI, 1993). In addition, the human SDN-POA contains thyrotropin-releasing hormone
(TRH) neurons (55), CCK (J.N. Zhou, unpubl. results), and glutamic acid decarboxylase
(Gao and Moore, 1996a,b), similar to what has been reported in rat (Simerly et aI, 1986).
Moreover, the SON-POA contains a high packing of the enkephalin precursor
preproenkephalin (Sukhov et aI, 1995) and indeed, enkephalin is one of the markers that
the rat and human SON-POA have in common (F.W. Van Leeuwen, unpubl. results).
The human SON-POA is identical to the intermediate nucleus as described by Braak
and Braak (1987). The first to describe the "intermediate nucleus" was Brockhaus (1942).
However, this term has become confusing, since Feremutsch (1948) described a different
"intermediate nucleus". He used this term for the accessory nuclei, i.e. the scattered cells
and islands of oxytocin- or vasopressin- containing neurons between the supraoptic and
paraventricular nucleus (Swaab, 1997). The extent of the confusion becomes clear,' e.g.
from Morton's paper (1961) using the term "intermediate nucleus" again by mistake, for
the accessory neurosecretory cells between the supraoptic and paraventricular nucleus,
but now referring to Brockhaus (1942)! Oaniel and Prichard (1975) used the term
"Preoptic Nucleus" for the human SDN-POA, but this name has not been used in the
literature since. Allen et al. (1989) did not confonn to the name SDN-POA, because they
found more than one sexually dimorphic nucleus in the hypothalamus and did not find a
significant sex difference in the SDN-POA (see below). However, they did not go back to
the original name of "intermediate nucleus" but gave this nucleus yet another name:
"Interstitial Nucleus of the Anterior Hypothalamus 1 (INAH-l)". Because of the
confusion about the term "intermediate nucleus" as described above, and due to the
growing evidence of immunocytochemical homology between the SDN-
POA/intermediate nucleus/INAH-l in human and the SDN-POA in rat we will continue,
in the present paper, to use the term SDN-POA.
A. B.
9Q
o
INAIi)'
IN.H"I
.r\, ~--I
Smm
Figure 2. Topography of the sexually dimorphic structures in the human hypothalamus. A is more rostral than
B. III, Third ventricle; AC, Anterior commissure; I, Infundibulum; LV, Lateral ventricle; OC, Optic chiasm;
SCN, Suprachiasmatic nucleus; SDN, Sexually dimorphic nucleus of the preoptic area (=SDN-POA); PVN,
Paraventricular nucleus; SON, Supraoptic nucleus; BNST-dspm, darkly staining posteriomedial component of
the bed nucleus of the stria terminalis; INAHI-4, interstitial nuclei of the anterior hypothalamus 1-4.
Morphometric analysis of the human SDN-POA revealed that the volume is more than
twice as large in young adult men as it is in women, and contains about twice as many
cells in men (Swaab and Hofman, 1988; Hofman and Swaab, 1989; Swaab and Fliers,
1985) (Fig. 3). The magnitude of the SDN-POA sex difference did not remain constant
throughout adulthood, but fluctuated with age (fig. 3). Recently we found stronger
androgen receptor and oestrogen receptor ex and ~ in the SDN-POA of men as compared
to women (Fernandez-Guasti et aI, 2000, offered; Kruijver et aI, 2000, offered). In males,
a major reduction in SDN-POA cell number was observed between the ages of 50 and 60
years, which resulted in a much less pronounced sex difference in cell numbers. In
females of over 70 years of age cell death was found to be prominent, dropping to values
SEXlJAL DIFFERENTIATION 85
which were only 10-15% of the cell number found in early childhood, so that it appears
that the sex difference in the SDN-POA increases again in old people (Hofman and
Swaab, 1989) (Figure 3).
50
Sexually Dimorphic Nucleu.
M~ 40
~
..
"E
.0 30
"
c:
male.
ii
U 20
«
0
Cl.
Z 10
0
'" female.
0 20 40 60 80 100
Alle(yr~)
Figure 3. Age-related changes in the total cell number of the sexually dimorphic nucleus of the preoptic area
(SDN-POA) in the human hypothalamus. The general trend in the data is enhanced by using smoothed growth
curves. Note that in males SDN-POA cell number steeply declines between the age of 50-60 years, whereas in
females, from the age of about 50 years, a more gradual cell loss is observed, which continues up to old age.
These growth curves demonstrate that the reduction in cell number in the human SDN-POA in senescence is a
non-linear, sex-dependent process (from Hofman and Swaab, 1989, with permission).
This sex difference in the pattern of aging, together with the fact that sexual
differentiation in the human SDN-POA only occurs after the 4th year of age (Swaab and
Hofman, 1988) might explain why Allen et al. (1989), who worked with a sample of
human adults containing a large number of middle aged subjects, did not find a
significant sex difference in the size of the SDN-POA (Swaab et aI, 1992). The age distri-
bution, however, does not explain why LeVay (Le Vay, 1991) was also unable to find a
sex difference in the volume oflNAH-l. It should be noted here that our original sample
for the SDN-POA measurements consisted of 18 women and 13 men (Le Vay, 1991). We
extended these observations to a sample of 103 subjects, the reference group being 38
females and 42 males (Swaab and Hofman, 1988) replicating the sex difference in the
young adult group. A more extensive work-up of the adults was performed in a
subsequent study (Hofman and Swaab, 1989). LeVay's data set consisted of no more than
6 females. It should also be mentioned that LeVay (1991) and Allen et al. (1989) only
measured the volume of hypothalamic structures. Volume is not only susceptible to
histological procedures and methods such as section thickness, but also to various pre-
and postmortem factors such as differences in agonal state and fixation time (Ravid et aI,
1992). It is, therefore, essential to include data on total cell numbers of hypothalamic
nuclei, since this parameter is not influenced by such factors. See Figure 3.
86 D. F. SWAAB. ET AL.
Other Human Hypothalamic Sexually Dimorphic Structures
Allen et al. (1989) described two other cell groups (INAH-2 and -3) in the preoptic-
anterior hypothalamic area of humans that were larger in the male brain than in the
female brain. It is unclear which nuclei in the rat are homologous to the INAH-2 and -3
because so far nothing is known about their neurotransmitter content. Since no immuno-
cytochemistry was performed it is not clear whether the nuclei should be considered as,
e.g., islands of the paraventricular nucleus (PVN) or bed nucleus of the stria terminalis, or
as separate anatomical entities. LeVay (1991) could not confirm the sex difference in
INAH-2, but did find such a difference in INAH-3. The discrepancy between LeVay's
(1991) data and those of Allen et al. (1989) in INAH-2 size can be fully explained by an
age-related sex difference in this nucleus. INAH-2 only shows a sex difference after the
child-bearing age and in a 44-year-old woman who had had a hysterectomy involving the
removal of ovaries 3 years prior to her death (Allen et aI, 1989). This seems, therefore, to
be an example of a sex difference depending on circulating levels of sex hormones, i.e. a
difference based on a lack of activating effects of sex hormones in menopause (see
further on) and not on the organizing effects of sex hormones in development.
Another clear sex difference was described by Allen and Gorski (1990) in what they
called the "darkly staining posteromedial component of the bed nucleus of the stria termi-
nalis" (BNST-dspm). The volume of the BNST-dspm was 2.5 times larger in males than
in females. We found a similar sex difference in the central nucleus of the BST (BSTc,
fig. 5). The BSTc is defined by its dense VIP innervation, which probably originates from
the amygdala and is characterized by its somatostatin fiber plexus and neurons, and
which is sexually dimorphic. The BSTc is 40% smaller in women than in men (fig. 6) and
contains also some 40% fewer somatostatin neurons. No connection was observed
between BSTc volume or somatostatin cell number and sexual orientation. In the
heterosexual reference group and a group of homosexual males a similar BSTc volume
and somatostatin cell number were observed. Moreover. the size and somatostatin cell
number of the BSTc were not influenced by abnormal hormone levels in adulthood
(Kruijver et aI, 2000, in press). See Figure 4.
The vasopressin-containing part of the suprachiasmatic nucleus (SCN), the clock of
the brain, showed a sex difference only in shape, but not in volume or vasopressin cell
number. The shape of the SeN was elongated in women and more spherical in men
(Swaab et aI, 1985). However, the vasoactive intestinal polypeptide(VIP)-containing
subnucleus of the human SCN was found to be twice as large in young men (10 to 30
years) as in young women, and contained twice as many VIP cells (Swaab et aI, 1994).
From the age of about 40 onwards this sex difference was reversed (Zhou et aI, 1995b).
These observations show again how important age is for sexual dimorphisms of the
human brain. It should be noted that sex differences in circadian time keeping have been
described (Ticher et aI, 1994).
The anterior commissure was found to be 12% larger in females, and the interthalamic
adhesion (or massa intermedia), a grey structure that crosses the third ventricle between
the two thalami, was present in more females (78%) than males (68%) (Allen and Gorski,
1991), confirming the old study of Morel of 1947. Among subjects with amassa
intermedia, the structure was on average 53% larger in females than in males (Allen and
Gorski, 1991}. The two latter observations point to a greater connectivity between the
cerebral hemispheres of women as compared to men.
Figure 4. Representative sections of the central nucleus of the bed nucleus of the stria terminalis (BSTc)
innervated by VIP. A: heterosexual man, B: heterosexual woman, C: homosexual man, D: male-to-female
transsexual. Bar = 0.5 mm; LV = lateral ventricle. Note that there arc two parts of the BST in A and B: a small-
sized medial subdivision (BSTm) and a large oval-sized central subdivision (BSTe). From (Zhou et ai, 1995e)
with permission.
Sex Differences in Hypothalamic Alzheimer Changes
In the mediobasal hypothalamus a striking sex difference has been reported in

neurofibrillary pathology associated with abnormally phosphorylated tau protein. The
pathology in the median eminence and infundibular nucleus is characterized by a dense
network of large dystrophic neurites with neurofibrillary tangles that are interspersed
among them. The terminal-like processes contact the neurohaemal vasculature of the
posterior median eminence and the adjacent infundibular nucleus (Fig. 2). The Alzheimer
pathology in the infundibular nucleus was identified in 79% of the males and in only 6%
of the females over the age of 60. The vessel-associated neurofibrillary lesions of the
mediob~sal hypothalamus develop independently of Alzheimer's disease-related
neocortical pathology (Schultz et aI, 1996). It is not known exactly what type of cells and
fibers are affected by this pathology.
An opposite sex difference in Alzheimer pathology was observed in the nucleus

basalis of Meynert (NBM), which is the major source of cholinergic innervation of the
neocortex and which is severely affected in Alzheimer's disease. The percentage ofNBM
neurons containing hyperphosphorylated tau as stained by Alz-50 was higher in females
than in males (Salehi et aI, 1998). This sex difference may be related to the higher
prevalence of Alzheimer's disease observed in women (Brayne et aI, 1995; Fratiglioni et
aI, 1997).
Functional Sex Differences in the Hypothalamus (for review see

Rhodes and Rubin, 1999)
One example of a structure that is influenced by circulating hormone levels, i.e.,

INAH-2, that shows only a sex difference in volume after the child-bearing age has been
mentioned before (Allen et al. 1989). Another example of functional age-related sex
difference is the hypothalamo-neurohypophysial system.
Although we did not find a sex difference in vasopressin neuron number, a sex
difference was reported in vasopressin plasma levels. Males have higher vasopressin
levels than females (Van Londen et aI, 1997; Asplund and Aberg, 1991). In addition it
was found that the posterior lobe of the pituitary is larger in boys than in girls (Takano et
aI, 1999). The sex differences are explained by the higher activity we found in
vasopressin neurons in the supraoptic nucleus (SON) in young males as compared to
females as determined by the size of the Golgi apparatus. The SON is the major source of
plasma vasopressin. In the course of aging, probably triggered by the decrease in
oestrogen levels in postmenopausal women, the neuronal activity in the SON gradually
increases in females, while it remains stable in males. The sex difference in neuronal
activity in the SON thus disappears after the age of 50 (Ishunina et aI, 1999; Fig. 5).
Consequently, this is an example of a hypothalamic system that shows no structural sex
difference but a functional sex difference instead. It is also an example of a sex difference
based on the "activating" (or in this case "inhibiting") effect of sex hormones. The
activation of neurosecretory vasopressin neurons in postmenopausal women was
confirmed by measurement of the cell size as a parameter for neuronal activity. The
minimum and maximum diameters were determined in order to estimate the volumes of
cell somata and cell nuclei in vasopressin neurons stained with an antibody against
human glycoprotein, a part of the vasopressin precursor, or a monoclonal anti-oxytocin
antibody in 15 men and 17 women ranging in age from 29 to 94 years. The vasopressin
neurons in the SON and paraventricular nucleus (PVN) appeared to be larger in young
men than in young women. In elderly women (>50 years old) vasopressin cell size
considerably exceeded that of young women.
In addition, vasopressin cell size correlated positively with age in women, but not in
men in both nuclei. Sex differences in the size of the PVN)vasopressin neurons were
pronounced on the left side and absent on the right, indicating the presence of functional
lateralization of this nucleus. No difference was found in any morphometric parameter of
oxytocin neurons in the PVN among the 4 groups studied. These data demonstrate sex
differences in the size of the vasopressin neurons, and thus in their function, that are age-
and probably also side-dependent, and the absence of such changes in oxytocin neurons
in the PVN (Ishunina and Swaab, 1999). The activation of vasopressin neurons in
postmenopausal women is probably mediated by a decrease in the presence of estrogen

receptor-~ as a possible mediator of inhibitory effects of estrogens and an increase in
estrogen receptor a as a possible mediator of stimulatory effects of estrogens in these
neurons (Ishunina et a1, offered).
Figure 5. Immunocytochemical staining of the Golgi apparatus in dl-SON neurons in a young woman (A,B)
and a young man (C,O). Note the clear difference at both low (B,O) and high (A,C) magnification between the
male and female patients. One subject with an ovariectomy fonowing an ovarian carcinoma (no. 80002) shows
a very intense and large Golgi apparatus (E,F). Scale bar: A,C,E=64 11m; 8,0.F=300 11m (from Ishunina et a1.
1999).
Another example of a sex difference based upon the activating effect of sex hormones
was'found in the mamillary body complex (MBC) that shows much stronger androgen
receptor staining in males than in females (Fernandez-Guasti et ai, 2000, offered). In a
follow-up study we have shown that this sex difference depends fully on the amount of
circulating androgens in adulthood, while the sex difference did not appear to be related
to sexual orientation or gender identity (Kruijver et ai, 2000).
Together, these data support the notion that sexual differentiation of the human
hypothalamus takes place after the perinatal period and before adulthood rather than
during mid-gestation, although it is, of course, possible that the pre- or perinatal
testosterone peak programs cell death a few years later.
90 D. F. SWAAB,ET AL.
HYPOTHALAMIC NUCLEI IN RELATION TO SEXUAL

ORIENTATION AND TRANS SEXUALITY
SDN-POA
A prominent theory about the development of heterosexuallhomosexual orientation is

that it develops as a result of an interaction between the developing brain and sex
hormones. According to Domer's (1988) hypothesis, male homosexuals would have a
female differentiation of the hypothalamus. Once we had found that the SDN-POA of the
hypothalamus of young male adults contains twice as many cells as that of females,
Domer's hypothesis concerning sexual orientation could be put to the test. In contrast to
this hypothesis, neither the SDN-POA volume nor its cell number in the hypothalamus of
homosexual men differed from that of the male reference group in the same age range or
from that of a heterosexual control group of subjects also suffering from AIDS (Swaab
and Hofman, 1988; Swaab and Hofman, 1990) (Fig. 6). The fact that no difference in
SDN-POA cell number was observed between homo- and heterosexual men did not agree
with the global formulation of Domer's hypothesis that male homosexuals have 'a female
hypothalamus'. Although LeVay's finding of a smaller INAH-3 in homosexual men and
heterosexual women agrees with Domer's hypothalamus, the observations in the SDN-
POA, the seN (Swaab and Hofman, 1990) and the presence of a sex difference in VIP
suPAACHlASMATlC NUCLEUS ~y OIMOf!PHIC NUClEUS
0.'
Figure 6. A: volume of the human suprachiasmatic nucleus (SCN) and sexually dimorphic nucleus (SDN) of
the preoptic area as measured in 3 groups of adult subjects: (I) a male reference group (n = 18); (2) male homo-
sexuals who died of AIDS (n = 10) and (3) heterosexuals who died of AIDS (n = 6; 4 males and 2 females). The
values indicate medians and the standard deviation of the median. The differences in the volume of the SCN
between homosexuals and the subjects from both other groups, are statistically significant. (Kruskal-Wallis
multiple comparison test, *p < 0.05; **P < 0.01; ***P < 0.001). Note that none of the parameters measured in
the SDN-POA (Fig. SA, 8) showed significant differences among the
3 groups (P always> .4).
neurons in the seN (Swaab et ai, 1994; Zhou et ai, 1995b) but an absence of a gay-
straight difference in these neurons (Zhou et aI, 1995a) do not support this idea and are
rather in favor of a "third sex", i.e., a "different" hypothalamus in homosexual men which
is neither similar to that in females, nor to that in male heterosexuals.
SUPRACHIASMATIC NUCLEUS SEXUALLY DIMORPHIC NUCLEUS
f?e1.~ Homo~ we\eN) ...

group ftXuaf$
(AIDS)
"XU.,
(AIDS)
Figure 6. B: total number of cells in the human SeN and SDN-POA. The seN in homosexual men contains 2.1
times as many cells as in the reference group of male subjects and 2.4 times as many cells as the seN in hetero-
sexual AIDS patients.
SUPRACHIASMATIC NUCLEUS
20
R~ HomO- Hetel'o-
group sexuals ~r,exuafs
(AIDS) (AIDS)
Figure 6. C: the number of vasopressin neurons in the human SeN (the human SON does not con-
tain vasopressin-producing cells). The SeN in homosexual men contains, on average, 1.9 times as
many vasopressin-(VP)producing neurons as the reference: group of male subjects and 3.G timl!s as
many VP neurons as the seN in heterosexual AIDS patients. Notice that the SeN of heterosexual
individuals who died of AIDS, contains less vasopressin cells than the subjects from the reference
group (from Swaab and Hofman, 1990, with permission).
seN
The first difference in the human brain relating to sexual orientation was found in the
vasopressin part of the SCN that was twice as large in homosexual men (Swaab and
Hofman, 1990). Our observation that the volume of the vasopressin part of the SCN in
homosexual men was 1.7 times as large and contained 2.1 times as many cells as the SCN
of the male reference group (Fig. 5) (Swl).ab and Hofman, 1990) also implied that the
difference in SCN volume could not be attributed to differences in shrinkage of
hypothalamic tissue during the histological procedure. The difference in the vasopressin
cells of the SCN in relation to sexual orientation seems to be rather specific, since the
number of VIP neurons in the SCN of homo- and heterosexual men was not different
(Zhou et aI, 1995a). The SCN is the clock of the human brain, and regulates circadian and
circannual changes (Moore, 1992; Hofman and Swaab, 1994). Differences in the SCN
between homosexual and heterosexual men may thus go together with differences in
circadian rhythms. It was reported that gay men arise and retire earlier than heterosexual
men (Hall and Kimura, 1993) (for possible circadian endocrine differences between
homosexual men and their heterosexual controls see also (Villette et aI, 1990) - although
not interpreted by the authors in this way).
This does, of course, not imply that the SCN, apart from its circadian function, could
not also be involved in sexual behaviour as was suggested by LeVay (1991). On the
contrary, there are many observations suggesting an involvement of the SCN in the
temporal regulation of reproductive processes. As long as 20 years ago, post-coital
ultrastructural changes indicating neuronal activation were observed in the SCN of the
female rabbit (Clattenburg et aI, 1972). It is also important to note that the activity of
SCN neurons increases suddenly around puberty (Anderson, 1981), which is indicative of
the addition of a reproductive function to the already matured circadian functions of the
rat SCN. In addition, efferents of the rat SCN innervate several regions that are known to
be involved in reproductive behaviours, e.g. the preoptic area, medial amygdala and bed
nucleus of the stria terminalis. The rat ovarian reproductive cycle is controlled by the
SCN, possibly by VIP fibres via direct innervation of luteinizing hormone releasing hor-
mone (LHRH) neurons (Van der Beek et aI, 1993). Several morphological sex differences
have been reported that support putative reproductive functions of the SCN. The SCN of
male rats contains a larger amount ofaxo-spine synapses, postsynaptic density material,
asymmetrical synapses compared to that of female rats. Their neurons also contain more
nucleoli than in females (GUldner, 1982; Giildner, 1983), indicating activation in males.
The sex difference in shape of the vasopressin subdivision of the human SCN (Swaab et
aI, 1985) and the sex difference in the number of VIP-containing neurons in the human
seN (Swaab et aI, 1994) is also consistent with sexually dimorphic functions. In seasonal
breeders VIP immunoreactivity in the seN fluctuates in relation to seasonal fluctuations
in sexual activity (Lakhdar-Ghazal et aI, 1992). The activation of c-fos in the seN by
sexual stimulation also points to a role of the seN in reproduction (Pfaus et aI, 1993).
The possibility of sex hormones playing a role in SCN development is enhanced by anti-
oestrogen treatment of the neonatal animal., although the role of sex hormones in this
context is possibly subject to species differences as shown by the work of Holman and
Hutchison (1991), who found that neonatal castration results in a 62% decrease in SCN
volume and the observation of S6dersten et al. (S6dersten and Eneroth, 1980), who
showed that the amplitude of the circadian rhythm in sexual behaviour, for which the
SeN is the substrate.
An interesting analogy to our observations on the enlarged SeN in homosexual men
and sexual orientation was recently observed by Bakker et al. (1993), who found that
male rats treated with the aromatase inhibitor ATD showed a partner preference for
female rats when tested in the late dark phase and a preference for male rats or no
preference at all when tested in the early dark phase. This is the first indication of the
involvement of the SeN in sexual orientation. In the same ATD treated "bisexual" rats
we found an increased number of vasopressin-expre:ssing neurons in the seN (Swaab et
aI, 1995). This supports the hypothesis that the increased number of vasopressin neurons
that we observed in the SeN of homosexual men (Swaab and Hofman, 1990) may be due
to a difference in the interaction of testosterone, aromatase, oestrogens, sex hormone
receptors and the developing brain.
Other Hypothalamic Differences in Relation to Sexual Orientation
The second anatomical difference in the hypothalamus according to sexual orientation,

after our observation in the SeN, was found by LeVay in the INAH-3 (1991). This
nucleus was twice as large in heterosexual men as in homosexual men. There is no
evidence for LeVay's assumption that INAH-3 would be homologous to the SDN-POA in
the rat. Recently, Fliers et al. (1994) found no TRH neurons in INAH-3, while they were
present in the SDN-POA/INAH-l. This supports the possibility that the human SDN-
POA/INAH-l is homologous to the rat SDN-POA (see before). Since no homology to
hypothalamic structures in experimental animals is known for INAH-3, we have to
conclude that the functional consequences, also of LeVay's finding, are at present far
from clear. A third idiosyncrasy according to sexual orientation was described by Allen
and Gorski (1992) who found that the anterior commissure was larger in homosexual
men than in (presumed) heterosexual men and women. A recent abstract reported that the
isthmus of the corpus callosum of gay men was 13% larger than that of heterosexual men
(Scamvougeras et aI, 1994), a similar result as the one reported for the anterior
commissure (Allen and Gorski, 1992).
Transsexuality and the BSTc
Transsexuals have, often from childhood onwards, the strong feeling of having been
born the wrong sex. Their desire to resemble the opposite sex is so strong that they are
even willing to undergo surgery to achieve this end. This gender identity problem has
been proposed to develop as a result of a disturbed interaction between the developing
brain and sex hormones (Domer, 1988). In view of the relationship between the
hypotheses on the development of gender and sexual orientation, it is interesting to note
that 60% of the male-to-female transsexuals are sexually orientated towards males and
that some 10% are bisexual. In no less than 95% of the cases are female-to-male
transsexuals sexually orientated towards women (L.J.G. Gooren, pers. comm.). The high
proportion of transsexuals sexually orientated towards their own genetic sex indicates
that indeed similar (but as yet unknown) mechanisms may playa role in the development
94 D. F. SW AAB, ET AL.
of both gender and sexual orientation. The search for structures that may be directly
related to gender identity, i.e. structures whose anatomy is "female" in genetically male
transsexuals, has so far led to our studying the central nucleus of the bed nucleus of the
stria terminalis (BSTc). A female-sized nucleus was found in male-to-female
transsexuals. The size of the BSTc was not influenced by sex hormones in adulthood and
was independent of sexual orientation (Fig. 4). Similar results were obtained when the
total number of somatostatin neurons was determined in the BSTc (Kruijver et ai,
offered). In the BSTc of one female-to-male transsexual a male volume and somatostatin
neuron number was found. Although the BSTc may be one of many structures involved
in gender feeling. These results do support the hypothesis that gender-identity develops
as a result of an interaction between the developing brain and sex hormones (Zhou et aI,
1995c).
Summary and Conclusions
Functional sex differences in reproduction, gender and sexual orientation and in the
incidence of neurological and psychiatric diseases are presumed to be based on structural
and functional differences in the hypothalamus and other limbic structures. Factors
influencing gender, i.e., the feeling to be male or female, are prenatal hormones and
compounds that change the levels of these hormones, such as anticonvulsants, while the
influence of postnatal social factors is controversial. Genetic factors and prenatal
hormone levels are factors in the determination of sexual orientation, i.e. heterosexuality,
bisexuality or homosexuality. There is no convincing evidence for postnatal social factors
involved in the determination of sexual orientation. The period of overt sexual
differentiation of the human hypothalamus occurs between approximately four years of
age and adulthood, thus much later than is generally presumed, although the late sexual
differentiation may of course be based upon processes that have already been
programmed in mid-pregnancy or during the neonatal period. The recently reported
differences in a number of structures in the human hypothalamus and adjacent structures
depend strongly on age. Replication of these data is certainly necessary. Since the size of
brain structures may be influenced by premortem factors (e.g. agonal state) and
postmortem factors (e.g. fixation time), one should not only perform volume
measurements, but also estimate a parameter that is not dependent on such factors as, i.e.,
total cell number of the brain structure in question. In addition, functional differences that
depend on the levels of circulating hormones in adulthood have been observed in several
hypothalamic and other brain structures. The mechanisms causing sexual differentiation
of hypothalamic nuclei, the pre- and postnatal factors influencing this process, and the
exact functional consequences of the morphological and functional hypothalamic
differences await further elucidation.
Acknowledgements
We would like to thank Ms. W.T.P. Verweij for her excellent secretarial work, Mr. G.
van der Meulen for the illustrations and Dr. J. Kruisbrink for her bibliographical help.
Brain material was obtained from the Netherlands Brain Bank (coordinator Dr. R. Ravid).
Financial support was obtained from the Ter Meulen Fund, KNA W.
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Questions for Dr. Swaab
Question - I would like to address this question to both of you actually and see if your
opinions on it - I am talking about the little boy with a micropenis who is otherwise 46-
XY who we are trying to determine what sex of rearing. In the past we used to sort of
SEXUAL DIFFERENTIA nON 101
think about those patients and try to determine what sex of reading would be most
suitable for them and these patients nonnally would have a testosterone stimulation tried
early on at 25 mg and if they failed that, be banged them with a higher dose at 50 mg and
we tried that for six to nine months of and if there is no response by the end of one year
of life we would after all of that androgen stimulation we say, "well, we do not think this
penis will ever grow" and we do have some patients that have been reassigned on the
basis of that. What have we done to their brains and does stopping the androgen, at that
point, reverse that or is this imprinting happen for life?
Answer - I would say send us the brains of these individuals, and we can give the answer
in ten years. But, even that would not, I think, give a clear answer because we are shown
these structural and functional differences. But for the majority of those differences, we
do not know what it means in functional tenns if it comes to sexual behavior and all as its
aspects. So the final answer should come, I think, from psychologists who are studying
those children and look at what you did to their behavior and to their cognition.
Question - Do you have any functional data on the vasopressin in terms of the
responsivity to osmotic or non-osmotic stimuli and do you know actually whether in fact
the changes in vasopressin cell numbers and presumably vasopressin granules might not
just simply reflect alterations in end organ function?
Answer - The functional data we've got is plasma levels of vasopressin and these are
clearly sexual dimorphic in the sense that they are twice as high in males than in females.
You can of course ask a question whether the osmotic circumstances under which the
people die are equal in all those people we studied, and we cannot answer this point, but
the rat data are similar. They show that the vasopressin secreting cells in males are much
more active than in females and that the plasma levels are higher. So in that sense, I
think there is no controversy and the higher activity is also related to higher blood levels.
Question - This meeting is, later on, going to be largely about the difficult decisions that
we as clinicians make and if we have to wait until all of our patients die before the tools
of science can tell us whether the brains are correctly orientated or not is a slight problem
for follow-up. I just wonder what your opinion is as fundamentally anatomists on the
data coming out from PET -scanning, showing the difference between the male and
female brain that the female tries to think with both halves of the brain at the same time
or the man lateralizes and so on. Have these developed sufficiently well for us to be able
to consider using them in our clinical practice to evaluate our success in gender
assignment?
Answer - I sincerely hope that nobody invited the two of us to solve ali of their problems
because we both would have immediately said no to this invitation. But we hope that you
will consider brain differentiation in the difficult decisions you have to make and that
means in my opinion, you should wait with reconstructions until the child can give the
answer whether it is male or female and this might come from playing behavior or
whatever; but the answer should come from the child, because the anatomy is not
reflecting directly behavior and we cannot conclude, from the anatomy at this moment,
whether the child will become a male or female later. So it would be an illusion to go for
PET-scanning or MRIs at this moment in order to decide whether the child is becoming
male or female. I can see for the future the use of scanning techniques, for instance in the
case of transsexualism. If there is indeed certain differences in the bed nucleus of the
stria terminal is it would be very interesting if this would be present already early in
development. Now the structure is only 2 cubic mm large so that means that it cannot be
visualized as confidence by means of MRI. The contrast for the different nuclei in
hypothalamus is not such that you can do that now, but perhaps in the future there might
be markers for this type of nuclei. For the bed nucleus of the stria terminalis,
somatostatin might be a good marker because it is full of some of the somatostatin
receptors. But then there is the problem that if you inject someastatin analogs, they do
not penetrate the blood brain barrier and will not get to the bed nucleus. So the hope is
for non-peptidergic analogs of this type of compound(s) in order to mark the nuclei and
perhaps then we will be capable to get to a solution which is enough to show those nuclei.
Comment - The corpus callosum had been reported to be larger in women than in men
and many studies failed to confirm that people measured it differently and so when Laura
Allen was with me she measured it by both procedures and found no size difference, but
the shape of the posterior component, the spleenium is more bulbous in women and more
tubular in men. And this is visible on MRls and I had a call from a clinical psychologist
in the Midwest who had a male to female transsexual before surgery and wanted me to
look at his MRI to tell him whether his brain was male or female. Obviously if we could
do that it would be wonderful. My response was that perhaps a series of these types of
markers might lead us with some confidence in the future. But certainly not in our
lifetime.
Comment - I just wanted to say that my proposal was that it would be used for
assessment on follow-up, not for determination in infancy and that is a completely
different matter. And your patient would have been very interesting to study before and
after and therefore, perhaps you should not have resisted the ideal of doing one initially,
but not using it to determine the treatment. And I think in the hair shirt atmosphere of
this Meeting and your proposal that we should wait until the child is of an age to
determine his or her sexuality themselves, one must not loose sight of the fact that
occasionally we might get it right and actually by determining the sex early in life, and
treating the thing from the baby properly, we might end up with a better, more adjusted
better sexually-orientated individual than if we had just put it to one side and waited until
it told us what it wanted to be.
Question - Two lines of questions, one with regard to both of your work with animals. It
seems that one of the recent developments in the field is a great interest in the whole
question of plasticity of the brains, for example the work on how even in the rat brain
there is exquisite sensitivity to the environment. I was wondering if you had any
thoughts about whether or not certain sexually dimorphic aspects of rat brains, and so on,
are more sensitive to environmental effects than others? My second question is, I have to
confess that I think both of you are presenting the human situation in a way that does not
give justice to its complexity. For example, with regard to research on congenital
adrenal hyperplasia, I think it is completely inadequate to give a reference to a study by

Earhardt and Baker that is 25 years old because there has been tremendous development
in study of women with CAH where we know a lot more about psycho-sexual
development in these women. I think one has to think about variable like their gender
identity, their gender role, their sexual orientation, the quality of their sexual life and
there has been a lot of advances in our understanding of the complexity of these women's
development. I think that if a human researcher like myself was just referring to rat
studies in 1974, you would be upset. Now, with regard to the question of Dr. Swaab's
comment of saying there is no evidence at all for social effects on gender identity
differentiation, I would just like to throw out two. We know from genetic females with
CAH that those who are raised as girls predominately develop a female gender identity,
but those genetic females with CAH raised as boys develop a '1lale gender identity and I
would argue that that is one line of evidence that supports the importance of the
environment. Secondly, both of you reference the famous Money and then the Diamond-
Simonds on case of ablatio penis, but neither of you mention the study by Bradley,
myself, and two other authors on a second case report of ablatio penis wherein mid-
adulthood this person had a female gender identity raised as a girl since around the age of
seven months and I think again that would be suggestive evidence for the complexity of
psycho-sexual differentiation. Two cases of ablatio penis, one instance where the person
reverted to living as a male where in our case the person is living as a woman and reports
contentment in living as a woman. So I think my point here is that we have to really
appreciate the multifactorial nature of the constructs that we are talking about. They are
very complicated and I think the situation was we are hearing how complex physical sex
differentiation is and I think the same respect has to be given to behavior sexual
differentiation.
Answer - Roger Gorski and I were sending faxes over and over during the last week or so
because we were desperate that we had to deal with the complexity of this matter in 25
minutes each and we agreed that we could only get over the principles and not the
complexity. In order to give an example of the complexity also as answer to the request
for MRI. If we simply measure brain width in transsexuals from which we know that the
bed nucleus of the stria terminalis is 50% of the size of which it should, brain size is
normal. And it means that for each part of the brain you have to develop a different story
and it is only a matter of when the hormones or other factors might hit the type of cells in
that structure where there will be male or female in the brain. It is a very, very complex
story and I am sure that we will not give a simple answer to the complex questions you
have raised this morning.
Answer (Dr Gorski) - Thank you for the criticism about the CAH. That just reflect my
lack of knowledge of the field as a rat person. I would think however that someone like
Milt Diamond (I should not criticize him since he is not here) your case of the successful
individual who lost her penis. Remember the quote that Diamond has simply says that,
"reassignment, removal of penis, etc. has never been successful". Every time I read that
slide to medical students or others I shutter because never is too profound because it is
very complex. And getting back to your first question, it is even more complex. You
mentioned the plasticity. I think the discovery of sex dimorphisms and the influence of
hormones has enhanced the view that the brain indeed plastic. In the hippocampus for
example, every 4 days with the estrous cycle of the rat there are significant changes in the
number of synapses on certain neurons. So the brain is highly plastic in terms of effects
of hormones and effects of the environment. In fact, I said earlier if you have a structural
difference in the brain that correlates with homosexuality, you do not know which came
first because our brain is sensitive to stress to environmental factors, etc. And like
several questions were raised this morning about what is the effect of testing the
androgen sensitivity of an infant before you decide how to raise him - are you doing
something. Well as brain scientist, when we do something to the animal we are changing
the brain.
Question - I enjoyed both of your papers and I really wanted to respond to your slide
quoting Milton Diamond. What I wanted to say is I think owe him a debt of gratitude to
have pointed out the mistake in John Money's work. I think he has gone too far and I
think that the support of some of his arguments for a moratorium are based on the same
weakness that we are accused on, and that is anecdotes. The second point is that I spent a
good deal of time talking with him at the last AAP Meeting. I wanted to find out from
him just what he would allow me to do surgically on the baby who presents with
ambiguous genitalia and he agreed that it probably was okay to do an abdominal
laporascopy or laporatomy to make a correct diagnosis and he agreed that it probably
would be okay to bring an undescended testis down to reduce the risk of malignant
degeneration or a potential infertility at a later point. But he felt it would be improper to
do anything to the external genitalia - anything to the superficial genitalia. And the more
I thought about this, the more difficulty I had with it because I felt that, while it may be
fine for the child to allow them to develop as they will develop over time, I think that we
have strong responsibility to parents to help them make decisions that are appropriate.
And it leaves parents of these kids in an untenable position. So I just throw that out - I
know there will be further discussion later; but it seems as though the parents get lost in
this morass.
Comment - Dr. Zucker who has spoken earlier, wrote that other article and it is
interesting there is other aspects that come out in his article and one I think that is
somewhat supportive of John Money's concepts. The different outcomes of these 2
patients with penis ablacio, one's gender and surgery was done later. In the first case
where the child identifies later as a male was done at 17 months. Toward the end of what
John Money's critical period which had varied but it ultimately I think became that 18
months where he preferred gender assignment should be done before. And in Dr.
Zucker's patient it occurred at 7 months. Again the ideal that perhaps we can in some
patients reassign gender if it is done early enough, because his was done at 7 months and
testes were removed at 7 months. The other concept though that is still confusing,
because we have to always think in terms of sexual dimorphism, is gender role, gender
identity and sexual orientation. Dr. Zucker's patient is bisexual, so does that mean if I
quote the article correctly, so we can effect certain things with a gender assignment. We
might affect orientation, we might affect role, we might not affect identity. So it is not an
all or none aspect in any of these three things and it may not be an all or none in any
adrenal cortical hyperplasia or any mixed
SEXlJAL DIFFERENTIATION 105
gonadal dysgenesis and so forth. The outcomes ar~~ going to be variable and of course it
is going to be variable on how the parents adjust and how fixed the parents are to that
gender assignment that their child is given and that is variable.
Comment - I will not mention names, but I am raising the issue of informed consent
because an individual that I know used to be in charge of the ambiguous genitalia and
denied absolutely that there was any possibility of hormonal effects on the human brain.
Why? Because in presenting. choices to the parents this physician had to make it
absolutely clear about what the risks, etc. were. And I personally find that totally
unethical. I do not know how you handle advising parents with all the complexities that
we have heard and the different outcomes. Again, a plea for a registry and solid data to
help clinicians in the future.
THE ENDOCRINOLOGIST'S APPROACH
TO THE INTERSEX PATIENT
Perrin C. White, M.D.

Division of Pediatric Endocrinology, UT Southwestern Medical Center, Dallas, Texas
INTRODUCTION
This chapter reviews the concepts underlying the initial evaluation and management of
the intersex child by the endocrinologist. By definition, such a child has lack of
concordance of various aspects of gender. These include chromosomal sex (46XX, 46XY
or other), gonadallreproductive sex (ovaries, Fallopean tubes and uterus vs. testes,
seminal vesicles, prostate gland and ejaculatory ducts), genital sex (vagina and clitoris vs.
penis and scrotum), and gender-specific behavior. Depending on chromosomal sex, most
patients may be classified as incompletely masculinized males (46 XV, male
pseudoherrnaphrodites or males with micropenis), excessively virilized females (46XX,
female pseudo hermaphrodites ), and patients with abnormalities of sex chromosomes such
as those with mixed gonadal dysgenesis. A large number of conditions may be associated
with intersex states (Table 1).
Pathophysiology of Intersex Conditions
Normal Sexual Differentiation
Early in gestation, the gonads are indifferent and bipotential (Figure 1, also see the
chapter by Hadziselimovic). During the i h week, the male gonads begin to differentiate
under the influence of a series of testis-determining genes (reviewed in (Parker et aI,
1999), also see the chapter by Villain). In contrast, the recently characterized signaling
molecule WNT-4 plays an active role in ovarian development (Vainio et ai, 1999).
Ovaries are recognizable at about 10 weeks. If there is no secretion of Mullerian
inhibiting substance, a glycoprotein factor synthesized by the Sertoli cells of the testis
(Lee et ai, 1993)( see the chapter by MacLaughlin), development of the Mullerian ducts
proceeds and female internal structures-the Fallopian tubes, uterus, cervix and upper
vagina-are formed. In contrast, development of male genital structures derived from the
Wolffian ducts, including the epididymis, ductus deferens, ejaculatory ducts, and
seminiferous tubules, requires high local concentrations of testosterone secreted from
Leydig cells of the testis beginning at about 7 weeks; in the absence of testosterone,
Wolffian ducts regress. External genital structures are also bipotential in early gestation,

108 P. C. WHITE
and differentiate as male under the influence of 5a-dihydrotestosterone (reviewed in

(W ilson et aI, 1993), which must interact with an intact androgen receptor (also see the
chapter by Imperato-McGinley)(Quigley et aI, 1995).
Genital ambiguity in genetic females is usually the result of exposure to excessive
levels of androgens. Virilizing congenital adrenal hyperplasia (CAH) is the most common
etiology, and 90-95% of CAH cases are caused by 21-hydroxylase deficiency (White et
aI, 2000). The fundamental defect among patients with CAH due to 21-hydroxylase
deficiency is that they cannot adequately synthesize cortisol (Figure 2). Inefficient
cortisol synthesis signals the hypothalamus and pituitary to increase corticotropin
releasing hormone (CRH) and corticotropin (ACTH), respectively. Consequently, the
adrenal glands become hyperplastic. But rather than cortisol, the adrenals produce excess
sex hormone precursors which do not require 21-hydroxylation for their synthesis. Once
secreted, these hormones are further metabolized to active androgens-testosterone and
dihydrotestosterone-and to a lesser extent estrogens-estrone and estradiol.
TABLE 1: Etiologies of Intersex States
Female pseudohermaphroditism
21-hydroxylase deficiency (I :15,000 births)
Salt wasting or simple virilizing forms
11 j3-hydroxylase deficiency (I: 100. 000)
3fJ-HSD deficiency (1:100.000)
Maternal/exogenous androgens: danazol, progestins
Structurall idiopathic
Male pseudohermaphroditism
Biosynthetic defects
I 7p-hydroxylase/l 7,20 lyase

3P-HSD
Lipoid adrenal hyperplasia
17 -ketosteroid reductase
Sa-reductase
Exposure to 5 a-reductase inhibitors
Androgen insensitivity (J :20,000)
Complete or partial
Gonadotropin insensitivity
Micropenis
Panhypopituitarism
Septo-optic dysplasia
Hypogonadotropic hypogonadism
Kallmann syndrome
Prader-Willi syndrome
Vanishing testes (may also cause ambiguous genitalia),
Other chromosomal syndromes affecting reproductive systems
Turner (I :2,500): 45 X, 45X/46XX mosaics, 46XXr, 46XXq-
Kleinfelter (1 : I ,000): 47 XXY
Mixed gonadal dysgenesis: 45X/46XY, 45X/47XXY
Other: Trisomy 13, Trisomy 18, Triploidy, 4p-, f 3q-
Female pseudohermaphroditism
THE ENDOCRINOLOGIST'S APPROACH 109
!
Sertoll cella
~I
•• •
!eYdig cells
Gonads
Follicle.
Oogonl?"'Y'°'
Wolffian
ducts
I• Rrresslon•
Mullerian
ducts ~I - I
Fusion
Vagina. ~. ~
External c!'
genitalia
Sf
,.
Ventral naxian of
phallus
Utricle
I IPro:?e
~I !
Urogenital
sinus Vagina
Vagina;eechB8
perinelJm
I I I I I I I ~
2 4 6 8 10 12 14 16 18 20 22 24
Weeks of gestation
Figure 1. Time course of prenatal sexual differentiation in male and female fetuses (adapted from Barthold et
ai, 1999).
Adrenal secretion of excess androgen precursors does not significantly affect male sexual
differentiation. In females affected with CAH, however, the urogenital sinus is in the
process of septation when the fetal adrenal begins to produce excess androgens; levels
of circulating adrenal androgens are apparently sufficiently high to prevent formation of
separate vaginal and urethral canals. Further interference with normal female genital
anatomy occurs as adrenal-derived androgens interact with genital skin androgen
receptors and induce clitoral enlargement, promote fusion of the labial folds, and cause
rostral migration of the urethral/vaginal perineal orifice. However, internal W olffian
structures such as the prostate gland and spermatic ducts are usually not virilized,
presumably because development of the W olffian ducts requires markedly higher focal
concentrations of testosterone than the external genitalia. Nevertheless, severely affected
females may occasionally have some development of typically male internal genital
structures.
Thus, the typical result in severely affected girls is ambiguous or male-appearing
external genitalia with perineal hypospadias, chordee, and undescended testes. The
severity of virilization is often quantitated using a five-point scale developed by Prader
(Figure 3) (Prader, 1954). Not all classic CAH females develop the same degree of
genital ambiguity. One might speculate that the physical signs of androgen excess are
dependent not only on direct adrenal secretion of androgen precursors, but also on the
efficiency with which such hormones are converted to more potent products, such as
dihydrotestosterone by peripheral enzymes such as Sa-reductase (Wilson et aI, 1993).
110 p. C. WHITE
Additionally, the concentration and transcriptional activity of androgen receptors may

playa further role in determining genital phenotype.
Approximately three-quarters of patients cannot synthesize sufficient aldosterone to
maintain sodium balance and are termed "salt wasters". This predisposes them to
episodically develop potentially life-threatening hyponatremic dehydration. Patients with
sufficient aldosterone production and no salt-wasting who have signs of prenatal
virilization and/or markedly increased production of hormonal precursors of 21-
hydroxylase (e.g., 17-hydroxyprogesterone), are termed "simple virilizers". Patients with
CAH due to 11 p-hydroxylase deficiency have similarly elevated secretion of adrenal
androgens, but synthesize aldosterone normally and do not have problems with salt
wasting. Instead, they are likely to become hypertensive due to elevated levels of
deoxycorticosterone and its metabolites.
Male Pseudohermaphroditism and Micropenis
Androgens are required to fuse the labioscrotal folds, to induce rostral migration of the
urethral meatus, and to increase the size of the phallus. Hence, androgen insensitivity, or
severe deficiencies of androgens that have been present since early in gestation, cause
ambiguous or female-appearing external genitalia. Usually Miillerean structures such as
the uterus, cervix and upper vagina are not present as the testes are able to secrete
Miillerean inhibitory substance. Thus individuals with these conditions have a short
vagina ending in a blind pouch.
The biosynthetic defects that cause genital ambiguity in males may be conceptually
divided into those that affect early and late steps of androgen biosynthesis (Figure 2).
Lipoid hyperplasia (a defect of cholesterol transport into mitochondria) (8tocco et ai,
1996) and severe 17P-hydroxylaseI17,20 lyase deficiencies (Yanase et aI, 1991) prevent
synthesis of any active androgens. Dehydroepiandrosterone, a very weak androgen, is
synthesized in 3P-hydroxysteroid dehydrogenase deficiency; this permits some degree of
phallic growth (Rheaume et aI, 1992). Androstenedione, a somewhat stronger androgen,
is synthesized in 17-ketosteroid reductase deficiency (Geissler et ai, 1994), and as there
are several isozymes with 17-ketosteroid reductase activity in otl).er tissues, some
testosterone is invariably synthesized, especially at puberty when circulating levels of
androstenedione increase . Finally, patients with Sa-reductase deficiency synthesize
entirely normal amounts of testosterone, but they cannot synthesize adequate amounts of
dihydrotestosterone, the most potent naturally occuring androgen. This enzyme is
expressed in genital skin (circulating levels of dihydrotestosterone are relatively low) and
it is required for normal development of the external genitalia. Virilization often occurs at
puberty nonetheless when increased levels of testosterone are secreted by the testis
(Wilson et aI, 1993). Milder or later-appearing deficiencies of androgen biosynthesis
may allow complete fusion of the labioscrotal folds and normal positioning of the
urethral meatus but subsequent growth of the phallus is suboptimal.
THE ENDOCRINOLOGIST'S APPROACH III
CY-P-17'!
I
~y~~
.. :~~"c;~~~;~~~. :,............. " .""" .. "." .. ".. ".. :

ICYP"TtAJ:.
: PREGNENOLONE .... 17·HYDROXY· DEHYDROEPI·'
131?-HSD r""
• I PREGNENOLONE
ow. ow . . . . " "
PROGESTERONE
ANDROSTERONE
..... ..... ...... " , .
17·HYDROXY·
:
:
!ANOROSTENEDIONE.
ICYP21 ~ PROrSTERONE : -l-------=r17if-HSD!

L ._ _ _ -'I OEoh. ".OEOXYCORnSOL :TESTOSTERONE .... :~--- .J
r::-:-:-::-:--=..
ICYP11 B2l--+-
CORTICOSTERONE . '
-l----jCYP11 ~"..:..:.j""""" S50R
r(,VD~OSTERONE
~ , __...t,.,o
CORTISOL
DIHYDRO-
L I_ _ _- '
'~YDROxy· TESTOSTERONE
icICYlYPP11111B~2~1 ::.:j:.=
CORTICOSTERONE
ALDOSTERONE
Mineralocortlcoids Glucocorticoids Androgens
Figure 2. Adrenal and testicular (denoted by dotted line) steroidogenesis. The pathways for synthesis of
progesterone and mineralocorticoids (aldosterone), glucocorticoids (cortisol) and androgens (testosterone and
dihydrotestosterone) are arranged from left to right. The enzymatic activities catalyzing each bioconversion are
written in boxes. For those activities mediated by specific cytochromes P450. the systematic name of the
enzyme ("CYP" followed by a number) is listed in parentheses. CYPII B2 and CYPI7 have multiple activities.
CYPII A, cholesterol side-chain cleavage enzyme; HSD, hydroxysteroid dehydrogenase; CYP21. 21·
hydroxylase; CYPll B2, aldosterone synthase; CYPll B 1. 11 ~·hydroxylase; CYP17, 17~-hydroxylaseI17.20
lyase; S5aR, steroid 5a-reductase.
Normal ~
• v Nor-mal rf
'va( W Y(J{ y(~{ ~

YCi)/ c5'
-L ~ -L --L- _L -L ~
Figure 3. Normal and abnormal differentiation of the urogenital sinus and external genitalia. Diagrams of
normal female and male anatomy flank a series of schematic representations of different degrees of virilization
of females. graded using the scale developed by Prader (figure lrom ref. Prader, 1954). Note that the uterus
(shaded) persists in virilized females even when the external genitalia have a completely masculine appearance
(Prader grade 5).
112 P. C. WHITE
Evaluation of Ambiguous Genitalia
Management of the child born with ambiguous genitalia presents a difficult challenge
to medical personnel. Insensitive and poorly informed statements made in the delivery
room subsequently may cause long-term psychological problems for the families of such
children. It is therefore important to refrain from assigning the sex until diagnostic
information can be gathered (Figure 4). Usually test results can be obtained within 24-48
hours and parents can be advised as to the child's chromosomal and gonadal sex and on
the anatomy of internal sexual structures.
In addition, the physician must keep in mind that intersex conditions may be
associated with life-threatening biochemical or anatomic abnormalities. In particular, the
most common cause of severely masculinized external genitalia in female, the salt-
wasting form of CAH due to steroid 21-hydroxylase deficiency, may cause
hyponatremia, hyperkalemia, hypovolemia and shock. In contrast, males with ambiguous
genitalia might have lipoid adrenal hyperplasia or a salt wasting form of 3~
hydroxysteroid dehydrogenase deficiency. Males with micropenis may have
panhypopituitarism, in which case they are at risk for significant hypoglycemia due to
low cortisol (from low ACTH) and low growth hormone levels, or they might have
congenital adrenal hypoplasia, in which case theycould have adrenal insufficiency.
Finally, patients with ambiguous genitalia are at increased risk from renal anomolies, or
they may have chromosomal syndromes with other associated anomolies (reviewed in
(Grumbach et aI, 1998).
XJX:( or
XfXX.Y
~dohenn-
.........__--' ..~~
aphrodite
I
,"'V'"'
Au.. .....1a
...........
I
Figure 4. Flowchart for initial evaluation of the infant with ambiguous genitalia.
History and Physical Examination
The gestational history should concentrate on potential exposure to agents that might
interfere with normal sexual differentiation. For a female infant with virilized genitalia,
these include progestational agents; whereas the mother of a male with incompletely
masculinzed genitalia might have been exposured to a 5 -reductase inhibitor through her
husbands' use of such an agent for alopecia or prostate enlargement. It should be
detennined whether an amniocentesis and karyotype have been performed. A family
history should elicit similar cases of genital ambiguity or cases of sudden death, which
could raise suspicion of undiagnosed salt-wasting CAH.
The physical exam should document the size of the phallus (clitoris or penis), the
degree of chordee, and the extent of fusion of the labioscrotal folds. The urethral meatus
should be identified, and there must be careful palpation for gonads in the inguinal canals
and labia or scrotum. Vital signs and other anatomic anomolies (if present) should be
documented.
Rapid Diagnostic Tests
In many centers, a fluorescent in situ hybridization (FISH) of interphase nuclei using

sex chromosome specific probes can be obtained within 24 hours. This technique
accurately counts sex chromosomes although it cannot detect trans locations or some
chromosomal fragments; these require a full karyotype on metaphase chromosomes. If a
reliable operator is available, a pelvic and abdominal sonogram can be performed in the
first 1-2 day of life, which will detect the kidneys and a uterus, if present; it may also
locate the gonads and determine the size of the adrenal glands. An MRI of the abdomen
is a more expensive alternative that requires sedati.on. A radiologic dye study is often
performed subsequently to further define the internal genitourinary anatomy.
At this point, the chromosomal and gonadal sex of the child are usually clear even
before the FISH is available. If gonads are palpable, it is very likely that the child is
genetically male, whereas demonstrating a uterus by sonography means that the child is
probably female. However, these tests usually do not identify the etiology of the intersex
condition, which requires further biochemical evaluation.
Biochemical Evaluation of the Female Pseudohermaphrodite
At the least, diagnostic tests should include measurement of basal serum 17-
hydroxyprogesterone, androstenedione and testosterone. Preferably a complete profile of
adrenocortical hormones is obtained before and one hour stimulation of the adrenal
cortex with cosyntropin (ACTH'_24., Figure 5). These assays should be deferred past the
first 24 hours of life. They will identify potential defects in adrenal steroidogenesis (i.e.,
CAH); 21-hydroxylase deficiency is identified by elevations in 17-hydroxyprogesterone,
whereas 11-deoxycortisol and II-deoxycorticosterone are high in 11 ~-hydroxylase
deficiency.
After testing is completed, the child's vital signs should be monitored for any
indication of adrenal crisis. It is rare for salt wasting crisis to occur before 7 days of life,
but many clinicians will obtain electrolyte measurements to assess hyponatremia and
hyperkalemia in CAH newborns during the first week. Plasma renin activity and
aldosterone are elevated in many normal infants and do not usually add much useful
information within the first days of life.
114 P. C. WHITE
Cosyntropin stimulation test

Measure steroids before and 60 1 after 250 meg
eye
of ACTH (1-24) IVIIM
I
~ __ ----l PREGNENOLONE 17-HYDROXY. DEHYDROEPf..
PREGNENOLONE ANDROSTERONE
f3j1:Aw. • t
L:~J_~ PROGESTERONE 17-HYDROXY· ANDROSTENEDIONE
PROGESTERONE
ICYP21 JI--:l-t- - - - I t -
. l1·DEOXY· 11·DEOXYCORTISOL
CORTICOSTERONE r:::-=-:-:-:::-:1
-1-·---iCYP11811
ALDOSTERONE CORTISOL
Figure 5. Cosyntropin stimulation test. Abbreviations as in figure 2.
Biochemical Evaluation of the Male Pseudohermaphrodite
In intersex males, it is necessary to test adrenal and gonadal function as well as

extragonadal androgen metabolism. As regards adrenal defects, II-deoxycorticosterone
and the ratio of pregnenolone to 17-hydroxypregnenolone are high in 17~-hydroxylase
deficiency, 17-hydroxypregnenolone and dehydroepiandrosterone are high in 3~
hydroxysteroid dehydrogenase deficiency, and all steroids are low in lipoid hyperplasia.
Defects in gonadal steroidogenesis are best evaluated after stimulation with human
chorionic gonadotropin (hCG, Figure 6). However, both 17-hydroxylase and 3~
hydroxysteroid dehydrogenase deficiencies affect both the gonads and the adrenal cortex,
and thus are often diagnosed by cosyntropin stimulation testing. Low levels of all
androgen precursors suggests 17~-hydroxylase/17,20 lyase deficiency or a generalized
defect in testicular function such as the vanishing testis syndrome or gonadotropin
insensitivity. A high ratio of androstenedione to testesterone is indicative of 17~
ketosteroid reductase (also called 17~-hydroxysteroid dehydrogenase) deficiency, and a
high ratio of testesterone to dihydrotestosterone is diagnostic of Sa-reductase deficiency.
The diagnosis of androgen insensitivity syndrome is suspected when a 46 XY patient
has ambiguous or female-appearing external genitalia despite normal or high circulating
levels of testosterone and dihydrotestosterone.
Patients with micropenis rarely have defects in steroid biosynthesis, but instead often
have low levels of gonadotropins (i.e., hypo gonadotropic hypogonadism). As
gonadotropins are usually higher in neonates than older children, these can be measured
directly. If they are low, or if there is any history of hypoglycemia, pituitary function
should be completely evaluated by measuring thyroid stimulating hormone and
thyroxine, baseline ACTH and cortisol. Growth hormone cannot be accurately measured
in random blood samples unless the patient is hypoglycemic, but levels of insulin-like
growth factor (IGF) 1 and IGF binding protein 3 are readily measured surrogates; growth
hormone can be measured after appropriate stimulation if suspicion of growth hormone
deficiency is high. Any patient with suspected hypopituitarism should have magnetic
resonance imaging of the head. Patients with hypogonadotropic hypogonadism should
have normal testicular function documented with an heG stimulation test.
HCG stimulation test

Inject 1500 IU hCG 1M on days 1,3,5.
Measure steroids on days 1 and 6.
ANDROSTENEDIONE
l -ir--17-13--H-SD--O,
TESTOSTERONE
~,-----1!S5aR
DIHYDROTESTOSTERONE
J
Figure 6. HCG stimulation test. Abbreviations as in figure 2.
Gonadal Biopsies
Patients with mixed gonadal dysgenesis, true hermaphroditism or unclear diagnoses

should undergo bilateral gonadal biopsies (the two gonads often are not identical in their
histology). Dysgenetic gonads have a high potential for malignant transformation and
will usually need to be removed in childhood.
Initial Medical Management
Patients with congenital adrenal hyperplasia require replacement of both

glucocorticoids and mineralocorticoids, usually with hydrocortisone and fludrocortisone,
respectively. Patients with panhypopituitarism usually require treatment with
hydrocortisone, thyroxine and growth hormone.
All males with ambiguous genitalia or micropenis in whom a male sex of rearing is
contemplated should have a 3 month therapeutic trial of monthly depot testesterone
injections to attempt to increase the size of the phallus during infancy. This may improve
social acceptability of the genitalia later in childhood and adolescence and/or may make
reconstructive surgery easier. In cases of suspected partial androgen insensitivity, this
treatment will also document the degree to which the patient is androgen responsive and
thus may provide useful information as to whether a male sex of rearing is feasible.
116 P. C. WHITE
Considerations for Sex Assignment
A team consisting of neonatologist, pediatric endocrinologist, urologist and preferably

an experienced social worker and/or child psychiatrist should promptly review the early
diagnostic data and make a recommendation to the family as to the sex of rearing and any
medical and/or surgical treatments. These recommendations should be based on both the
current state of knowledge of psychosexual development in intersex individuals and the
feasibility of surgical correction. Although all available options should be reviewed with
the family, these recommendations should be as unequivocal as possible.
In general, the recommended sex' assignment should be that of the genetic/gonadal
sex, if for no other reason than to retain the possibility of reproductive function. This is
especially true for females with CAH who have normal internal genital structures and
potential for child-bearing. An exception to this rule might be the genetic female with
completely male appearing genitalia, especially if the child has been raised as a male for
more than a few months. Such children will need to be castrated at puberty to avoid
feminization.
Conversely, genetic males with completely female appearing external genitalia
(usually due to complete androgen insensitivity syndrome but also seen with severe
testosterone biosynthetic defects) should be raised as female as the potential for
reconstruction of male genitalia is poor. They, too, need to be castrated at or before
puberty to avoid malignant transformation of the testes. However, males with 17-
ketosteroid reductase or Sa-reductase deficiency should usually be reared as males as
they have normal levels of androstenedione or testosterone, respectively, and often
virilize significantly at puberty.
Corrective Surgery
This topic is reviewed in more detail elsewhere in this volume. Whether, how and
when to intervene surgically in the correction of genital anomalies is the subject of
continuing debate (Wilson et aI, 1998). Some adult patients with CAH and other intersex
conditions who are unhappy with their gender assignment, as well as some physicians,
have advocated postponing genital surgery until the affected individual is able to provide
informed consent for cosmetic genital surgery, and select the gender with which he/she
will be most comfortable (Diamond, 1999). It is not clear, however, whether families
would readily accept the idea of raising a child with indeterminate gender and/or
ambiguous genitalia, whether children would then be psychologically traumatized due to
lack of societal acceptance of such conditions, and whether such children would be able
to develop an unambiguous gender identity at all.
It must also be recognized that recommendations for sex assignment are to some
extent culture specific. In cultures that value infant boys over girls, parents may strongly
resist rearing a female with ambiguous genitalia as a girl, and many girls with severely
virilized external genitalia will be raised as males.
According to self-assessment surveys among sexually active women with CAH who
have had genital surgery, most are able to have satisfactory intercourse. Reoperation is
frequently required to achieve satisfactory results. As surgical and medical treatment

regimens have improved in recent years, more women with CAH have successfully
conceived spontaneously, completed pregnancies and given birth (Premawardhana et aI,
1997)(Powell et aI, 1995). Most often delivery is by cesarean section due to an
inadequate introitus, but vaginal delivery is possible in some cases.
Gender Role and Identity
The influence of prenatal sex steroid exposure on personality is controversial

(reviewed in (Meyer-Bahlburg, 1999)(Meyer-Bahlburg, 1999; Wilson, 1999». In
considering this question, it is important to distinguish between gender role, sexual
orientation and gender identity.
Gender role refers to gender-stereotyped behaviors such as choice of play toys by
young children. For example, parents of young girls with CAH often report that their
daughters prefer to play with trucks as compared with dolls, and tend to be tomboyish
later in childhood. Indeed, low interest in maternal behavior, beginning with infrequent
doll play in early childhood and extending to lack of interest in child-rearing for older
girls and women, is a recurring theme in CAH research (Berenbaum, 1999).
Sexual orientation refers to homosexual versus heterosexual preferences. In many
studies, a small but significant percentage of adult women with CAH have been actively
homosexual or bisexual or have an increased tendency to homoerotic fantasies. These
characteristics occur more frequently in women with the salt-wasting form of 21-
hydroxylase deficiency.
Gender identity refers to self-identification as male or female. Spontaneous gender
reassignment back to male has been reported in cases of males with penile trauma who
were raised as females or male pseudohermaphrodites raised as females (Diamond,
1999), especially in cases of Sa-reductase or 17-ketosteroid reductase deficiencies, in
which the brain may be exposed to high circulating levels of androgens (reviewed in
(Wilson, 1999)). Conversely, female-to-male transsexuals may have relatively high levels
of androgens and a high incidence of polycystic ovary syndrome (Bosinski et ai, 1997).
However, self-reassignment to the male sex is unusual in women with CAH (Zucker et aI,
1996). When it occurs, it may be related to delays in gender assignment or genital surgery
or to inadequate suppression of adrenal androgens with glucocorticoid therapy (Meyer-
Bahlburg et aI, 1996). Severely virilized females are more likely to be raised as males in
cultures that value boys more highly and/or in third world countries in which the
diagnosis is likely to be delayed. There have been few studies directly comparing
psychosexual functioning in severely virilized genetic females with CAH raised as
women or men, but it does not appear that those raised as men are psychologically better
adjusted than those raised as women.
The uncertainty concerning the effects of prenatal and postnatal effects of androgen on
gender identity and gender role extends not only to the female CAH population, but also
to male pseudohermaphrodites of other etiologies. The role of external genital anatomy
before and after genital surgery in fueling problems relating to gender is unclear
compared to the roles of prenatal hormone exposure, rearing by the family and
118 P. C. WHITE
community attitudes. Unfortunately, much of the data in this area is anecdotal (reviewed
in (Meyer-Bahlburg, 1999), and see the chapter by Meyer-Bahlburg).
Families of intersex patients should be assessed for emotional health. The initial
screening will most likely be done by the pediatrician and pediatric endocrinologist.
Parents should be offered psychologic counseling soon after the diagnosis is made.
Intermittent assessment of family functioning may be a useful tool in predicting future
problems. Children should, as they mature, be repeatedly informed about their condition
by parents and physicians in a sensitive and age-appropriate manner. When
psychotherapy is undertaken, medical and psychiatric caregivers should maintain
communication so that both are aware of the patient's and family's status. Unfortunately,
many locales lack mental health professionals with experience in counseling patients and
families with intersex conditions.
Although psychosexual development of intersex patients is incompletely understood,
patients' families should receive anticipatory counseling. For example, counselling of
parents of girls affected with CAR should initially address the high likelihood that such
girls will exhibit tomboyish behavior, masculine play preferences and perhaps, when
older, a preference for a career over domestic activities. In the contemporary United
States, these preferences usually have a high degree of social acceptance, considering the
increased availability of and interest in girls' competitive sports as well as the many
women who work. Parents should also be reassured that the majority of (but not all) girls
with CAR function heterosexually, although they may require repeated genital surgeries
in order to have satisfactory intercourse. The endocrinologist and/or mental health
professional (depending on inclination and experience) caring for the adolescent girl with
CAR should address sexual orientation, both fantasized and actual. The patient should be
reassured that some degree of attraction to other girls, although it does not always occur,
is a "normal" feature of her condition. A discussion of psychotherapy for homosexuality
is beyond the scope of this review, but it should be accepted by health care professionals
that a minority of women with CAR may be most comfortable as homosexuals and that
such individuals should be helped to come to terms with their situation. Adult patients
should also be made aware of relevant patient advocacy groups.
REFERENCES
Barthold JS, Gonzalez R. Intersex states, in: Pediatric Urology Practice, ET Gonzales, SB Bauer, eds.,
Lippincott Williams & Wilkins, Philadelphia, pp. 547-578, 1999.
Berenbaum SA. Effects of early androgens on sex-typed activities and interests in adolescents with congenital
adrenal hyperplasia. Horm Behav 35(1 ):102-110, 1999.
Bosinski HA, Peter M, Bonatz G, Arndt R, Heidenreich M, Sippell WG, Wille R. A higher rate of
hyperandrogenic disorders in female-to-male transsexuals. Psychoneuroendocrinology 22(5):361-380,
1997.
Diamond M. Pediatric management of ambiguous and traumatized genitalia. J Urol 162(3 Pt 2):1021-1028,
1999.
Geissler WM, Davis DL, Wu L, Bradshaw KD, Patel S, Mendonca BB, Elliston KO, Wilson JD, Russell DW,
Andersson S. Male pseudohermaphroditism caused by mutations of testicular 17 beta-hydroxysteroid
dehydrogenase. Nat Genet 7:34-39, 1994.
Grumbach MM, Conte FA. Disorders of sex differentiation, in: Williams Textbook of Endocrinology, 9/"
Edition. JD Wilson, DW Foster, HM Kronenberg, PR l.arsen, eds., W.B. Saunders, Philadelphia, pp. 1303-
1426,1998.
Lee MM, Donahoe PK. Mullerian inhibiting substance: a gonadal hormone with multiple functions, Endocr
Rev 14(2):152-164,1993.
Meyer-Bahlburg HF, Gruen RS, New MI, Bell JJ, Morishima A, Shimshi M, Bueno Y, Varga I, Baker SW,
Gender change from female to male in classical congenital adrenal hyperplasia. Horm Behav 30:319-332,
1996.
Meyer-Bahlburg HF, Gender assignment and reassignment in 46, XY pseudohermaproditism and related
conditions. J Clin Endocrinol Metab 84:3455-3458, 1999.
Meyer-Bahlburg HF. What causes low rates of child-bearing in congenital adrenal hyperplasia? J Clin
Endocrinol Metab 84(6):1844-1847,1999.
Parker KL, Schedl A, Schimmer BP. Gene interactions in gonadal development. Annu Rev PhysioI61:417-
433,1999.
Powell DM, Newman KD, Randolph J. A proposed classification of vaginal anomalies and their surgical
correction. J Pediatr Surg 30(2):271-275,1995.
Prader A. Der genitalbefund beim ppseudohcrmaphroditismus femininus der kengenitalen adrenogenitalen
syndroms. He/v Paediatr Acta 9:231-248, 1954.
Premawardhana LD, Hughes lA, Read GF, Scanlon MF. Longer term outcome in females with congenital
adrenal hyperplasia (CAH): the Cardiff experience. Clin Endocrinol (Oxf) 46:327-332, 1997.
Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS. Androgen receptor defects:
hIstorical, clinical, and molecular perspectives. Endocr Rev 16(3):271-321, 1995.
Rheaume E, Simard J, Morel Y, Mebarki F, Zachmann M, Forest MG, New MI, Labrie F. Congenital adrenal
hyperplasia due to point mutations in the type II 3 beta-hydroxysteroid dehydrogenase gene. Nat Genet
1239-245, 1992.
Stocco DM, Clark BJ. Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev
17(3):22 I -244, 1996.
Vainio S, Heikkila M, Kispert A, Chin N, McMahon AP. Female development in mammals is regulated by
Wnt-4 signalling. Nature 397(6718):405-409, 1999.
White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev
21(3):245-291,2000.
Wilson BE, Reiner WG. Management of intersex: a shifting paradigm. J Clin Ethics 9(4):360-370,1998.
Wilson JD, Griffin JE, Russell DW. Steroid 5 alpha-reductase 2 deficiency. Endocr Rev 14(5):577-593,
1993.
Wilson JD, The role of androgens in male gender role behavior. Endacr Rev 20:726-737, 1999.
Yanasc T, Simpson ER, Waterman MR. 17 alpha-hydroxylase/I 7 ,20-lyase deficiency: from clinical
investigation to molecular definition, Endocr Rev 12:91- 108, 1991.
Zucker KJ, Bradley SJ, Oliver G, Blake J, Fleming S, Hood J. Psychosexual development of women with
congenital adrenal hyperplasia. Harm Behav 30(4):300-318,1996.
Questions for Dr. White
Question - I was wondering if you had any thoughts on males with complete androgen
insensitivity. What happens to those patients when they are stimulated with high doses of
testosterone to determine if they are indeed complete androgen insensitivity and they
show no response in penile growth - what happens to their brain as far as imprinting in
120 P. C. WHITE
concerned - do they lack androgen receptors in the brain, too or are we going to imprint
their eNS?
Answer - The studies of individuals with complete, and I emphasize the words complete
androgen sensitivity, is that they tend to have normal - they tend to actually - in one
sense, have a better female gender identity than, let us say, females of 21 hydroxylase
deficiency. They have maternal feelings. There have been many cases of women with
complete androgen insensitivity who have successfully adopted. And I usually tell those
kids when I see them later is, as far as I am concerned, they are girls. They don't have a
uterus, their chromosomes may not be those of a girl, but they as persons are girls and
they should by large feel comfortable with that assignment.
Question - If you were to see a patient in your hospital tomorrow, that presents with a
micropenis as a newborn, before you made the diagnosis of a complete androgen
insensitivity are you going to stimulate or give some androgen?
Answer - By definition, you are talking partial. If you say, how do you distinguish
between complete and partial androgen insensitivity - yes your correct is that as nobody
is doing the genetic test and even if they did, is there an uninformative in the majority of
cases is you have to give some androgens and look to see what happens. But by
definition that is partial and I agree that intermediate syndromes are those where often as
you really cannot make an unequivocal optimal gender assignment within the first couple
of days of life for those reasons.
5a-REDUCTASE-2 DEFICIENCY AND COMPLETE
ANDROGEN INSENSITIVITY: LESSONS FROM
NATURE
J. Imperato-McGinley, M.D.
Weill Medical College of Cornell University, New York, NY 10021
5a-REDUCTASE-2 DEFICIENCY
The importance of the hormones testosterone and dihydrotestosterone in the male is

best illustrated by studies of the inherited condition of male pseudohermaphroditism due
to 5ex-reductase-2 deficiency (Imp.;:rato-McGinley et aI, 1974,1976;Fratianni and
Imperato-McGInley, 1994; Walsh et aI, 1974). Testosterone is converted to
dihydrotestosterone by two known Sex-reductase isoenzymes 5ex-reductase-l and
5ex-reductase-2. It is defects in the 5ex-reductase-2 gene that cause male
pseudohermaphroditism. The syndrome of 5ex-reductase-2 deficiency results in a
selective decrease in dihydrotestosterone. The clinical and laboratory findings of this
syndrome have defined specific roles for testosterone and dihydrotestosterone in male
sexual differentiation and development. The unusual psychosexual aspects of this
clinical entity have helped to define the role of androgens in gender identity formation.
The phenotypic characterization of subjects homozygous for 5ex-reductase-2
deficiency, includes striking ambiguity of the genitalia with a clitoral-like phallus;
severely bifid scrotum and pseudovaginal perineoscrotal hypospadias. Due to severe
genital ambiguity, many affected children are believed to be girls and reared as girls from
birth. On occasion, more masculinized subjects have been described who have a blind
vaginal pouch which opens into the urethra; a penile hypospadias or even a penile urethra
(Imperato-McGinley et aI, 1974, 1976; Fratianni and Imperato-McGInley, 1994; Walsh et
aI, 1974). Under the influence of testosterone there is normal internal Wolffian
differentiation with seminal vesicles, vasa differentia, epididymides and ejaculatory
ducts. On occasion the testes may be located in the abdomen, but are usually found in the
inguinal canal or scrotum (Imperato-McGinley et aI, 1974,1976;Fratianni and Imperato-
McGInley, 1994).
With puberty substantial virilization results (Imperato-McGinley et aI, 1974,1976;
Fratianni and Imperato-McGInley, 1994). There is: (1) increased muscle mass and
deepening of the voice B musculature is particularly prominent in Dominican, New
Guinean and Turkish affected males, and they are equal in height to their unaffected
siblings; (2) substantial growth of the phallus with mgation and hyperpigmentation of the
scrotum; (3) inguinal testes which descend into the scrotum at puberty have been
observed in some patients; (4) libido is preserved and patients are capable of erections;
and (5) gynecomastia is not present in adulthood. Normal sperm concentrations have
been reported in subjects with descended testes. An affected male from the Dominican

Edited by Zderic et aI., Kluwer Academic/Plenum Publishers, 2002
122 J.IMPERATO-MCGINLEY
kindred successfully fathered children (2 pregnancies, 3 children) via intrauterine

insemination of his wife with his sperm; two affected brothers from Sweden have also
been reported to father children (Katz et aI, 1997;Nordenskjold et aI, 1998).
Dihydrotestosterone-deficient adult subjects have less facial and body hair than their
non-affected male relatives. Significant male pattern baldness has never been observed
in these patients (Imperato-McGinley et aI, 1974, 1976;Fratianni and Imperato-McGInley,
1994). Additionally, the prostate in affected males is non-palpable on rectal examination
and rudimentary on both transrectal ultrasound and MRl visualization. Prostatic volumes
are the size of prepubertal males, approximately one tenth that of age-matched, normal
controls (Imperato-McGinley et aI, 1997).
The biochemical characteristics of 5a-reductase-2 (5-RD-2) deficiency have been well
defined. They are: (1) decreased levels of plasma DHT; (2) normal to elevated levels of
plasma testosterone (T); (3) an increased T to DHT ratio at baseline and/or following
Hcg stimulation; (4) decreased conversion of T to DHT in vivo, with conversion ratios of
T to DHT of <1 %; (5) reduced 5a-RD activity in genital tissue slices and cultured
fibroblasts; (6) normal metabolic clearance rates of T and DHT in affected adult males;
(7) decreased production of urinary Sa-reduced androgen metabolites resulting in
increased 5BI5a urinary metabolite ratios (Imperato-McGinley et aI, 1974; Peterson et aI,
1985; Imperato-McGInley et aI, 1990); decreased plasma and urinary 3a-androstanediol
glucuronide, a major metabolite of DHT (Can et aI, 1998); and (8) a global defect in
steroid Sa-reduction as demonstrated by decreased urinary 5a-reduced metabolites of
both C-21 steroids and C-19 steroids other than testosterone (i.e. cortisol, corticosterone,
I1B-hydroxyandrostenedione and androstenedione)( Imperato-McGinley et aI, 1974;
Peterson et aI, 1985; Imperato-McGinley et aI, 1990). Although the defect is generalized,
only the deficiency 5a-reduction of testosterone appears to be of clinical significance.
Cloning of 5a-reductase type 1 and type 2 genes have made it po.ssible to define the
molecular genetic defects in patients with 5a-reductase-2 deficiency. Both 5a reductase
genes are composed of 5 exons and 4 introns. The gene encoding the enzyme 5a-
reductase-2, has been localized to chromosome 2, and is the major isoenzyme present in
genital tissue and prostate. The enzyme is optimally active at an acid PH and is sensitive
to the 5a-reductase inhibitor, finasteride. To date, over thirty-one mutations in the 5a-
RD-2 gene have been identified as responsible for male pseudohermaphroditism
including gene defects in the three largest kindreds of male pseudohermaphrodites with
5a-RD deficiency in the world -- Dominican, New Guinean and Turkish (Thigpen et aI,
1992). Mutations are found throughout all 5 exons and range from a single point
mutation to a deletion of all five exons found in the New Guinean kindred. (Wilson et aI,
1993). Mutations have been shown to effect substrate or cofactor binding. The substrate
binding domain involves amino acid sequences in the amino terminal of the enzyme,
while cofactor binding appears to be located in the carboxy terminal (Wilson et aI, 1993).
The 5a-reductase-1 gene ~ localized to chromosome 5, and is normal in patients with
inherited 5a-reductase-2 deficiency. The enzyme is active at an alkaline pH, is expressed
in low levels in the prostate, and is relatively insensitive to the 5a-reductase inhibitor,
finasteride. Its role in human physiology remains to be determined (Thigen et aI, 1993).
Both isoenzymes are transiently expressed in the skin and the scalp of newborns, but
5a-REDUCTASE-2 DEFICIENCY 123
only the type I isoenzyme is expressed postpubertally (Thigen et aI, 1993). The data
suggest an interaction between the two genes in the development of baldness.
Gender Identity and Gender Role
Subjects with 5a-reductase-2 deficiency often change gender role during or after
adolescence, verifying our initial reports (lmeprato-McGinley et aI,
1974, 1979b 1981 ;Fratianni et aI, 1994). In the Dominican community, the female sex of
rearing was never reinforced through castration and subsequent female hormone therapy.
As such, at puberty a change in gender from female to male occurred in the vast majority
of subjects from the older generation who were unambiguously reared as females
(Imeprato-McGinley et ai, 1974, 1979b 1981 ;Fratianni et ai, 1994). The affected subjects
were often the objects of derision and in the vernacular of this rural community are
known as Amachihembra == A half man / half woman ==; or Aguevedoce == Apenis at 12.
Psychosexual data was available for 18 subjects, 17 of whom successfully changed
gender. Between 7 and 12 years of age, patients who were raised as girls began to
experience considerable anxiety over their lack of breast development, and often began to
be sexually attracted to girls. They became convinced of their male gender identity over
the next several years and were masturbating, experiencing morning erections and
nocturnal emissions. The change in gender role occurred on average at 16 years of age,
with a range of 14-24 years. In three subjects, a gender role change did not occur until
they were in their 20s. Admittedly, the fear of being stigmatized and anticipation of
harassment by local villagers caused some subjects to hesitate at the prospect of changing
gender roles, in some cases until they were confident of their ability to defend themselves
(Imeprato-McGinley et aI, 1974,1979bI981;Fratianni et aI, 1994). We have continued to
observe the behavioral characteristics of these subjects for over twenty years, and have no
doubts as to their male behavioral pattern in adulthood.
The social and psychosexual development of New Guinean male
pseudohermaphrodites with 5a reductase-2 deficiency of the Sambian tribe has been
recorded by field observation of Dr. Carlton Gajdusek over three decades (Gasdusek,
1964,1967-1976,1977; Farquhar et aI, 1981). The Sambian tribal culture of the eastern
highlands is rigidly gender segregated. While women are the caretakers, men are hunters
and warriors. Traditional pubertal male initiation rites include ritualized fellatio among
males, and first stage initiates to men of premarital age. Gender segregation was allegedly
enforced by death to any female who accidentally witnessed these imtlatlOn rites
(Gajdusek, 1977). In the past, some New Guinean 5a-RD subjects were raised as girls
until puberty, whereupon they made the stormy transition to male (Gajdusek, 1977).
Today, however, the condition is usually recognized in childhood, if not at birth by
experienced midwives. Because of the gender change observed in affected Papua New
Guineans, the term Aturnim-man= from the Melanesian pidgin was incorporated into the
Sambian lexicon. This term connotes gender transformation and the implicit cultural
belief that these anatomically ambiguous individuals are innately, and biologically,
driven to masculinize (Herdt and Davidson, 1988).
124 J. IMPERATO-MCGINLEY
Some observers believe that the New Guinean pseudohermaphrodites were regarded
as a third sex and were motivated to change to a male gender role in a pragmatic attempt
to adapt to their Amale-admiring= society (Herdt and Davidson, 1988). However, it has
been the experience of Carlton Gadjusek as well as our own, that these subjects clearly
regard themselves as male. To support this, three affected New Guinean Sa reductase-2
patients specifically requested and obtained genital correction in adulthood so that they
could be, as they stated, complete men (Imperato-McGinley et aI, 1991, and unpublished
data).
Gender change has been noted in affected subjects from many countries including
individuals from the Dominican Republic, unrelated to the large pedigree, New Guinea,
Turkey, Mexico, Cyprus, Algeria, Italy, Lebanon, Brazil, Saudi Arabia, UAE, Sweden,
etc.( Imperato-McGinley et aI, 1974,1976,1980,1991;Fratianni and Imperato-McGInley,
1994; Akgun et aI, 1986;Wilson et aI, 1993;Mendonca et aI, 1996;Mulaisho et aI,
1990;Mendez et ai, 1995;AI-Attla, 1996;Savage et aI, 1980;Ivarsson et aI, 1988;Patel et
aI, 1986;Hochberg et aI, 1996). It appears from the observed natural history in subjects
with this inherited condition, that if puberty is permitted to occur spontaneously without
surgical and hormonal or societal reinforcement of the female sex of rearing, then a male
gender identity, although discordant with the sex of rearing, will prevail. Under these
circumstances it appears that the extent of androgen (i.e., testosterone) exposure of the
brain in utero, during the early postnatal period, and at puberty, has more of an effect in
determining male gender identity than does sex of rearing and sociocultural influences.
Though maintenance of a female gender identity post pubertally has been reported in
some some subjects, long-term psychosexual follow-up, which is critical to the
determination of gender, is not available in most subjects. For example, we studied a 65-
year-old male pseudohermaphrodite with a 5a-reductase deficiency born in southern Italy
and raised as a girl. The subject emigrated to the United States at age 16. Lengthy
psychosexual evaluation revealed that the subject identifies as a male. He has a male
gender identity, but because of family pressure has retained a female gender role
(Imperato-McGinley et aI, 1980).
Normally the sex of rearing and testosterone imprinting of the brain act in unison to
determine the complete expression of the male gender. However, subjects with 5a
reductase-2 deficiency demonstrate that in a laissez-faire environment, when rearing
(female) is discordant with the testosterone mediated biological sex; the biological sex
prevails if normal testosterone activation of puberty is permitted to occur. From the data
it appears that the extent of testosterone exposure of the brain in utero, in the early
postnatal period, and at puberty has greater impact in determining male gender identity
than the female sex of rearing. Theoretically, masculinization of the brain occurs under
the influence of testosterone, during the prenatal and/or neonatal period and together with
the activation of a testosterone-mediated puberty, a male gender identity develops,
overriding the female sex of rearing. This experiment of nature emphasizes the
importance of androgens, which act as inducers and activators in evolution of male
gender identity in man (Imperato-McGinley et aI, 1974,1976,1979b,1981;Fratianni and
Imperato-McGinley, 1994).
It is understandable that gender identity might coincide with the sex
of rearing. In subjects with inadequate testosterone production or action, if adequate
androgen imprinting has not occurred, the sex of rearing becomes the predominant factor.
Such individuals are a testimonial to the malleability of human beings in the acquisition
of gender identity but do not approximate the normal sequence of events (Imperato-
McGinley et aI, 1974,1976,1979b,1981;Fratianni and Imperato-McGInley, 1994;
Diamond, 1965).
It has been proposed that the gender identity becomes fixed by 18 months to 4 years of
age, at the time of language development (Money et aI, 1955a,bI974). At this time a
child becomes aware of his or her gender. However, being aware of one's gender and
being unalterably fixed in that gender are two separate issues. It appears that the
development of gender identity in man is continually evolving throughout childhood,
becoming fixed with puberty.
There are reported cases of 46XY subjects with ambiguous gentalia who have
successfully changed gender from female to male after the proposed critical period
(Chapman, 1951;Burns et aI, 1960;Ghabrial et aI, 1962; Berg et ai, 1963;Brown et aI,
1964;Teter et ai, 1965;Dewhurst et aI, 1969;Stoller, 1964). In many cases, the gender role
change also occurred during adolescence. Adequate hormonal evaluation would be
important in these cases, as they also appear to challenge both the theory of the
immutability of gender identity after the age of 3 or 4, and the sex of rearing as the major
factor in determining male gender identity. We evaluated the patient reported by Stoller
(Stoller, 1964) and found that this patient, who had changed gender, had the enzyme
defect, 17B-hydroxysteroid dehydrogenase (Imperato-McGinley, 1979). This was the
first report of a subject with 17B-hydroxysteroid dehydrogenase deficiency to have
undergone a gender change. Since then a gender change from female to male in genetic
males with 17B-hydroxysteroid dehydrogenase defect has been reported by others (RosIer
and Kohn, 1983).
In humans, environmental or sociocultural factors are not solely responsible for the
formation of a male gender identity. Subjects homozygous for 5a-reductase-2 deficiency
demonstrate that androgens make a strong and defi.nite contribution. Analogous to the
induction of the male phenotype from an inherent female phenotype, the formation of
male gender identity in man also appears to be at least partially induced by androgens
from an undifferentiated and/or inherently female nervous system.
Dilemma over Sex of Rearing
Affected subjects with 5a-reductase-2 deficiency who change gender identify behave
as normal males in adulthood despite the trauma of having been raised in the wrong sex.
Consequently early childhood diagnosis of the condition followed by surgical correction
of the external genitalia and correction of cryptorchidism if present is desirable. With
successful genital repair, the parents can be assured that (1) the child will have a male
puberty with normal male psychosexual development; (2) he will be equal in height to the
normal males in his family; (3) there will be growth of the genitalia, an increase in
muscle mass, and deepening of the voice; (4) gynecomastia will not be a concern; (5)
fertility in this condition has been reported.
To enlarge the phallus and facilitate hypospadias repair, newborns with this condition
can be treated by administering dihydrotestosterone cream (Imperato-McGinley,
1997;Carpento et ai, 1990). The rationale behind the treatment is replacement of the
deficient hormone dihydrotestosterone to induce phallic growth; theoretically growth that

would have occurred in utero and in the postnatal period. It should be remembered that
administering dihydrotestosterone after the critical period of sexual differentiation in
utero will stimulate phallic growth but will not correct the genital defect, as sexual
differentiation occurs during a critical period in utero.
Most patients described in the literature, have perineoscrotal hypospadias and,
therefore, surgical correction is difficult. However, despite this concern, surgical
correction of the genitalia is feasible and made easier if enlargement of the phallus can be
accomplished with dihydrotestosterone cream administration. The quality of any surgery,
however, is dependent upon the expertise of the surgeon and is clearly only to be carried
out by an expert in hypospadias repair.
The most serious debate involves the management of subjects who are raised as
females and diagnosed as having Sa-reductase deficiency in the peripubertal period and
postpubertal period. After careful psychiatric evaluation often subjects will be found to
identify as males and should be encouraged to take their place as males in society. It
should be noted that pseudovaginal perineoscrotal hypospadias has been successfully
repaired in adulthood in subjects with this condition. There are other subjects who
identify as male and who will change gender role with time if they can deal with the
social pressures of family, friends, etc. (unpublished). Thus, whether or not a gender role
change will occur in an individual with Sa-reductase deficiency at this time is obviously
dependent upon a host of social and cultural factors which might either consciously or
subconsciously suppress or foster the change. All these factors must be considered by the
patient's physician, as well as the psychiatrist working in concert with the patient and the
family (Imperato-McGinley, 1997).
The Syndrome of Complete Androgen Insensitivity
Introduction
Subjects with complete androgen insensitivity (CAIS) have an inherited disorder at the
androgen responsive target areas, which results in failure to respond to androgens. As a
consequence of complete androgen umesponsiveness in utero, an affected child is born
with female genitalia and raised as a girl. They are phenotypic and psychosexual
females. Owing to normal MIF secretion by the testes in utero, Mullerian derived
structures are absent or rudimentary. Consequently, the uterus and cervix are absent,
although occasionally rudimentary structures are present. Thus, the presence of an XY
karyotype and testes in an individual with a complete female phenotype is highly
suggestive of CAIS. During puberty there is breast development but scant to absent
pubic or axillary hair. The adolescent female usually presents to the doctor with the
complaint of primary amenorrhea. Occasionally presentation is earlier in childhood if
masses are palpated in the inguinal canal (Imperato-McGinley et aI, 1992,1992;Quigley
et ai, 1995,1995).
Sa-REDUCTASE-2 DEFICIENCY 127
X-linked inheritance in complete androgen insensitivity was first suggested by

pedigree analysis. The basis of the syndrome is due to defect(s) in the androgen receptor
gene located on the long arm of the X-chromosome close to the centromere Xqll and
Xq12. The androgen-receptor gene has been found to contain eight exons, A-H, which
code three domains of the androgen receptor: the amino terminal section, whose role is
theoretically modulatory (exon A); the DNA binding domain (exons Band C, coding first
and second zinc finger motifs, respectively); and androgen binding domain (exons D-H).
In general these defects range from complete, partial deletions to single base substitutions
involving all exons, although most involve the androgen binding domain (Imperato-
McGinley et ai, 1992 ;Quigley et ai, 1995).
In complete androgen insensitivity, the levels of testosterone are frequently elevated
and correlate often with elevated LH levels. LH levels correlate well with the histologic
findings of Leydig cell hyperplasia. The elevated LH and the Leydig cell hyperplasia
appear to be due to the umesponsiveness of androgens at the level of the hypothalamus
and/or pituitary. The serum LH levels, however, are not in the castrate range, probably
owing to the negative feedback effect of estrogen on the hypothalamus and/or pituitary
(Imperato-McGinley et aI, 1982). Castration results in further elevation of gonadotropin
levels. FSH levels are normal to elevated (Imperato-McGinley et aI, 1982). Urinary
estrogens and plasma estradiol levels are in the high-male to low-female range and an
increased production rate of estrone and estradiol, due to substantial amounts of estradiol
secreted by the testes, has been described. The elevation of estrogen production, together
with androgen umesponsiveness results in an unopposed estrogen effect and is the most
likely explanation for breast development at puberty (Imperato-McGinley et ai, 1992,
1992; Quigley et aI, 1995).
Cognitive
Subjects with androgen insensitivity have been shown to demonstrate a significantly

lower performance on subtests of spacial ability on the Wechsler Intelligence Scale than
either control males or control females from the same kindred (Imperato-McGInley et aI,
1991). Since subjects with complete androgen insensitivity are raised as females and
have a totally female psychosexual orientation, their cognitive performance, therefore,
may reflect their sex roles, as a reflection of social experience and values placed upon
them. However, this consideration does not explain their significantly lower overall
performance when compared to control females from the same kindred on the perceptual
organization factor and subtests of spatial ability. This exaggerated female pattern of
performance suggests an effect of androgen umesponsiveness. Furthermore, the fact that
there was no differences in performance between AI subjects that were gonadectomized
post pubertally and those that were not, suggests that the effect of androgen
umesponsiveness on the brain occurred earlier, most likely prenatally or in the early
postnatal period (Imperato-McGInley et ai, 1991).
In another study of cognitive function in subjects with complete androgen
insensitivity, AI subjects from seven umelated families were studied (Masica et ai, 1969).
A modest but consistent and significant tendency toward superiority of Verbal
128 J,IMPERATO-MCGINLEY
Intelligence Quotient over Performance Intelligence Quotient was found in 15 subjects

with complete androgen insensitivity, a finding similar to their 10 female sibling controls.
The androgen-insensitive subjects demonstrated a lower score on the perceptual
organization factor when compared to the verbal comprehension factor. The lowest
scores were also for the visuospatial subtests comprising the perceptual organization
factor. There were no comparison data, however, of AI subjects with control males or
females on these subtests.
Studies of men with congenital idiopathic hypogonadotrophic hypogonadism have
demonstrated an impairment of spatial ability, as demonstrated by a poor performance on
the block design subtest of the WAIS (Hier and Crowley, 1982). The deficit does not
correct with administration of exogenous androgens in adulthood and does not occur in
subjects who develop hypo gonadotrophic hypogonadism following puberty (Hier and
Crowley, 1982). Such findings also suggest that androgens exert a primary organizing
effect on the brain, in the prenatal and/or early postnatal period.
Management
Subjects with complete androgen insensitivity are phenotypic and psychosexual

females and are reared as girls. It is known that 2%-25% of patients with complete
androgen insensitivity can develop testicular neoplasms. However they occur only rarely
before age 25. The optimal time for gonadectomy as far as psychological adjustment is
concerned is still a matter for debate. If a child is diagnosed as having this condition,
traditionally she is usually observed until puberty is completed and breast development
has occurred naturally. Subsequently she is referred to a surgeon for gonadectomy. It
should be realized that waiting for the postpubertal period to perform the gonadectomy
means that it will often be done during the tumultuous years of adolescence or shortly
thereafter. Gonadectomy performed at this time, however, has the advantage of
involving adolescents or young adults in the decision making.
It can be argued that gonadectomy should be performed in childhood, thus avoiding
the difficulties of yet another psychologic adjustment during adolescence. However some
adult patients with this condition, who were gonadectomized in childhood feel that
gonadectomy should have been delayed to early adulthood, when they could have been
more actively involved in the decision making; thus making the psychologic adjustment
easier (Gooren, 1997). However, if a child has an inguinal hernia and is brought to a
physician for this reason and the diagnosis is made, consideration should be given to
perform gonadectomy at the time of hernia repair, thereby avoiding a second operation.
It is important that the patient learn about their condition from their doctor, in a careful
and sensitive way. This will avoid severe psychologic problems. Therefore it is critical
that the doctor develop a good relationship with the patient and the parents. The timing of
the information from initiation of the topic to a detailed discussion must be
individualized: psychologic counseling is needed for the patient and the family as well.
Cyclic estrogen replacement therapy or estrogen with progesterone should be
prescribed at the appropriate time to prevent osteoporosis and to maintain breast
development. For most patients, the vagina is of sufficient size to allow normal coital
function. In cases where the vagina is too small, it can be enlarged with vaginal dilators,
thereby avoiding reconstructive surgery. Vaginal surgery is rarely required.
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Walsh PC, Madden J, Harrod MH, Goldstein JL, MacDonald PC and Wilson JD. Familial incomplete male
pseudohermaphroditism, type 2: decreased dihydrotestosterone formation in pseudovaginal perineoscrotal
hypospadias. New Engl J Med 291 :944, 1974.
Wilson JD, Griffin JE, Russell OW. Steroid 5a-reductase-2 deficlency. Endocr Rev 14:577-593, 1993.
Questions for Dr. Imperato-McGinley
Question - Can you comment on the genetics in the5-alpha reductase deficiency, because
although you showed a large kindred, the sporadic nature in which other patients show up
seems to suggest a spontaneous mutation rate that is appreciable in this entity?
Answer - Yes, obviously there is - there are, let me see - the Turkish, the New Guinean,
the Dominican, the Schiit Muslim - and those are the largest, those are the 4 kindred and
there are at least 40 to maybe more mutations that involve all exons. Most of them are
missense mutations, single base mutation. The New Guinean have a total deletion of all
5 exons, so it is just like the androgen insensitivity syndrome and I guess like all the
enzymatic defects there is no one major deletion for this disease. It involves all exons
and it ranges from, not only missense mutations, but insertions of 1 or 2 base pairs, to the
entire deletion for both the androgen receptor gene and the 5 alpha reductase gene.
Question - Thank you for a masterly exposition. I was delighted, first of all, with the
amount of anecdotal detail it contained, because I think that is going to remain very
central to this meeting; however much we would like it not to be. I'd just like to take
issue with you on one point and then ask you a question. I would have to nit-pick on the
question of the normal sperm count. I do not know about New York, but the robust
European ejaculate would contain somewhere between 500 million to a million sperm.
Although your sperm concentration may have been normal with it's small volume, the
sperm - the ejaculate from that person contained only a very small quantity of sperm;
therefore, his sperm production must have been very small.
Answer - I am sorry, you are absolutely correct. The count was normal but if you put it
into the terms of total ejaulcate, it would be diminished.
Response - Exactly, so sperm production is not normal. The question I would really like
to ask you is how do you diagnose 5 alpha reductase deficiency unambiguously in the
newborn period? The androgen resistant or the 5 alpha reductase patients are our most
difficult group. And they are the group in which we have to decide which way to go in
terms of sex of rearing or surgery. And it seems to me as an amateur endocrinologist,
that diagnosing 5 alpha reductase deficiency in the newborn and infant period is actually
quite difficult to do it with certainty.
Answer - Well, you have no time unless there is a family history and you know the
genetics and you can go right quickly to do genetic screening. But if it is a denovo case
the only way to do it is bio-chemically and there, two things have to be done which are
often missed by endocrinologists and everyone. You can give HCG, as Dr. White
mentioned, raise the testosterone, the DHT will not keep up with the rise in the
testosterone and the ratio will be, in most of the cases, much greater than 18. The best
way is to correlate that with urinary metabolites of cortisol, because the androgen
metabolism in infancy is not the same as an adult - you cannot look at androsterone. We
did those studies with Shackleton's group using gas chromotography and mass
spectroscopy, and you look at tetrahydrocortisol, 5 beta and 5 alpha. Now why do I say
you should correlate the ratio in the plasma with the urine? Because patients with
complete androgen insensitivity since the 5 alpha reductase enzyme is partially androgen
responsive can have abnormal T to DHT ratios, and in that large kindred that I showed
you, and I did not have time to go through the bio-chemical studies we have done over
the years, some of those patients, but not all of them, had ratios as abnormal as patients
with 5 alpha reductase. But they have normal urinary C-19 and C-21 cortisone
metabolites. So I would combine the two, urine and the plasma.
5a-REDliCTASE-2 DEFICIENCY 133
Question - And therefore you are able to say quite certainly that this patient has or has not
a degree or complete 5 alpha reductase deficiency?
Answer - Yes, fortunately it is a universal abnormality, so it affects cortisol metabolism

whereas the androgen insensitivity that pertains to the 5 alpha reductase that is making
androgen only and it is mild - so that you can - the ratios are normal versus abnormal in
the cortisone metabolites.
Question - I am not absolutely clear why the complete androgen insensitivity babies do
not get androgen imprinting of the brain in utero. Is it because they have got no brain
receptors either?
Answer - That is the theory. I do not know - it is very different from the animal model -
that is for sure. But there is certainly, in looking at these patients no signs of any
masculinity and I think that is worldwide. And it has never been any as far as I know -
maybe someone in the audience has evidence - even the published literature that one case
that was actually a Hopkins case that was carried as androgen insensitive when we re-
evaluated that case and that was prior to the syndrome being known - that one case that
did not adjust to a female gender was actually a 5 alpha reductase patient.
Question - So there isn't actually any evidence that they do not get imprinting it is the
supposition based in the fact that they don't exhibit android behavior.
Answer - That's right. Most of gender identity theories are just that - they are theories
and hypotheses. To be either accepted or refuted.
Question - I think it relevant to mention another population of androgen insensitive

patients reported by Dr. Ressler, and these patients have 17 beta HSD deficiency and as a
group they also spontaneously change from the female sex to the male sex at puberty.
And given the society that they were a part of they tended to do this with relative ease.
Answer - We do not classify those as androgen insensitivity. Those patients have 17

keto steroid reductase deficiency which is the last step before testosterone and that is the
other condition where gender identity change with puberty has been reported. I have a
slide of a patient that was evaluated by Dr. Fred Stoller at UCLA who has since deceased
who is a very well known Psychiatrist with an interest in gender identity disorders. Can
you go back maybe 2 slides and I will show you this case? This is a patient that Dr
Stoller evaluated long before and I found it through the Index Medicus, that's before
Medline. And I asked him we could evaluate the patient. This child was born and raised
as a girl and in fact Dr. Stoller sent me the birth certificate and he actually had written
this case up and published it. But then at age 14 suddenly, and the Mother claimed no
matter what she did this child would not behave as a little girl, would not grow her hair.
Despite sending this child to Parochial School for discipline, she could do nothing to
change this young girl's behavior. Then at age 14 the patient dropped out of school. Dr.
Stoller was asked to see this child in consultation by a General Internist who then sent the
child to an Endocrinologist because he noted testes and he noted an enlarged phallus.
Stoller then did the very brave thing at that time because he was supposed to be telling
the child if you were a girl we'll take care of this. He listened to this child and then he
wrote, "There is some biologic influence that is causing this child to actually have a male
gender identity." And after the interviewing he decided to tell the child, right then and
there one day on the spot, and he describes it rather dramatically. The change was almost
immediate. The child become very happy. The Mother moved away to another
neighborhood as this child finished high school and then college and then veterinary
school and has since married. We later on contacted Dr. Stoller and Dr. Goodwin, who
was the Urologist and did the evaluation. This was a case of 17 keto reductase
deficiency.
THE CHOP EXPERIENCE WITH CLOACAL
EXSTROPHY AND GENDER REASSIGNMENT
Stephen A. Zderic, Douglas A. Canning, Michael C. Carr, Christine

Kodman-J ones, Howard McC. Snyder
Division of Urology, The Children's Hospital of Philadelphia
INTRODUCTION
We have chosen to emphasize our experience with the management of patients with
cloacal exstrophy, because this is the primary patient population in which gender
reassignment has been utilized at this center over a 30 year span. These 11 reassigned
patients comprise a cohort with similar medical and surgical issues, and their progress
may be contrasted with 4 males who were not gender reassigned as well as 7 genetic
females. By gender reassignment, we are specifically referring to the assignment of a
female gender for a neonate with an absent phallus who has a normal XY karyotype and
functional testes in the absence of any endocrine disorder. Other reasons for gender
reassignment within this department have included aphalia, ablatio penis, and one case of
classic exstrophy. In addition to these extremely rare cases, the division has
accumulated a substantial experience in gender assignment in intersex cases with the
most common diagnosis being congenital adrenal hyperplasia. It has always been the
philosophy of this division to assign a gender consistent with the chromosomal and
gonadal sex whenever possible.
Historical Perspective
Cloacal extrophy remains one of the most devastating birth defects that pediatric
surgeons and urologists are called upon to manage. It remains a rare diagnosis, occurring
with a frequency of 1 in 200,000 to 400,000 live births. Even within a major referral
center such as Children's Hospital of Philadelphia, this would average out to 1 new case
per year. These neonates present with a multitude of problems brought about by
herniation of the urinary bladder and hindgut through the abdominal wall. Complicating
this are an associated omphalocele which is often quite large, orthopedic malformations
(club feet), and vertebral anomalies such as lipomeningocele or myelomeningocele.
Short gut complicates the management of these patients, and accounts for why it was not
until 1960 that Rickham reported the first survivor (Rickham, 1960). Prior to this report,
these neonates lingered for a few months before succumbing to dehydration,
malabsorption, sepsis, or neurological sequelae. Over the past 40 years a combination of
advances in neonatal anesthesia and intensive care medicine, hyperalimentation, pediatric

136 S.A. ZDERIC, ET AL.
surgical techniques, and antibiotics have resulted in a dramatic change in the prognosis
for these patients (Howell et ai, 1983). A once nearly uniformly fatal condition now has
series reporting survival rates of 90%.
In addition to the problem of severe short gut which often requires prolonged total
parenteral nutrition (TPN), these patients have difficulty with water reabsorption. The
preservation and tubularization of all available hindgut to create a colostomy has allowed
for better preservation of hydration status as opposed to a simple ileostomy (Hussman et
ai, 1988). While some authors have reported attempts at rectal pull through surgery, the
association of this anomaly with spina bifida and the liquid (or at best soft) stools
characteristic of short gut syndrome have made a well placed end colostomy the preferred
method for management in most series. The wet perineum and associated skin
breakdown lead one older patient in this series to seek a reversal of the pull through and
conversion to a colostomy. Mitchell's description of gastric augmentation also
represented a significant advance in terms of achieving continent reconstruction in this
complex population with short gut (Carr and Mitchell, 1996) in whom assocaited spina
bifida makes spontaneous voiding unlikely (Hussman et ai, 1999).
Antenatal Diagnosis
Today it is possible to achieve an antenatal diagnosis of cloacal exstrophy assuming

that screening sonography is carried out as a first step. Austin (Austin et ai, 1998) has
reviewed the ultrasound findings that are characteristic of cloacal exstrophy, and should
trigger further
investigation. The observation of absent bladder filling on multiple observations should
raise the possibility of an exstrophic condition. When this finding is combined with a
large midline anterior abdominal wall defect and or vertebral findings, the index of
suspicion for cloacal exstrophy should increase. Minor findings included lower extremity
defects, renal anomalies of number or position, hydronephrosis, ascites, hydrocephalus,
thoracic narrowing, single umbilical artery, and pubic/pelvic diastasis. In combination
these criteria allowed for an accurate diagnosis by ultrasound alone in 19122 cases
(Austin et ai, 1998). Today the techniques of maternal fetal MRI and if needed fetoscopy
can enhance diagnostic accuracy. Increasing utilization and experience with antenatal
maternal fetal MRI, along advances in MRI technology, should obviate the need for more
invasive fetoscopy.
The advances in antenatal diagnosis have both medical and social ramifications. It has
been our impression at this large referral center, that the incidence of cloacal exstrophy
has declined; the last neonatal presentation at CHOP was in 1996. However screening
sonograms in the community have lead to several maternal fetal MRI evaluations, and
once the diagnosis was established, these families have opted for termination of
pregnancy. Ultimately as the techniques of fetal surgery are refined, some of these
families may opt for antenatal therapy (see next section). At present, the clinical
significance of these new developments is that unsuspected presentations of cloacal
exstrophy are more likely to take place in situations where families have limited or no
access to health care. In France, women are reimbursed to undergo several prenatal
sonograms, and the incidence of unexpected presentations of major congenital anomalies
THE CHOP EXPERIENCE 137
has dropped. In the United States, we surmise that unsuspected major diagnoses such as
cloacal exstrophies will primarily present among younger or single parents with limited
access to health care both before and after the pregnancy. This will increase the need for
services to help meet the special needs of these children.
Antenatal Therapy
To date no center has reported any attempt at management of cloacal exstrophy in

utero, nor is it likely that this will be undertaken any time soon. However what seems
impossible today, may well become feasible in 20 years. Since Harrison first advocated
the concept of fetal therapy, major strides have been made (1980). These advances have
been made possible because of major advances in imaging and greater accuracy in
antenatal diagnosis, extremely careful patient selection, and a major commitment to
detailed scientific laboratory investigation preceding any human trials. Adzick has begun
a major effort to improve outcomes in spina bifida via antenatal intervention early in the
second trimester (Olutoye and Adzick, 1999). While the Vanderbilt series has reported
no improved urologic outcomes following antenatal intervention for spina bifida
(Holzbeierlein et aI, 2000), these interventions were done at a later time point in
gestation. Solving the problems of in utero spina bifida will be an important step in the
path to management of cloacal exstrophy. All would agree that this field is in its infancy,
and work is ongoing at several major centers. The diagnosis and management of intersex
disorders in utero has already been reported for congenital adrenal hyperplasia (Shapiro
et ai, 1989), and are described in detail in Chapter 13 by Rintoul and Crombleholme.
Principles of Neonatal Management
In the traditional management approach taken at CHOP, major emphasis was placed
on neonatal closure, and every effort was made to achieve a closure of cecum, bladder,
and resolution of the omphalocele in one stage. In the last 20 years, this was often
achieved in the first 48-72 hours of life by joint general surgery and urology teams. The
general surgical team begins the case by dividing the colovesical fistula and closing the
colon with a tubularization of all distal hindgut to fc)rm a colostomy. The omphalocele is
then excised to initiate the abdominal wall closure. At this point the urology team takes
over and dissects the hemibladders free while preserving their vascular pedicles. The
bladders were then approximated circumferentially with the bladder neck being left wide
open and draining to the perineum. In four cases the omphalocele was so large as to
preclude primary closure, and a silo closure was utilized. Three of these four patients
survived long term, while one patient succumbed to sepsis in the neonatal unit. In three
surviving cases, the silo closure was applied with the hemibladders being left intact to
allow for delayed bladder reconstruction once the omphalocele was closed. In one
surviving case, the hemibladders and the lower abdominal wall were closed, and then the
silo was applied.
There have been a total of 24 patients operated on at CHOP with a diagnosis of cloacal
exstrophy, with 22 long term survivors. One patient succumbed to sepsis after
application of a silo to manage a huge omphalocele, and a second patient died at 2 years
of age from hepatic failure felt to be secondary to TPN. The current age distribution for
these long term survivors is as follows: 9 adults, 3 adolescents, and 10 prepubertal
patients.
Continence Status
As of this time, 20/22 patients are left with a permanent colostomy. It is anticipated
that at some point one of the patients with a pull through will undergo conversion to a
colostomy as occurred with one other patient in the series. There are 6 patients who are
left with a permanent urostomy, and who are unlikely to opt for continent reconstruction
now or in the future. There are now 6 patients that have undergone continent
reconstruction, and 10 await reconstruction. It has been our practice to defer on any
complex urinary tract reconstructions in this setting until such time as the social situation
is optimal and the patient exhibits a high degree of motivation and enthusiasm for clean
intermittent catheterization.
Gender Management Issues
Gender issues for these patients were managed with the traditional approach in which
reassignment was viewed as the better option for those genetic males with diminutive and
widely separated corporal bodies. Clearly this was not an issue for genetic females, but
it was of major concern for genetic males whose anatomy left them functionally aphallic
in a majority of cases. It is important to address the two main underlying themes that
dictated this approach in years past: 1)penile reconstruction for patients with cloacal
exstrophy was deemed unfeasible and 2) the neonate was assumed to be gender neutral.
In contrast to patients with the classic exstrophy epispadias complex, the phallus in
patients with cloacal exstrophy does not exist. At the time of presentation these patients
present with two very widely separated dimminutive corporal bodies, and very often
these are not symmetric in size. Glanular development is very rudimentary. The wide
pubic diastasis (secondary to the massive abdominal wall defect and associated visceral
herniation) further complicates the problem of penile reassembly. The poor results early
on with attempts at phallic reconstitution in these patients lead several authors to
conclude that neonatal orchiectomy, and gender reassignment would be a better
alternative for these patients (Tank and Lindenauer, 1970). Hussman et al (1989)
described followup in a cohort of 8 genetic males with cloacal exstrophy managed by
assignment to a male gender with a two stage phalloplasty. Of these 8 patients 4 were
prepubertal and could not be effectively assessed for long term outcome. Of the 4
postpubertal males, 3 were left with a phallus insufficient for vaginal penetration, and all
4 had phallic lengths and dimensions well below the norm. These 4 patients required
psychological counseling and support. The authors concluded that gender reassignment
was a valid option for genetic males with cloacal exstrophy, which represented a better
alternative. These authors presented a very different picture for these male patients than
that reported by Reilly and Woodhouse in their long term followup of men with
micropenis who were well adapted and sexually active (Reilly and Woodhouse, 1989).
These assumptions concerning gender reassignment for genetic males with cloacal
exstrophy were made based upon the best evidence available at that time. Supporting this
approach were studies by Money who suggested that the neonate was gender neutral, and
that a child's gender identity was primarily determined by the sex of rearing. In effect this
theoretical concept held that nurture would win out over nature. In a well documented
case of ablatio penis gender conversion was performed at 18 months of age, and early
results appeared in the literature suggesting a good outcome (Money, 1975). However, it
was not until nearly 20 years later that this patient's ultimate unsatisfactory long term
outcome came to light, with the patient having reassumed a male gender (Colapinto,
1997; Diamond and Sigmundson, 1997).
Based on the concept of neonatal gender neutrality as well as the poor prognosis for
creating a functional phallus in these patients, the approach taken at CHOP in the past
was to recommend neonatal gender reassignment. Within our series of 24 patients, there
were 17 genetic males, 12 of whom are being raised in a female role following
orchiectomy (Figure 1). The timing of the orchiectomies was in the neonatal period in all
except 2 cases. Two deaths occurred within this group of patients leaving 17 patients
followed by us on a long term basis. There have been no new presentations of cloacal
exstrophy at out institution over the past 5 years.
CHOP Cloacal Exstrophy Series

24 Patients
7 Females 17 Genetic Males
5 males 12 raised in female role
4 survivors 11 survivors
Figure 1. The distribution of patients presenting at CHOP with a diagnosis of cloacal exstrophy and their
subsequent gender management.
Gender Outcomes
Despite their immense medical challenges, these patients and families have done
remarkably well from an overall social perspective. Among the older patients,
employment is the rule rather than the exception. During the elementary and high school
years, attendance at school mirrors that for other complex medical conditions. Other
studies have demonstrated good psychological outcomes with good social adjustment for
classic as well as cloacal exstrophy patients (Montagnino et ai, 1998)
For the 11 surviving patients who have been gender reassigned, long term
psychological followup is available for 8 patients as of this time. For 1 adult patient, long
term assessment will be difficult due to severe developmental delays. Within this group
of 8 gender reassigned patients, there are at least 3 instances where gender identity has
been questioned by the patients themselves. One patient at the age of 7 years has
reassumed a male gender identity. Among 2 older patients in their late 20s and early 30s,
there have been questions of gender identity that have not lead to gender reconversion.
One patient has been chronically depressed, and has required extensive pyschiatric
support, including an acute hospitalization following suicidal ideations. Both patients
have experienced significant gender identity issues, and are most comfortable in lesbian
relationships. While they have had sexual relationships with males, none has been
sustained.
For the younger gender reassigned patients, gender identity issues are beginning to be
voiced by some children and parents. It is clear that for most of these children, toy
preferences, dress style, and general interests will pursue more male stereotypical
patterns. However there is a wide range over which these behaviors and interests are
noted. It may be anticipated that within this series one or two more patients may seek a
change in gender over the next 10 years, based on our recent experience as well as that of
Reiner (Chapter 11).
It is equally important for psychological followup to be obtained for those patients in
whom gender reassignment was not performed. The longest followup in our series is
with a genetic male for whom multiple reconstructive procedures were performed over 30
years ago. Today he is left with an ileal loop urinary diversion, a colostomy, and
functional aphallia. Despite severe spina bifida and scoliosis erotic sensations are intact
and he is orgasmic. He has been followed by a psychiatrist and psychologist for many
years, and has been referred for sex therapy. While understanding all his options, he at
this point again wishes to consider phalloplasty. Severe circulatory problems would
preclude an osteomyocutaneous flap (Sadove et aI, 1993) (McRoberts and Sadove,
Chapter 18). He has however been evaluated on several occasions for the radial forearm
flap approach (Jordan, 1998,Chapter 17). In speaking with this patient one can sense his
frustrations with his current situation; his male gender identity is fully intact. He is well
read, and has openly volunteered to us that he feels he would not have fared any better in
a female gender role. He has also stated that he would not want his child to go through
what he has in life, and would recommend termination of pregnancy if he were in this
situation.
There are three other genetic males in the series who were not reassigned, but the
oldest is just now approaching mid adolescence. Our feeling is that none of these males
will have a functional phallus. Further long term followup will reveal how these boys
adjust to their condition. Reasons for maintaining these patients in a male gender ranged
from family refusal to the preference of the attending surgeon who felt that a phalloplasty
might be feasible. It is clear however that creating a functional phallus for males with
cloacal exstrohpy represents a major surgical challenge despite the new advances in
exstrophy surgery (Mitchell and Bagli, 1996). Ultimately we suspect that these 3
patients will also be candidates for total phallplasty based on the radial forearm flap
technique (Jordan, Chapter 17).
Conclusions
In 1997, Diamond and Sigmundson reported on the long term followup of a patient
who had undergone gender reassignment secondary to ablatio penis. This gender
reassignment had been carried out at 18 months of age, and in the early prepubertal years
the patient was reported to be doing well. However upon reaching the adolescent years,
the patient stopped his estrogen supplements, and upon further counseling, and learning
his medical history, adopted a male gender identity. The impact of the Joan/John case is
best described in the patients own words (Colapinto, 1997). It must be acknowleged that
not all of these cases have produced such unsatisfactory long term outcomes (Bradley et
ai, 1998). Diamond has argued that there has never been a successful case of gender
reassignment, and has provided evidence to suggest that in the nature nurture
controversy, nature will win out. In several publications and lecture, he has advocated
the assignment of a working gender, and deferral of any irreversible surgery until the
child is able to verbalize his/her own gender identity (Diamond, 1997, 1998, 1999). Nor
is Diamond alone in his conclusions; Reiner has also advocated the notion of minimal
surgical intervention until such time as the child's gender identity may be assessed (1997,
Chapter 11). From an ethical perspective, McCullough also feels that this approach has
merit (Chapter 10).
There is growing evidence to support the notion that the brain is a sexual organ
(LeVay, 1994) whose differentiation is affected by androgens. In a rat model, Gorksi has
shown that antenatal androgen exposure will affect the size of the sexually dimorphic
nucleus of the preoptic area (SDN-POA) (Gorski, Chapter 5, 1978) (Woodson and
Gorski, 1999). Analagous to these alterations in th~: hypothalamus, are the changes that
have been documented in human brains in the autopsy series of Le Yay (1991) and Swaab
(Chapter 6,1985). Growing evidence supports the notion that androgen exposures in
women with congenital adrenal hyperplasia produce changes in gender typical behaviors
when these patients are compared with a control peer group (Meyer-Bahlberg, Chapter
12), although these changes fall across a spectrum even when the patients are staged for
the degree of virilization. Males with cloacal exstrophy have testes that are histologically
normal (Matthews et ai, 1999), and consequently it is not surprising that most of these
patients will exhibit some degree of CNS virilization. Whether this pattern of CNS
imprinting is exclusively due to androgen effects is not certain, for there is now evidence
for transcription of the SRY genes within the brain (Lahr et aI, 1995; Mayer et aI, 1998).
As with CAH, these findings fall across a spectrum with some patients merely exhibiting
some interests in traditional male activities and toys, while others reach a point where
they seek a change in gender role. Reiner has estimated that up to 50% of genetic males
who underwent gender reassignment in the neonatal period may ultimately revert back to
a male gender.
A major factor in the long term outcome analysis of these patients is the objective
assessment of a non-biased observer. The relationship between patient, family, and
surgeon is a special one, and there is little doubt that this affects the way in which
142 S.A. ZDERIC, ET AI.
information about gender role can be accurately collected. Pleasing the physician with
reports that make things seem better than they really are is a real phenomena which can
be observed whenever subj ective criteria are being assessed. Anecdotal stories of the
gender reassigned patient who arrives at the office in a pink dress, but who refuses to
wear dresses otherwise lend credence to this notion. This phenomena is bothersome to
some physicians who would argue that "my patients would never lie to me." In fact
major problems will be reported to the treating physicians, but it is the more subtle
findings that are often not reported or expressed in the best possible light. We must
never forget that physicians and parents together made difficult decisions, and as such
parents will have a vested interest in seeing a "good outcome". In this instance doctor
pleasing is not lying, it merely reflects hopeful thinking and a desire on the part of
parents to put these difficult decisions into the distant past. The best way to assess these
kinds of subjective findings is to have patients assessed by a non-biased observer. In this
setting parents and patients will be more inclined to reveal more of their concerns and
fears. Clearly such long term assessments are going to be best handled by psychiatrists or
psychologists.
In light of these findings, we have initiated a program at CHOP where all patients with
cloacal exstrophy are assessed by both their surgeon as well as a psychologist. Families
are being made aware of the current shifts in thinking about gender reassignment, and the
kinds of problems that patients are now reporting. On a yearly or semiannual basis
families and patients will meet with the psychologist to assess how overall development
is progressing, and in particular how gender identity is developing. The goal is to initiate
these dialogues in early childhood and identify patients and families who are
experiencing difficulties. It is also important to facilitate the process for those patients
who would opt to revert to a male gender.
Counseling of both family and patient are important in the management of patients
with cloacal exstrophy irrespective of whether or not neonatal gender reassignment was
performed. There is occasionally some resistance on the part of families to seeing
psychologists or psychiatrists because of the stigma associated with mental health issues.
This is unfortunate because the real goal of such sessions is to help families cope with the
immense social and emotional burden of caring for a child with multiple special needs
and thus achieve their maximal potential. Once the process has begun, most families
appreciate the opportunity to discuss their child's development.
In summary, it has become clear that gender reassignment for cloacal exstrophy has
not proven to be a panacea which resulted in well adjusted females with less
psychological and psychiatric morbidity than their non-reassigned counterparts. There
can be no question that many genetic males who remained in a male role have been left
functionally aphallic and unhappy, and a growing number of these men are seeking
phalloplasties. The techniques for phalloplasty have undergone major improvement over
the past 10 years, and there are now several patients with cloacal exstophy who have
undergone such reconstructions. There are a number of genetic males who were
reassigned to a female gender role who have found that their lives could not continue in
that direction, and who have subsequently have reverted to life in a male gender. These
findings are occurring at a time when antenatal diagnosis has resulted in fewer neonatal
presentations of patients with cloacal exstrophy. As more centers see fewer such cases,
it is important that the outcome for gender reassignment for this condition be reported.
Given the new advances in exstrophy surgery and phalloplasty techniques, we foresee
that fewer cases of gender reassignment will be performed in this setting.
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exstrophy. J Ural 160: 1179-1181, 1998.
Bradley SJ, Oliver GO, Chernick AB, Zucker KJ. Experiment of nurture: ablatio penis at 2 months, sex
reassignment at 7 months, and a psychosexual followup in young adulthood. Pediatrics 102:e9, 1998.
Carr MC, Mitchell ME. Continent gastric pouch. World J Ural 14: 112-116, 1996.
Colapinto J. The true story of John Joan. Rolling Stone, pp. 54-97, Dec 11,1997.
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Questions for Drs. Zderic and Kodman-Jones
Question - I assume most these patients, whether XX or XY had a vaginoplasty and I am

wondering how they did accepting that and at what age was vaginoplasty performed and
how did they cope with that?
Answer - In most instances that was done around the time of continent reconstruction.
Question - When have you thought about telling your patients that they have a Y-
chromosome?
Answer - Dr Jones - That is the question of the hour, isn't it? I guess that is why we are
contributing information to try to determine when is the best time. But I think earlier
than adolescence tends to be my first thought about this, before a lot of anxiety is being
generated about their own development. I do not have a good answer for that. I am
wondering what other hospitals have developed a protocol to that.
Question - The high frequency of depression is striking in the cases you described so
well. We know most from animal experiments and from observations in human beings
that stress during development might organize the hypothalamic pituitary adrenal axis
adrenal in such a way that CRR hormone from the hypothalamus is produced in higher
amounts for the rest of their lives. If CRR is injected into the brains of animals you can
change this in movement and sexual behavior and eating behavior, just like the symptoms
of depression. I wondered whether you tested for an elevated activity of the
hypothalamic pituitary adrenal axis for instance by the dexamethasone suppression test
in order to see whether this was the cause of the higher rate of depression because it
might be treated.
Answer - Dr Zderic - We have not done any of that kind of medical work-up to look at
depression, but obviously that is an area that needs to be addressed. I We are not alone in
wrestling with the issue of when to talk with patients about their exact diagnosis. And I
think Dr. Imperato-McGinley's statement was that you have to give information in stages
and you have to know the patient. The biggest thing is how much time it takes, which
makes such discussions difficult in a busy surgical clinic. Hence we feel it is crucial to
have a team in place and emphasize the important role of the Psychiatrists and
Psychologists.
Comment - And the Social Worker, which is what I am. I am the Social Worker at The
Hospital for Sick Children in Toronto and I have been working with the Intersex
population for the last 10 years. What we have been doing - we have not done the
outcome studies to see how this is working and a lot of my thinking has come from work
that I did previously and on-going in adoption around issue of secrecy and also
explaining issues of identity to kids. And what I do, I am the time-saver for those busy
doctors and nurses in the clinics because I get involved and spend hours and hours and
hours at the beginning when babies are born and then on-going through the years as well.
Helping the parents right from day one starting to think about how they are going do to
some of the explanations and how they can construct their story using life books, telling
the story and they can practice when they have an infant because you can make all sorts
of mistakes in your story and the child doesn't know what you are talking about and you
can practice those really difficult stories that you have to tell. Also, that you add to it at
developmentally appropriate stages and you need to get to the chromosomes before the
kids discuss it in school and you need to be in touch with what's going on with Sex
Education in the school, both from the playground and the formal kind of stuff because
you have to be ahead of it and then work with the Interdisciplinary Team to pull it all
together. The people who are buying into that seem to be really, really coming along
writing these stories and it is going to be interesting to see how that works out.
Question - Let me ask you this - do you find it is better that discussion of their
chromosomes, or their genetics, is best done as a team with a doctor in the room as well
as a parent so that the burden of this discussion is not with the parents or do you do it a
number of different ways, given the situation.
Answer - I think it has to be worked out with the family, but what works best for them
and what I have been doing with a number of kids, particularly with partial and androgen
insensitivity syndrome, is over the years working with the family, getting to know the
child and sort of gradually working towards that. And some parents say, "I just can't do
this", or some have chosen to bring the child to the hospital and in between clinics talk
about that and then bring the child in with the professionals to get further information and
then more follow-up right afterwards. That seems to be working well. Supporting the
parents and really including the parents as part of the team, I think is extremely important
and one of the things I have done over the last 2 years is run an Internet support group for
exstrophy parents and we have got, people probably from lots of your centers, we now
have about 100 from 9 different countries and there are Intersex discussion that go on
there for the cloacal exstrophy and parents are really working with each other as well
about what works. And I think that is really important.
Question - 1 have a question for Dr. Kodman-Jones. We have seen a remarkable amount
of depression in our children with cloacal exstrophy, but I was wondering if you can
comment on how much is just due to the general health problems of these individuals
versus how much is due to the gender dysmorphia that you see?
Answer - Dr Kodman-Jones - Of course I cannot break it down into percentages, we are

not that good yet, but 1 think it is a combination of a number of different factors. How
well the surgical and medical apsects are going is a contribution. If it is going well than
that is less of an issue. If there is a lot of gender activities that seem to be confusing to
the parents, that can contribute. I think there are multiple avenues that contribute to that
depression - I don't think there is one factor at all.
Dr William Reiner - Chris, can I comment on that a little bit? I am not going to preempt
anything I am going to deal with tomorrow, but I think it is important to realize that the
kids with cloacal exstrophy and the kids actually with classical exstrophy as well have
questions that are far greater than just gender identity. They have a much broader
identity problem. And I think that is very important to realize and when you deal with
the gender identity issues, they are part of that whole identity spectrum. I think the
second point is there's no doubt when a depression is very, very high in the cloacal
exstrophy group it seems to be very commonly precipitated when the kids recognize what
their genetic gender is and when they seek to be that gender. It needs to be treated and it
needs to be treated vigorously and it is just as treatable as anybody's else's depression.
By the way, the same is true of the classical exstrophy kids although it has nothing to do
with their gender, it doesn't appear to be related to their gender. It appears to happen
very commonly in adolescence and DSM-4 criteria are met for major depression and at
least 30% of the kids I have dealt with in adolescence in a classical exstrophy group,
which numbering about 50 or 60 right now, in terms of full evaluations. It is probably
higher than that in the cloacal group, but I actually haven't counted them out so I cannot
give you an actual number. I think that you have to look at it from a broad perspective. I
think the aspect of being programmed, if you will, by the early experiences in terms of
their hypothalamic pituitary adrenal axis, I think, is probably very, very true. One
hundred percent of these kids I have evaluated so far have an anxiety disorder - 100%.
And that is not a minor point, anxiety disorders are extremely impairing. And that is true
to the cloacal and the classical exstrophy group - 100% I have evaluated, somewhere
counting all the kids probably in the neighborhood of 100 now - exstrophy kids, and they
all have an anxiety disorder. The minimal anxiety disorder I have picked up, and this is
in these personalities that are extremely laid back, easy to deal with and the kind of
people that can tolerate the big one in Los Angeles and kind of walk around and help pick
up the pieces, they have the very least in anxiety disorder around medical and surgical
procedures. I think those are all things to consider.
Comment - This is a purely anecdotal, but I have 8 cloacal exstrophy patients, 10 years
and older. Four of them have not been gender reversed and they are psychosocially very
well adjusted. Two of them are married and one is engaged to be married and they are
already first class individuals. The 4 younger ones are all gender reversed. They have all
aged around 10 years of age - 3 of them are very much tomboys and I have apprehension
about all of those.
A FRAMEWORK FOR THE ETHICALLY JUSTIFIED
CLINICAL MANAGEMENT OF INTERSEX
CONDITIONS
Laurence B. McCullough, Ph.D.

Baylor College ofMedicine, Houston. Texas
INTRODUCTION
The clinical management of intersex conditions, as well as related conditions that are
not, strictly speaking intersex conditions, has become a matter of considerable
controversy within the medical profession and in the lay public (Diamond and
Sigmundsen, 1997; Colapinto, 1997; Diamond, 1999). This controversy has a number of
origins, including complaints from patients who have been assigned what they experience
as discordant gender identity, coverage in the lay press of individual cases (such as
John/Joan) (Colapinto, 1997) and activism on the part of organizations such as Intersex
Society of North America. It is worth noting that a recent number of the Journal of
Clinical Ethics was devoted to consideration of intersex conditions, in response to the
public controversy and to different views and approaches that have been expressed in the
medical literature (Howe, 1998;Groveman, 1998;Wilson and Reiner, 1998;Crouch,
1998;Chase, 1998;Schober, 1998; Kipnis and Diamond, 1998; Preves, 1998). The
current controversy appears to have created sharp divisions within the clinical
community. These divisions apparently affected the preparation of above-referred
number of the Journal of Clinical Ethics, because some physicians with experience in
the clinical management of intersex conditions declined to contribute (Howe, 1998).
This chapter attempts to provide a framework for the ethical justification of the
clinical management of intersex conditions. This chapter therefore begins with a primer
on ethics, ethical justification, medical ethics, and bioethics. The chapter then turns to
the clarification of the concepts of sex, gender, normality and abnormality, the nature of
the physician as an authority and in authority, and decision making about the pediatric
patient, because they concepts playa central role in the current controversy and because
lack of clarity about these concepts has contributed, perhaps in preventable ways, both to
generating and sustaining the controversy about the clinical management of intersex
conditions. An ethical framework for clinical judgment and the offering and
recommending of gender assignment is then set out and provided ethical justification.
One crucial feature of this framework is the concept of a trial of assignment of
phenotypic sex and gender-role assignment. Possible criticisms of this concept are
considered in a separate section. The chapter closes by identifying the implications of the
proposed ethical framework for the future clinical management of intersex conditions.

Edited by Zderic et af., Kluwer Academic/Plenum Publishers, 2002
150 L. B. MCClJLLOlJGH
A Primer on Ethics, Ethical Justification, Medical Ethics and

Bioethics
The clinical management of intersex conditions is controversial clinically and

ethically. Before turning to a consideration of that ethical controversy we first need to
define ethics, ethical justification, medical ethics, and bioethics.
Ethics has been understood for centuries in world philosophy as an intellectual
undertaking that is best defined as the disciplined study of morality. Morality
encompasses our actual beliefs, attitudes, and norms about human behavior and character.
Morality has many sources in contemporary societies, especially multicultural societies
such as the United States. These sources include, non-exhaustively, the ways in which
we are brought up by our parents and extended families, family and ethnic traditions,
religions, regional and national influences, and law. It takes but a moment to appreciate
that these sources of morality can frequently be in conflict, e.g., as they are regarding
abortion in many countries, and that these different sources of morality can both unite
and sometimes divide people, e.g., as in the former Yugoslavia now splintered by what
appear to be intractable religious, ethnic, and national animosities.
As an intellectual tradition, ethics in Western philosophical thought has its origins in
the recognition that morality can be improved and often needs to be improved. We begin
to move in the direction of improving our morality when we ask, not how we are actually
behaving toward each other, but how we ought to be acting toward each other and by
asking, not what current standards of character (or lack of same) happen to be, but what
our character ought to be (McCullough and Chervanak, 1994). Asking these questions in
a rigorous way leads to critical evaluation of and improvement in current morality.
Ethics, as the disciplined study of morality, therefore asks two general questions:
1. What ought to be the behavior of individuals toward each other, of institutions
toward each other, of institutions to individuals, and of individuals to institutions? That
is, what are obligations that should shape our moral lives and the moral lives of
institutions, so that we and our institutions act better and therefore improve the human
condition?
2. What ought to be the virtues in our moral lives, those traits or habits of character
that dispose us as a matter of routine to discern our obligations and to act on them
routinely? That is, what obligations ought to shape our moral lives, so that we improve
our characters and therefore the human condition?
Ethics achieves intellectual discipline in addressing these two questions by
undertaking the clear, consistent, and coherent analysis of those concepts relevant to the
evaluation and critique of human behavior and character and then developing arguments
using these concepts to determine how we ought to act and what character we ought to
cultivate in our moral lives (McCullough and Chervanak, 1994). The intellectual
discipline of ethics requires those who reason about what morality ought to be to reach
only those conclusions supported by argument. Appeals to "gut feelings," various forms
of authority, the superiority of one's own or one's institution's policies and practices and
other strategies of assertion without the benefit of argument have no place in ethics.
Ethical justification means that one has followed the intellectual discipline of ethics by
first getting clear about relevant concepts and then making arguments. An ethically
justified approach to a morally controversial topic therefore requires both conceptual
A FRAMEWORK FOR THE ETHICALLY JUSTIFIED 151
analysis and argument. Both of these core methodological tools of ethics are used in this
chapter.
Medical ethics concerns what morality ought to be in medicine (Beauchamp and
McCullough, 1984; McCullough and Chervanak, 1994). First, medical ethics inquires
into the obligations of physicians and health care institutions (both those that deliver and
those that pay for health are) to their patients and other health care professionals, the
obligations of patients to physicians, health care institutions, and society, and the
obligations of society to physicians, health care institutions, and patients in the form of
health care policy and asks what these obligations ought to be (Beauchamp and
McCullough, 1984; McCullough and Chervanak, 1994). This chapter focuses on the
question of the obligations of physicians to patients with intersex conditions and to their
parents. Second, medical ethics inquires into the character of physicians, the virtues that
they should cultivate so that they can discern and routinely fulfill their moral obligations
to their patients, to health care institutions, to other health care professionals and to
society (Beauchamp and McCullough, 1984; McCullough and Chervanak, 1994). This
chapter also focuses on the virtues that physicians should cultivate in response to the
current controversy over the clinical management of intersex conditions. Chief among
these virtues is what the eighteenth-century Scottish physician and medical ethicist, John
Gregory (1724-1773), called diffidence: the studied indifference of the scientifically
disciplined investigator to his or her own beliefs and the consequent openness to the need
to modify one's beliefs and practices on the basis of new evidence (McCullough,
1998a,b).
Medical ethics has deep roots in the literate traditions of medicine all around the
world, going back to ancient times (Jonsen, 1999). In Western medicine this literate
tradition originates in Ancient Greece, in the Hippocratic Oath and the texts that
accompany it. This tradition continues through the Early Christian, Medieval, and
Renaissance periods. Medical ethics as a secular undertaking, based on the results of
rational inquiry and therefore open to and binding on all who submit to the intellectual
discipline of rational discourse (conceptual analysis and argument, as explained above),
dates especially from the Enlightenment. Professional ethics, i.e., accounts of what the
obligations and character of the physician ought to be as a professional, someone
committed primarily to the protection and promotion of the patient's interest rather than
self-interest, also dates from the Enlightenment (McCullough, 1998a,b). This chapter
flows directly from this Enlightenment tradition.
Bioethics is a term coined in the late 1960s and early 1970s to name what its
proponents then took to be a new field of inquiry into what the ethics of health care
professionals ought to be, what ethical considerations should guide biomedical and
clinical research, and what ethical considerations ought to guide health care policy
«(Jonsen, 1998b; Reich, 1993,1994). Without realizing it at the time, the field of
bioethics continued the Enlightenment tradition of medical ethics. The historical roots of
bioethics in Enlightenment medical ethics have become better understood as bioethics
had begun to attend to the history of medical ethics before bioethics. Medical ethics can
be understood now to be part of the larger field of bioethics This chapter should
therefore be understood to be a contribution to the literature ofbioethics.
152 L. B. MCCULLOUGH
Clarifying Key Concepts
Concepts of Genotypic and Phenotypic Sex in Humans
In the literature on the clinical management of intersex conditions there is more than
one concept of sex, including, non-exhaustively, gonadal sex, reproductive sex,
chromosomal sex, genetic sex, and chromosomal sex (Wilson and Reiner, 1998). As a
consequence, discussions of determining and assigning sex are at risk of disabling
ambiguity. Our first task, therefore, is to seek clarity about the different senses in which
sex in humans can be conceptualized. I will attempt such clarification within the current
scientific paradigm of medicine, molecular medicine. Molecular medicine, which has
replaced the infectious disease paradigm of the past century-and-a-half, understands
human health and disease to be the function of the interaction of genes and environment.
Environment is understood to be everything other than genes, from the immediate
biochemical environment of genes up to and including the complex behaviors and
institutions that characterize human societies. Engel's classic model of health and
disease as biopsychosocial phenomena beautifully anticipated the emergence of
molecular medicine in the 1980s (Engel, 1980). As we shall see, a biopsychosocial
approach to sex and gender plays a central role in this chapter.
Genotypic Sex
In the molecular medicine paradigm sex can be conceptualized in genetic terms, which
can be called the genotypic concept of sex: sex as a function of the genetic constitution of
an organism. There are two further levels at which genotypic sex can be understood:
chromosomal and genomic. The concept of chromosomal sex dominates the literature on
intersex conditions, in part because this is the most common and currently least expensive
way in which to identify genotypic sex. Thus, XX chromosomes are taken to define
female chromosomal sex, while XY chromosomes define male chromosomal sex. The
concept of chromosomal sex tends to be dimorphic, sorting all human beings into one of
two chromosomal sexes. This dimorphic conceptualization of sex has been enormously
influential on the literature on intersex conditions. Indeed, the very term, "intersex"
reflects the dimorphism of chromosomal sex (which in turn, reflects the dimorphic
concept of sex in society generally).
The basic science and, with increasing rapidity, the clinical science and practice of
molecular medicine have moved beyond the concept of chromosomal sex to the concept
of genomic sex (Jordan and Vilain, 2001). Chromosomal sex, as it were, tells us only the
anatomy of genes - where they are to be found - but not their physiology, their
function. A functional concept of genotypic sex therefore requires the concept of
genomic sex. The phenotypes of sex are more a function of genomic sex than they are of
chromosomal sex. Moreover, at the level of genomic sex it seems clear that sex is not
dimorphic. Jordan and Vilain report elsewhere in this volume on the apparent
translocation of genomic material between the X and Y chromosomes (Jordan and Vilain,
2001). Thus, from the genomic perspective, sex displays just the sort of variance that the
concept of biologic variability predicts. That is, from the genomic perspective, there are
multiple genotypic sexes in human and other mammalian species. The dimorphic
concept of sex is not scientifically adequate for mapping this genomic variance and its
phenotypic expressions and therefore should be abandoned.
This has important implications for the clinical management of intersex conditions.
First, conditions such as hermaphroditism and pseudohermaphroditism, as well as
congenital adrenal hyperplasia, should not be understood as intersex conditions, but
rather as expected variance of genomic sex. Genotypically and genomically, these may
be third or fourth or nth sexes. Second, therefore, dimorphic concepts of sex should be
abandoned as conceptually inadequate scientifically and clinically. The concept of
dimorphic sex has contributed to the current controversy over the clinical management of
intersex conditions, but does not contribute to its solution. Third, the nomenclature of
"intersex" conditions should be abandoned in favor of the nomenclature of the biological
variability of sex as a human trait that originates in the biologic variability of genomic
sex. The human genome exhibits variance that is predicted by evolutionary biology and
genomic sex is no exception. (For convenience sake, "intersex" is used in this chapter.)
Phenotypic Sex
Phenotypic sex concerns the expression of the genome in human traits, including both
anatomic and physiologic traits. These traits include the presence gonads, which has
been characterized as gonadal sex. These traits also include the functional status of the
penis, vagina, and other body parts in sexual behavior, which can be called genital sex.
Genital sex plays a major role in sexual satisfaction. Yet another sense of phenotypic sex
involves whether gonads are functional and presence and functional status of other
reproductive organs such as the penis, vagina, and uterus, which has been characterized
as reproductive sex. The production of hormones that contribute to virilization or
feminization count as yet another sense of phenotypic sex, which can be called hormonal
or endochrine sex. There is also increasing evidence of sex differentiation of the brain,
discussed elsewhere in this volume, which has been characterized as brain sex (Goy,
1980;Gorski, 200 I ;Swaab et ai, 200 I). There is also a general appearance in body type
of phenotypic sex (which can sometimes be very ambiguous indeed), which can be called
body-type sex. Thus, phenotypic sex includes at least gonadal sex, genital sex,
reproductive sex, hormonal or endochrine sex, brain sex, and body-type sex.
As expected with any human trait and especially with complex traits, phenotypic sex
exhibits considerable variability. Indeed, this variability defines intersex conditions in
that the different senses of phenotypic sex do not align consistently as "male" or
"female," but as a mixture, e.g., a virilized female body-type sex. Again, these should be
conceptualized in terms of third, fourth, and nth phenotypic sexes, rather than as intersex.
That is, the diverse observed alignments of the different senses of phenotypic sex should
be treated as expressions of variance in traits that we label sex traits. As a consequence,
no one of these different senses of phenotypic sex should be taken as the defining sense
of phenotypic sex. Failure to follow this rule has helped to fuel the current controversy
about the clinical management of intersex conditions. Any adequate ethical framework
for the ethically justified clinical management of intersex conditions must take the
variability of genotypic and phenotypic sex into account.
Concepts of Gender, Gender Roles and Gender Identity
Gender, gender roles, and gender identity are complex biopsychosocial traits that are
multifactorial in origin, just as the molecular medicine paradigm predicts. Gender is a
biopsychosocial phenomenon constituted only in part and never in whole by genotypic
and phenotypic sex. Gender really is an abstract concept, under which fall two more
concrete concepts gender role and gender identity.
Gender in the sense of gender roles involves the construction of social roles and of
self-identity by individuals, families, cultures, and societies in response to and under the
constraints of the biologic variability of genotypic and phenotypic sex. The range of
possible responses and the role of sex in constraining that range are not well understood.
It does seem clear that Money's claim about the considerable plasticity of sex and gender
needs to be modified; the constraints imposed by biology may not be quite so plastic
(Money, 1975; 1995a; 1955b). Gender roles in different societies exhibit different levels
of variability. Gender roles are psychosocial phenomena. One key feature of gender roles
concerns sexual orientation. Cultures and societies have developed variable norms about
which forms of sexual orientation are morally acceptable, which are not, and which are of
uncertain moral status. That is, the morality of sexual orientation varies within cultures.
In a multicultural society such as that of the United States we should expect there to be
competing accounts of the morality of sexual orientation. And indeed there are.
Gender in the sense of gender identity involves the construction or perhaps discovery
- it is not clear which description is accurate; perhaps both are - of one's self-identity
in response and under the constraints of genotypic and phenotypic sex, expected gender
roles in one's culture and society, and no doubt other factors. Sexual orientation, sexual
satisfaction, and fertility (which is valued with considerable variability) are components
of gender identity. In the open societies of liberal democracies individuals are free to
accept, reject, and modify the social roles and identities available to them from the
various cultures that make up these complex societies. Individuals will therefore vary
considerably in the value that they give in their self-identity to the various senses of
phenotypic sex and to sexual orientation. Thus, some will place a paramount value on
reproductive capacity whereas for other individuals this will be a relevant but not
overriding component of their gender identity.
It must be appreciated that medicine and surgery can assign only phenotypic sex and a
gender role, but not gender identity. Of the senses of phenotypic sex, surgery can assign
only gonadal, genital, and reproductive sex, usually not completely. Given these
limitations, the gender role assigned may be unstable, even fragile. Physicians, parents,
and society cannot and therefore should not expect to assign irrevocably gender identity
in all cases, given the variability of gender identity that is apparent among individuals
with intersex conditions. Obviously, there can be discordance between expected gender
roles and individual gender identity. Narrow and inflexible expectations about social
roles on the part of a culture or society constitute an important source of the current
controversy about the clinical management of intersex conditions, because an
individual's self-declared gender identity may be deeply discordant with the assigned and
therefore expected gender role and its expected gender identity, resulting in sometimes
severe psychosocial disorders (Reiner, 2001).
One possible explanation of this discordance is the following. When genotypic sex
and all of the senses of phenotypic sex align (as they most often do), we should not
expect gender identity to vary a great deal. Thus, we observe individuals who self-
identify as boys and girls, and then men and women - although, importantly, with
variations regarding sexual orientation, sexual satisfaction, and fertility. Assigning
gender roles in these cases can and should be undertaken with considerable confidence.
Thus, the now notorious case of Joan/John teaches us that assignment of such individuals
in the future to male gender roles reflects this alignment and thus decreases the risk of
discordance of assigned gender role and self-declared gender identity later in life,
whereas assignment to female gender roles of these patients increases the risks of such
discordance.
When genotypic sex and the various senses of phenotypic sex align variably, we
should expect variability in gender identity. Assigning phenotypic sex and a gender role
in these cases should not be undertaken on the basis that we can predict with high
reliability in all cases the risks of discordance between assigned gender role and self-
declared gender identity later in life. Which gender role ought to be assigned in such
cases is an ethical issue at the heart of the current controversy and will be addressed later
in this chapter in a proposal for a trial of assignment of phenotypic sex and gender role.
Normal and Abnormal Conditions
Are intersex conditions normal or abnormal? This is an important question for

clinicians, as well as for parents and society. There different answers to this question,
however, based on different ways in which "normal" and "abnormal" are understood
(Engelhardt, 1995).
One sense of normal and abnormal is statistical or observed normality and
abnormality, a sense of these two terms that identified by Francis Bacon, the first
Western formulator in the modem period of the scientific method (McCullough,
1998b;Bacon, 1875). In its statistical or observed sense, the normal refers to events that
most commonly occur and the abnormal to rarely occurring events, with the latter
expressed in contemporary science in such terms as the number of standard deviations
from the mean (i.e., the most common). In these sense of the terms, intersex conditions
are part of the observed but low frequency biologic variability of human genotypic and
phenotypic sex and are statistically abnormal. From this descriptive sense of intersex
conditions as abnormal no norms of judgment or behavior follow, because statistical
abnormality is not always disvalued. For example, rarely occurring genius levels of
intelligence or the extraordinary manual skills of a particular surgeon are highly valued.
Indeed, their statistical abnormality is sometimes lamented.
In the clinical setting, the terms "normal" and "abnormal" refer to anatomic and
physiologic conditions that are either healthy or diseased, respectively. Health and
disease are not simply descriptive terms; they are also value-laden. They reflect value
judgments about anatomic and physiologic conditions and whether they are acceptable to
us or not. Thus, "disease" is used in clinical judgment and discourse to name anatomic
and physiologic conditions that are disvalued because they involve increased risk of
premature or unnecessary death, unnecessary pain and suffering, or loss of structure or
function required in life tasks that we value. As a value-laden concept, "disease" goes
beyond description of a condition as statistically abnormal to a judgment that it is
disvalued and should be altered in the direction of the valued condition of health
(Engelhardt and Wildes, 1995).
Among intersex conditions, congenital adrenal hyperplasia (CAH) counts
unequivocally as a disease, because it involves an increased risk of premature death and
because CAH can be treated effectively and safely with medical intervention. Preventing
a premature and unnecessary death at a low iatrogenic cost to the patient is among the
core obligations of physicians in medical ethics.
Other intersex conditions are not unequivocally diseases. They do not involve
increased risk of premature death, nor of unnecessary pain and suffering (in and of
themselves). Assignment of phenotypic sex and a gender role when the different senses
of genotypic and phenotypic sex align variably involves the risk of discordance between
assigned phenotypic sex and gender role, on the one hand, and later, self-declared gender
identity, on the other hand. This discordance can become a source of suffering, including
psychosocial disturbances diagnosable in the nosological nomenclature of psychiatry and
psychology (Reiner, 2001). That is, the iatrogenic risks of phenotypic-sex and gender-
role assignment may be non-trivial. Given the poor state of outcomes data for
discordance of gender-role assignment and self-declared gender identity, these iatrogenic
risks are difficult to quantify and therefore to predict. It follows that any claim that
current medical or surgical management of intersex conditions, including those that are
immediately life-threatening, involve acceptable iatrogenic risks cannot at the present
time be supported by outcomes-based evidence. Intersex conditions once their life-
threatening aspects have been managed medically cannot, therefore, be regarded in well-
formed clinical judgment as uniformly abnormal in the value-laden sense, i.e., as
pathologies that should be promptly treated. Moreover, current forms of clinical
management of intersex conditions may result in non-trivial iatrogenic biopsychosocial
harm to patients.
Concepts of the Physician as an Authority and the Physician as an

Authority
In decision making about patient care with patients and, in the case of the pediatric
patient, with the patient's parents or guardian, it is important for the physician to
distinguish between his or her role as an authority in the decision-making process and as
someone who is in authority in that process (Engelhardt, 1995). In his or her professional
role the physician is expected to be an authority, i.e., knowledgeable about the nature and
purposes of medical and surgical management, the outcomes of such management, and
also of their benefits and risks.
That is, the physician is an authority about matters over which physicians can justifiably
claim scientific and clinical competence. When there is reliable clinical judgment that a
condition is a pathology and that a particular intervention has unequivocal clinical benefit
for the patient, as in the case of medical treatment of CAH, the physician as an authority
should recommend such intervention or present it as medically "indicated." When
clinical judgment that a condition is a pathology is poorly supported by outcomes data or
when the benefits and risks of various courses of intervention are little or poorly
documented, then the role of the physician as an authority in the clinical decision-making
process diminishes. Therefore, at most, clinical interventions can be offered to the
patient or parents in such circumstances, but .lli~ recommended. Given the poor
outcomes data for the surgical assignment of phenotypic sex and gender role, surgical
assignment to a particular phenotypic sex and gender role should rarely if ever be
recommended; to do so exceeds the scientific and ethical justification required for
physicians to recommend intervention, namely, that in the population of patients treated
with a particular intervention the benefit-risk ratio is demonstrably and significantly
favorable.
This problem is compounded by the fact that both phenotypic sex and gender roles are
assigned. We saw above that the dimorphic concept of genotypic sex, which still
strongly influences the medical and surgical literature, is conceptually inadequate to
analyze current genomic information about sex chromosomes. Moreover, phenotypic sex
in intersex conditions exhibits variance such that the different senses of phenotypic sex
do not always align on one sex. Gender roles involve value judgments - about preferred
sexual orientation, level of sexual satisfaction, or the relative value that should be given
to fertility - about which physicians have no more expertise than non-physicians. Thus,
the status of the physician as an authority of about appropriate phenotypic-sex and
gender-role assignment, after the medical management of life-threatening conditions, is
variable, at best.
To be in authority in the clinical decision-making process means that one has the final
say about whether medical or surgical intervention that has been offered or recommended
should be performed. It is a very stable matter of American medical law and medical
ethics that the adult, competent patient is in authod!y in the clinical decision-making
process (Faden and Beauchamp, 1986). More than 80 years ago, Judge Benjamin
Cardozo (Schloendorff, 1914) made this abundantly clear in the landmark case of
Schloendorff v. Society of New York Hospital: "Every human being of adult years and
sound mind has a right to determine what shall be done with his own body; and a surgeon
who performs an operation without his patient's consent commits an assault." The only
exception to thus rule are emergencies, i.e., clinical circumstances in which the patient's
life or health is in danger and there is no time to obtain consent before death or serious
loss of health can be prevented by clinical intervention (Schloendorff, 1914). Respect for
such self-determination (the legal concept) or autonomy (the concept from medical
ethics) means that the adult, competent patient is in authority. Gender identity is
determined by the individual. Assignment of phenotypic sex and a gender role in
intersex patients does not result in assignment of the concordant gender identity in a
currently unknown percent of cases, eliminating a role for the physician as being in
authority about which gender identity an individual ought to have.
For pediatric patients, the concept of pediatric assent has recently been developed and
endorsed by the American Academy of Pediatrics Committee on Bioethics (1995). The
child should participate in clinical decisions at a level commensurate with his or her
developmental capacity. The child's preferences should be elicited and taken seriously.
If those preferences are justifiably to be overridden, i.e., in cases in which the child's life
is at stake, then this must be explained to the child. Infants, obviously, do not possess
developmental capacity to participate in the clinical decision-making process; their
parents act as in authority over them. Insofar as the child does possess developmental
capacity to participate in clinical decision making (starting about age 5 or 6), then his or
her parents should negotiate decision-making authority with their child.
The concept of pediatric assent has important implications for the clinical management
of intersex conditions. Reiner reports in this volume that self-declaration of gender
identity in children born with cloacal exstrophy can occur as early as six or seven years of
age (Reiner, 2001). Moreover, for this condition, genotypic and phenotypic sex tend to
strongly align, making assignment of the opposite gender role a clinical course fraught
with unnecessary and preventable iatrogenic psychosocial risk. While it may be easier
surgically to assign a male child with cloacal exstrophy to female gonadal sex and female
gender role, the benefit-risk ratio of doing so appears to be unfavorable. The child's later
predictable preference about gender identity plays a decisive role in this evaluation. The
same can be said of surgical management of micropenis and clitoromegaly.
When self-declared gender identity occurs just before or during puberty, the case for
pediatric assent becomes stronger. It is well accepted that adolescent patients should play
a major and sometimes determining role in clinical decision making, especially in the
management of chronic conditions - among which intersex conditions should be
included. It follows from this that no irreversible assignment of phenotypic sex and of
gender role should be undertaken when the outcomes in terms of gender identity vary: the
physician should anticipate and preserve the role of the adolescent and adult the
adolescent will become as being in authority about which gender identity the patient later
declares.
The concept of pediatric assent has another important implication for the clinical
management of intersex conditions. Consistent with their developmental ability to absorb
and cope with such information, children should be taught about their condition and its
management, supported in their coping with this information, and gradually given an
increasing role of being in authority about is subsequent clinical management. It is worth
noting that Money and his colleagues were more than three decades ahead of their time in
this respect, in their calling for just such education and support of children with intersex
conditions (Money, 1955a,b). Some in the bioethics literature have attributed to Money,
if only by implication, the practice of some physicians of withholding information and
support from children, a practice diametrically at odds with his recommendations
(Drager, 1998).
Summary of Key Concepts and Their Implications for the Clinical

Management of Intersex Conditions
In summary, genotypic sex varies and affects the variance of phenotypic sex. The
variation in phenotypic sex affects the variation in gender roles and gender identity.
Societal gender roles may be and often are in the United States and other countries less
variant than the variability of genotypic and phenotypic sex and of gender identity. In
cases of intersex conditions physicians and surgeons cannot assign genotypic sex. They
A FRAMEWORK FOR THE ETHICALLY JUSTIFIED ]59
can only assign phenotypic sex, and then usually only some and not all senses of
phenotypic sex. Surgical assignment of phenotypic sex can sometimes leave unaffected
non-alignment of the senses of phenotypic sex or, even more problematic, non-alignment
of genotypic and assigned phenotypic sex. Gender roles can be assigned by physicians,
parents, families, and society, but not, with finality, gender identity. Physicians are
unequivocally an authority about the life-threatening nature of CAH and justifiably
recommend immediate medical intervention once a diagnosis has been confirmed.
However, no surgical management is required to address this medical emergency. For
intersex ('onditions in which genotypic sex and the various senses of phenotypic sex align
variably, outcomes in terms of gender identity and in terms of the iatrogenic psychosocial
risks of sex and gender-role assignment exhibit a variance the full range of which is
currently undocumented by outcomes studies. Clinical judgment to date has been
informed only by small series case studies or, even worse, anecdotal case reports.
These are, at best, insecure foundations for the physician as an authority in the clinical
decision-making process and do not provide adequate scientific or ethical justification for
recommending surgical intervention as "indicated." Thus, in these cases the physician is
at best weakly an authority about phenotypic-sex and gender-role assignment and is even
more weakly authority about gender-identity assignment, given the variability in
outcomes.. It is a standard rule of medical ethics that when the outcomes of conditions
that are not life-threatening vary and the clinical management of these conditions
involved unknown incidence and severity iatrogenic biopsychosocial risk in the affected
population, no clinical course that is irreversible should be undertaken. This is a variant
on the well known admonition to do no harm, or the ethical principle of non-maleficence
(Beauchamp and Childress, 1994). Parents are in authority in the clinical decision-
making process until their child is able to participate in that process in a developmentally
appropriate fashion. Adult, competent patients are in authority over their gender identity
and children have a measure of being in authority over this matter as developmentally
appropriate - perhaps as early as five or six and certainly as early as adolescence.
An Ethical Framework for Assigning Phenotypic Sex and Gender

Roles
The preceding ethical analysis and argument have established that, with the exception
of the medical management of CAR, physicians are, at best, variably an authority about
the clinical management of intersex conditions and that the ethically justified clinical
management of these conditions turns on the observed variability with which alignment
of genotypic sex (which is still poorly understood scientifically), the various senses of
phenotypic sex, and ultimate gender role and gender identity occurs and on the incidence
and severity of the biopsychosocial risk of discordance between genotypic sex, assigned
genotypic sex, assigned gender role, and self-declared gender identity. This risk can
occur as a result of both intervention to assign phenotypic sex and a gender role and from
non-intervention.
The lack of adequate outcomes data from current approaches to the clinical
management of intersex conditions only compounds the problem of the variability of the
physician as an authority. In the absence of outcomes data the medical and bioethics
literature has relied on small series of cases or on anecdotal case reports such as that of
Joan/John. Reliance on anecdotes or small case series has led in some of the bioethics
literature to the strategy of calling into question clinical management strategies on the
basis of a single counterexample or a handful of counterexamples. The case of Joan/John
(Colapinto, 1997), for example, has loomed large, even though it is not about intersex
conditions at all. Clinical judgment should not be based on anecdotal case reports or
small case series because of the lack of scientific rigor such an approach affords; a
handful of cases with opposite outcomes turns clinical judgment in the opposite direction.
Scientifically disciplined clinical judgment should as much as possible be based on
outcomes data; hence the calls for creation of a registry. In medical ethics, arguments
should also be based on outcomes and not anecdotes. The issue for medical ethics is
whether, on balance for all patients with intersex conditions various management
strategies produce a greater balance of biopsychosocial benefit over harm for a
population of patients. Unfortunately, the bioethics literature has relied exclusively on
anecdote and arguments from counterexample, from which no general ethical framework
can be developed with any confidence.
Until the outcomes have been gathered and rigorously analyzed, physicians and
surgeons should take a diffident approach (in Gregory's sense (McCullough, 1998a,b»
toward current practice. And, until outcomes have been gathered and analyzed, an
ethically justified framework for the clinical management of intersex conditions must be
based on a rigorous process of decision making, in order to form clinical judgment
rigorously under the discipline of the concepts that have been set out and clarified in the
previous section of this chapter. Such an ethical framework takes the form of questions
that must be carefully addressed in a particular order. In this way the physician can work
toward the goal of achieving a disciplined inquiry into what the clinical management of
intersex conditions ought to be, so that that management can be continuously improved
for the benefit of patients. It is worth emphasizing that the child's biopsychosocial well
being should be the primary focus of this ethical framework, because the child is the
patient - not the parents and certainly not society (especially when a society's inflexible
expectations for gender roles and gender identity are themselves part of the problem).
Parents of a patient are moral co-fiduciaries of their child with the child's physicians and
other members of the health care team responsible for their child's care.
A process-oriented ethical framework for the ethically justified clinical management
of intersex conditions addresses the following six questions.
1. With what frequency do genomic and chromosomal sex align?
2. With what frequency do genotypic and the various senses of phenotypic sex align?
3. With what frequency do assigned phenotypic sex and gender role align?
4. With what frequency does discordance occur between assigned phenotypic sex and
gender role, on the one hand, and later, self-declared gender identity, on the other hand?
5. With what frequency does psychosocially manageable discordance (with clinical
support as necessary) occur between assigned phenotypic sex and gender role, on the one
hand, and later, self-declared gender identity, on the other hand?
6. With what frequency does psychosocially unmanageable, psychosocially
pathologic discordance occur between assigned phenotypic sex and gender role, on the
one hand, and later, self-declared gender identity, on the other hand?
7. How reversible (including level of iatrogenic biopsychosocial risk) is surgical

assignment to phenotypic sex?
This framework should be used for the management of all intersex conditions. In the
case of CAH, this framework should be used for the clinical management of the child's
intersex condition after the medical emergency has been addressed.
The first three questions address the biologic variability of genotypic and phenotypic
sex and the biopsychosocial variability of sex, gender roles, and gender identity. When
scientific evidence from outcomes studies support the claim that the genotypic and
phenotypic sex and self-declared gender identity as an outcome of sex and gender roles
align with a frequency approaching 100%, the physician ought to act as an authority in
the clinical decision-making process and recommend the appropriate surgical assignment
to phenotypic sex and the psychosocial assignment to gender role.
When this alignment, based on rigorous analysis of outcomes data and new
information from scientific research into genotypic and phenotypic sex is rigorously
judged to exhibit variability, then the physician ought to act as an authority in the clinical
decision-making process and present the alternatives and what is known about the
benefits and risks of each, without making a recommendation. If one gender identity
occurs with greater frequency than another, then assignment to the requisite phenotypic
sex and gender role ought to be proposed as a trial of management, with the parents
informed that the child's self-declared gender identity may turn out to be discordant.
This trial should be undertaken with all of the psychosocial support of the patient,
siblings, and parents described by Reiner in this volume (Frader and Caniano, 1995), as
well as the support described presciently by Money four decades ago (Money, 1955a,b).
When a trial of assignment to phenotypic sex and gender role is employed as the
clinical management strategy, the fourth through sixth questions must be addressed.
They should function as rules for continuing the trial. If the answer to question four is
no, then the trial should continue. If the answer to question five is a high frequency, the
answer to question six a very low frequency, and the answer to question seven a high
level of reversibility, then the trial should continue. As the frequency of psychosocially
unmanageable discordance rises, then serious consideration should be given to stopping
the trial and exploring with the patient at that point his or her own already self-declared
but not announced or emerging gender identity.
To the extent that addressing the seventh question leads to serious doubts about the
reversibility of surgical assignment to phenotypic sex, such surgical management should
be postponed. Here I differ from Diamond, who has called for a moratorium on surgical
management (Diamond and Sigmundson, 1997; Diamond, 1999;Kipnis and Diamond,
1998). In this way the principle of non-maleficence or "do not harm" is implemented and
serious or pathologic psychosocial discordance between genotypic and phenotypic sex,
on the one hand, and self-declared gender identity, on the other hand, is maximally
prevented. There is a relevant analogy here to genetic testing of children for disposition
to disease. Based on the concept of pediatric assent, one could argue that, if the onset of
disease typically occurs in adult years or adolescence, then the child should not be tested.
Doing so could cause harm to the child as a consequence of parental knowledge. More to
the point, the information most directly affects the child later, when that individual can
assent or consent for himself or herself whether such testing should occur. Gender
identity appears to be self-selected or self-declared (for reasons not well understood) as
162 L. B. MCCllLLOlIGH
the child develops. The ability to select a gender identity not consistent with the assigned
gender role should be preserved to the greatest extent possible. Therefore, surgical
assignment to phenotypic sex should eschew irreversible outcomes to the greatest extent
possible.
Parents need especially to be supported in this respect. They need to do their best to
be open to their child's self-declared gender identity and to the possibility that it will not
be consistent with the assigned phenotypic sex and gender role (Reiner, 2001). The
advantage of the concept of a trial is that it includes the possibility of events not coming
out as expected or desired. Reminding parents of this aspect of the clinical management
of intersex conditions will be crucial for their psychosocial support.
The most effective way to conduct this strategy of trial of assignment of phenotypic
sex and gender role is to do so as a multi-center protocol that will bring such a trial under
the most rigorous scientific management. This approach would also require approval by
each participating center's Institutional Review Board, which would help to achieve
rigorous ethical management of such a trial. Research methods do exist for surgical
innovation and they could result in a substantive ethical framework (Frader and Caniano,
1995), i.e., a framework that ideally would provide an algorithmic approach to
assignment of phenotypic sex and gender role. If we can achieve such a result, then the
controversy about the clinical management of intersex conditions would become part of
the history of medicine.
Trial of Assignment of Phenotypic Sex and Gender-role Assignment:

Critique and Response
One major critique of the proposal of a trial of assignment to phenotypic sex and
gender role is that it might have harmful consequences. This is a possibility, but current
approaches and practices also may have harmful consequences. A multi-center trial
would have the significant advantage of being able to identify and quantify both
beneficial and harmful consequences, which cannot be done in the current circumstances
of an unmanaged approach to innovation in the clinical management of intersex
conditions. Surgical specialties have tended to innovate in a scientifically unmanaged
fashion (Jones and Fletcher, 1995). While this has resulted in some remarkable advances
in surgery, it has also resulted in surgical procedures that, when examined rigorously,
turned out not to be beneficial in the population of patients treated with that surgery. A
trial of management of intersex conditions might produce sirrular results. Thus,
diffidence requires that current practices should not be regarded, especially by their
adherents, as scientifically or clinically privileged. Without outcomes data, such a
privileged status cannot be claimed in a scientifically and clinically convincing fashion.
The current unmanaged innovation in the clinical management of intersex conditions
has also produced the current situation in the clinical management of intersex conditions
in which ethical "argument" is by case series, or anecdote. Medical ethics should be
population-based, just as scientific medicine should be (McCullough, 1999). The current
case-based approach in the bioethics literature has not significantly advanced either
ethical analysis or argument about the clinical management of intersex conditions. Thus
a critique based on one poor outcome from a trial would not count against such a trial,
because the issue in the evaluation of any trial is the overall result in terms of the balance
of clinical benefit over harm for the entire population of patients in the trial.
Finally one might object to the proposal of a trial of clinical management of intersex
conditions by appeals to authority or rhetorical questions such as "How could you
propose a trial of assignment of phenotypic sex and requisite gender role to anxious and
concerned parents?," as if the obvious answer were that no one in good conscience could
do so. The force of the preceding ethical analysis and argument is that the intellectual
discipline imposed on clinical judgment and decision making by medical ethics obligates
physicians and surgeons to offer such a trial and challenges the intellectual and moral
authority of those who act as, or invoke, authority figures in medicine to oppose such a
proposal. Moreover, those who oppose the concept of a trial of management then must
oppose current approaches and practices. They, in effect, are also trials of management.
Worse, they are poorly designed, managed, and evaluated.
Conclusions
This chapter has attempted to follow the requirements of the intellectual discipline that
ethics imposes on clinical judgment and decision making. First, this chapter undertook
an analysis of key concepts to make them clear so that their ethical implications for the
clinical management of intersex conditions could be rigorously identified. Second, these
concepts were then used to develop a framework for the ethically justified clinical
management of intersex conditions. This framework is necessarily process oriented and
will remain so until sufficient outcomes data are available to make the transition to a
more substantive, algorithmic framework. The proposed trial of assignment of
phenotypic sex and requisite gender role, if conducted in a multi-center design, would be
instrumental in producing just the data required to make this transition. In this way, the
medical ethics of the clinical management of intersex conditions would appeal to
population-based information about outcomes, not the current appeals to anecdotes, small
case series, authorities, and rhetorical questions. The well being of patients, who cannot
consent to their management, should not be put at risk by continuing the current
scientifically and ethically unjustifiable clinical management of intersex conditions.
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Questions for Dr. McCullough
Question - You made some suggestions as to how to deal with confidentiality issues if
there is going to be a delay in gender assignment?
Answer - Confidentiality in terms of who would have information about this?
Response - Right now when an Intersex baby is born, there is tremendous care in most
neonatal nurseries to keep things extremely quiet until the sex of a baby is assigned and if
this is something that is going to be delayed, even to the point where we can get pediatric
assent which might mean years in certain cases, how do you suggest we deal with the
whole issue of confidentiality?
Answer - Oh, you would tentatively assign gender? You mean you would assign a
working gender and we would have to rethink our conceptual framework around this.
Why should we be trapped by our current inflexible frameworks in this area if as it seems
to be the case, it is causing considerable psychosocial harm to children who cannot
consent to be put at that risk. So you would assign a working gender and then you would
not tell anyone else - any other parents in the nursery anything about that child, but that
should be standard practice already.
Response - I am just trying to take this to the next level, to an extreme of the suggestions
that there might be more than two sexes - stuff that is being brought up on the Internet.
Answer - Oh, I think the biological evidence is in. There are more than two sexes. There
are. There is mostly male, there is mostly female. We saw yesterday the spontaneous
transposition apart of the chromosome, that may explain that some person is something
third. But we already have more than two genders. That is already the case, either by
self identity, or by sexual orientation, we already know that. Now our society does not
necessarily accept that, but the facts are in. People have constructed multiple genders
already.
Response - I am just suggesting, as we all know, that this an incredibly difficult neonatal
problem and practically when you are dealing with parents who want to know the sex of
the baby. I think if a registry and working together is going to be determined, the issue of
confidentiality in the neonatal nursery is one that has to be addressed, also.
Response - Right, but confidentiality ought already to be strict in that setting. Is the
problem that people gossip with other parents and who share information about other
children that they should not be going and is the problem of laxed practice of respect for
confidentiality, which my hypothesis would be that is a problem that if you checked you
would find that you have to address first. Remember parents of children born with
chronic problems have to face many adjustments. Their expectations for their child have
to be adjusted. The challenges of intersex are more disturbing because of the
psychosocial response in our society. But children born very prematurely, parents are
facing all kinds of decisions - they have to adjust in not having had a perfect baby. I
think we have a lot of experience with how to help parents cope with that information.
But confidentiality should be strict already, it should not be a problem.
Response - It is, I am just suggesting that the spectrum now is an incredibly worrisome
one because on one hand this used to be considered a pediatric emergency where a
decision had to be made immediately as to whether or not this was a boy or a girl and on
the other hand we are hearing Dr. Diamond's recommendations in print that there should
be a moratorium on any kind of surgical determination of sex and I am saying that this is
a psychosocial issue that has to be addressed.
Answer - I agree, but it is not an emergency and so there are problems with the approach
that I propose. I have no doubt about that and I am sure I have not identified all of them.
The problems of the approach that you are taking I think are much harder to manage than
the two options that I have given you.
Question - I very much enjoyed what you had to say. I want to pose an issue for you that
I think really is an ethical one for us physicians. Your concept of physicians having
variable authority is an intriguing one. When, 25 years ago, I went into medicine doctors
made a lot of decisions, including life and death without reverting to any other outside
authority. Clearly I think that was perhaps over-stepping the bounds of what was
appropriate. The difficulty we have practicing today is that physicians really have lost so
much authority that it becomes now the other side of the coir.. It is difficult to support
families because people don't any longer trust often what doctors say to them and I
would just ask you to advise us on how do we cope with that when we are facing these
issues with a small baby? Do we need to invoke a committee that has to meet altogether
with a family? How do we overcome this issue of lack of trust in physicians today?
Response - That is a really hard question. I am tempted to use one of my Baylor medical
student's answers to tough ethical questions - your beeper goes off and you are
summoned to manage a metaphysical crisis in a far away place and will not be back for
two months and that is my beeper going off right there. Let me try to think it through this
way. In the critical care set, I think the problem shows up this way - what has happened
to critical care the last several, maybe 10 years I think, it is hard to get a handle on this as
I have thought back through it - physicians and nurses have started offering things to
families because they are in the unit, not because they make sense and good clinical
judgment. And so patients have gotten used to asking for things, even if they don't work.
So that is one problem and that is physicians have lost control of the consent process in
many settings, not just here. If you had good consensus clinical judgment to guide you
here, even better the results of a multi-center triaL, you could get back in charge of the
consent process because you could layout with great confidence the future consequences
of a given action. And you could help parents really think that through. Innovating
without protocol sets you up to have little authority. And remember where you are
speaking about matters where lay perspective counts heavily for the nature of the value
laid in judgment of normality and abnormality, parents are right to challenge you that you
are not the final authority in making these judgments. This is shared authority and those
boundaries would then have to be very carefully worked out. Now in the United States
we have the additional problem under Managed Care of patients withdrawing trust. This
is being documented now in the literature. I teach courses regularly for the American
College of Physician Executives, 100 physician managers at a time. When I ask them
how many think patients are withdrawing trust, usually 90% of the hands are going up.
Why is that? Because patients are not confident that the reason that you are doing, or not
doing things, in Managed Care is to protect your own incomes as physicians or whether
you really know what you are doing. And so we have this problem that society is not
confident that physicians really know what they are doing. By the way, even when the
evidence is in society rejects it. So in 1996 the issue arose in the Presidential Election
about so-called drive-by deliveries. And the data came out that Fall showing that for
single and uncomplicated vaginal deliveries an extra 24 hours in the hospital, not only
added no benefit it added risk of nosocomial infection. They should be sent home in 24
hours. The Congress of the United States rejected that science and mandated an extra
day. So the distrust is actually taking on scientifically irrational forms, so it has to be
addressed.
Question - What is the antidote to the distrust is giving parents reliable information. And
that is precisely the position you find yourselves not able to be in uniformly. And I take
it as much as you yourselves would like and so what I try to do is propose what I thought
were two reasonable solutions to address that problem. But, recognize this is a shared
authority. What gender ought a sexually anomalous human being be is not something we
may not be able to answer with great confidence prospectively. We may only be able to
answer that with very low levels of confidence and that means that sharing decisions with
parents becomes even more appropriate. I really feel there is a history and by the way,
invoking the language of an emergency is precisely a way to claim authority. Once it's
seen that these are not emergencies that claimed sole authority becomes suspect and so I
am trying to sort through this history as best I can. But I think it is a very complicated
story plus we have the movement of alternative medicine now in our society where the
scientific paradigm from which you explain health and disease is now rejected by people,
or at least put in competition with other paradigms for which there is minimal or no
scientific evidence. One of the disturbing features of that movement is the people in it
are all the most well educated people in our society. They are university and college
graduates. By the way, this is an old problem in the history of medicine. It has been
around before so we may be at the end of what historians of medicine call the success of
modern allopathic and osteopathic medicine of gaining intellectual authority in the
explanation of health and disease. We may be entering an era in which you are going to
have to compete much more vigorously than you did in the past. The bottom line I think
is the less you can say with confidence what the consequences of various alternatives are,
the more your authority rightly is going to be challenged by parents. So what I am try to
suggest the urgent problem is to get that first problem addressed, then we can negotiate
the boundaries of authority with more confidence. Does that address your question? It
was a hard question and I did my best to answer it.
Response - I think I agree in what you say, but I think it lacks a dimension that I am
concerned about and that is to equate protocols with a science really implies that science
is purely the organization in classification of knowledge. But innovation does not come
through protocols and I fail to see how innovation won't be stifled by a "science" that is
driven by protocol. For example, Koch's postulates, I think, were very good in that it
permitted infectious disease to make advances, but it overlooked a lot of the diseases that
were truly infectious because they couldn't be proven by limitations of Koch's postulates.
So protocols, I think, are limitations and that true innovation comes from accidents and
serendipity not through driven protocols. So how does that fit in? How do you keep
innovation going by insisting that we follow protocols for a treatment of our patients?
Answer - That's a really good question. I think what we need in areas of medicine where
innovation is crucial is we need to be working hard on when the innovation should be
subject to clinical trials, because you then can standardize it or if there are variances, test
the variance. But then get the outcomes data, which if you don't have you cannot get
adequate consent from anyone for the procedure. Because the informed consent process
requires you as someone who wants to do surgery on me to say here are the expected
consequences of this procedure. Here are the one we call benefits and here are the ones
we call risks or harms. If you are not in the position to do that the informed consent
process is disabled from the start. That is the problem, maybe I should have put it with
prolonged innovation, without protocol. Some innovations actually crucially get new
ideals tried out and the research process accommodates that, but the evaluation of the
innovation, so that the informed consent process can move forward, especially in
contested areas of normality and abnormality without the trial or some strong consensus
process, the consent process for parents is disabled. And that belps address your concern
that patients and parents are withdrawing trust. Because they get a sense that you cannot
give them the very information that they need. Which gender ought I to assign to my
child? Very complex moral biopsychosocial judgment. So I am not an advocate of
stifling innovation, I am an advocate of trying to prevent the problems, let's call it of
unmanaged innovation. Maybe that is the better way to have put it. By the way, the
history of surgery is filled with the failure of innovation. Maybe more often than the
successes.
Response - But that's the way it always worked. That the way evolution works. The
failures die and your progress is marked only by accepting only that which survives and
you are going to have a lot of failures, but that's the way you move forward.
Response - Dr McCullough - Evolution works by a series of accidents which human

beings give some value to ... survivorship and death. Evolution is a blind, stupid,
unknowing process that just goes on. Medicine is not a blind, stupid, unknowing process.
And if patients die as a result of innovation, who are children who cannot consent, you
have major ethical problems that have to be addressed. And if innovation has to be
restrained to protect unconsenting patients from urmecessary harm, then the logic of the
protection children is that we restrain the innovation. Do we loose some potential
benefits? Yes. Is that an understood aspect of research ethics? Yes. Is it accepted in all
the developed countries that have very sophisticated techniques for the public regulation
of research and the answer is yes. The benefit that you might gain is not worth any harm
that you have caused the patients. That's the logic of research ethics. So I have to I am
by the force and logic of concepts to disagree with you, and it is not always a happy thing
to have to do.
Response - Thank you for that presentation. I certainly, and most of us, agree with your
demand that we consider the ethical considerations of children and their parents and that
we share authority. I would take issue with a blanket statement, however, that we not do
any form of surgery which we, in our informed way, might consider helpful to the child
and the family in any condition until there is informed consent. By so doing in a child
with congenital adrenal hyperplasia, for instance, you are putting that family at
tremendous social risk by not allowing the family to share that child with a caretaker or
any other member of the family because of the ambiguity of the genitalia. You are also
putting the child at medical risk by allowing the child to be, to develop
hematometrocolpos or some form of obstructed uropathy. Therefore, making a blanket
statement that delaying any surgery, even be it cosmetic surgery, until the time of
informed consent of the child is not an appropriate statement.
Response - I tried to be clear because this I think is where I disagree with Dr. Diamond, if
I understand his recent proposals correctly. It is irreversible - surgery's irreversible
consequences in the absence of well-documented or consensus benefit for the patient, so
it strikes me - you have given an example where surgery would be permitted under the
account that I have tried to give. But there are many areas of surgery where the outcomes
are not so clear and the risks of non-surgical intervention are manageable. Those are the
ones that I was more concerned about. If I heard you correctly, you described a clinical
circumstance where the outcomes of non-surgical intervention are unmanageable and
very risky for patient and parents. That would not be one of the kinds of surgeries I
would want to stop. Provided that we have good solid evidence or strong consensus
judgment to support that clinical judgment. It is not all surgeries, it is surgeries without
reliable respected benefit for the patient.
Question - Thank you for the masterly exposition. I think only a fool would challenge
you in this context. But as a simple surgeon, I think I must pick you up on some points.
Some of which have been addressed already in a different form. I think the first is your
rejection of the anecdote. I think that you would agree that the management is a highly
complex issue which involves different social structures, different family structures and
therefore treatment has to be individualized. If treatment is individualized than the
anecdote has to be part of its evaluation. You can't escape the anecdote if you
individualized treatment. My second point would be to define emergency only as
something which is life threatening. And that is a point I think that Terry was making as
well. And the third was that you said we should not do anything irreversible without
expected benefit. What we should also not do is allow something irreversible to occur.
And that may be of importance in terms of changes which take place in the brain, patterns
of behavior because we do not intervene.
Response - There were three questions, I think. First is the place of anecdote and the
need to individualized treatment. I take seriously the molecular medical paradigm of
medicine that patients are to be managed under the appropriate population to which they
should be assigned by rigorous diagnostic prosthesis. That is good science. There is no
science of any individual. One of my colleagues at Baylor is a cardiovascular surgeon
who tells me that bypass surgery involves over 1,150 steps that must be performed in
precisely the correct order and he understands the variation from patient to patient. But it
is by populations of patients, not by individuals. So I think that the scientific paradigm of
medicine challenges that cherished assumption about individualizing care. Much that is
individual about the patient is less relevant than what population the patient belongs to.
Now is there a risk of impersonal care in this environment? You bet there is, and we
have to think through it very carefully. By the way, these ideals do not originate with me.
Dr. Gregory, when he is instructing his students two hundred years ago at Edinboro,
made this very same point that we have to be careful of this. Now if you accept the
anecdote than you got a problem. The Joan/John case refutes the value of all surgical
management of intersex conditions, because that one disastrous outcome is impermissible
on your account of not allowing any disastrous outcomes. So that is the advantage of
population-based thinking and population-based evaluation is that one disastrous
outcome does not necessarily mean that the intervention was ethically impermissible
because it might be one disastrous outcome out of ten thousand and that would be an
acceptable outcome. So the problem is I disagree with you. I think there is a real serious
conceptual disagreement between us and it maybe in the surgical specialties this is a
special problem. But the concept of individualizing care, if you listen to your colleagues
in basic science doing the molecular genetics of disease, they want you to put patients in
the appropriate population and devise managing strategies and evaluate them for that
population so that we can evaluate outcomes and approve the quality of healthcare that
we offer the patients. But if you accept the individualization then you have argument by
anecdote and the bioethics literature if full of this and you will get unresolvable
controversy and I think what happens is it gets personalized quickly. And then you have
attacks on the person making the argument rather than a attacks on the argument itself.
So I think there are risks associated with the way you want to go. We need to think them
through.
I think my definition of emergency is standard and as I read the literature preparing for
this talk I was struck by the intellectually not always discipline use of the concept of
emergency here. And I began to wonder was this used as a way to assert sole authority in
matters where perhaps sole authority should not be asserted. That there are major
sequelae for chronic illness in the future, does not make for an emergency now. So that
there are major problems in the future that we have to anticipate and manage, does not
make something an emergency now. So I think my definition of emergency is standard.
The concern about not intervening that we know would have irreversible consequences
and we can document that, than I take it the condition of equipoise would not be met and
so that would not be included as an arm of a trial. Again, we would have to think that
through looking at the outcomes as best as we could identify them. So there I think I
would be obliged to agree with you. If we could, either with consensus or strong
consensus or outcomes, we could say that non-intervening for particular condition is
known to produce irreversible serious unmanageable or barely manageable psycho-social
consequences or biopsycho-social consequences then equipoise is not met and you would
not put that into an arm of the trial.
Question - Dr. McCullough, again, a marvelous exposition and I have been provoked by
two issues that you have brought up. One is the physician authority, which has a very
fascinating area of how we are empowered with authority and in the decision-making
process. And the other that you just mentioned was the issue of what constitutes an
emergency in the sense of let's talk about children that are diagnosed antenatally with
congenital anomalies or with intersex states. Dr. Fekete' presented a very provocative
case yesterday of a child albeit with a number of deformities, but with the issue at hand
was really the intersex state and the child was terminated late in the pregnancy. At that
point, the issues that are involved that are provocative to me is the authority of the
individuals making that decision, of course, and the lack of decision-making process of
the affected infant who has no responsibility and no recompense or no ability to retort.
Answer - The way to think about the management of these conditions in the antenatal
period, and this comes from work I've done with Dr. Frank Shermanack who is Chief of
Obstetrics at the New York Hospital Cornell Medical Center. I have been working with
Frank for about 16 years now in obstetrics ethics. The concept we have developed and
deployed is the concept of the fetus as a patient. And what we have argued is that the
viable fetus is a patient and that when there are conditions in the third trimester that are
lethal or that eliminate the possibility or nearly eliminate the possibility of any central
nervous system development, termination of pregnancy in the third trimester is permitted
but not otherwise. And we just recently published a piece in the British Journal of
Obstetrics and Gynecology criticizing the practice in the United Kingdom of third
trimester termination for trisomy 21, which provoked the Editor of the Journal to write an
editorial to accompany our article and one other. Obviously an area of great controversy
and an area of sharp difference, by the way I think, between obstetric ethics in the United
States and obstetric ethics in the UK. and the European continent. And a matter of
international controversy in current obstetrics ethics to which we should all respond by
taking standard scholarly approaches and examining the arguments. I listened to Dr.
Fekete's case. That struck me as a multiply anomalous fetus and I was not sure if it had
so many anomalies that the sum of them was a condition incompatible with existence. In
which case under the criteria that Dr. Sherman and I propose that third trimester abortion
was legitimate. But if you had Intersex conditions in the third trimester and that's all you
found that would not meet the criteria that Dr. Shermanack and I have defended in the
literature, so we would be obliged to take the position that termination of pregnancy
would not be permitted - before viability, if they could be detected that early and I am not
sure. I have asked Dr. Shermanack about this by ultrasound. He is not sure he can say
that, but if that were the case than, on our account if a woman is not obliged to give the
pre-viable fetus the moral status of being a patient and so what happens in a termination
of pregnancy is that she withdraws that status and so at least the physician is not killing
the patient. Now abortion kills a human being. There is no doubt about that, but it is
independent moral status is disputed without anyway to settle that dispute that has been
going on for more than 2000 years. When philosophers cannot figure something out after
2000 years, it is because it cannot be figured out. That is the conclusion that I reach.
That is enough time for the winning argument to appear. And it has not and every time
someone thinks they've got it, it gets refuted in the next issue of the Journal in which it
appeared. So one thing we can be clear on is when the fetus is a patient. So if a
physician is willing to perform a pre-viable first or second trimester termination of
pregnancy for intersex conditions, if that is a good enough reason for the physician, than
Frank and I would argue that that is ethically permissible. But that takes us into the area
of obstetric ethics.
Comment & Question - Yes, we should know more about the outcomes of what we have
been doing for years. And we are guilty for not knowing that. At the same time it is an
experiment doing nothing. We know less about that and the morality of doing nothing
and knowing less about it and exposing the patient to something we do not know the
outcome. We only know the outcome in the old literature where something is an
individual case series of a patient, and those patients ended up living like hermits and
isolating themselves from society because they were different. When you limit what you
can do if there is moratoriums in specific areas - is the moratorium on the gonads? Is the
moratorium on cosmetics? Do we leave ovaries and testes? Can we take the testicle out
or the ovary out? How do we decide that? We do not know what body image to change
- if you let/allow for androgenization during puberty, how much does the psyche adapt to
one's own body image? Dr. Meyer-BahlburgiBahlberg has shown this in patients that
stop taking their androgen suppression who have adrenal cortical hyperplasia. We know
that these patients - should we continue suppressing their adrenal glands because they are
making - if they are not salt losers, they can live lives without salt lose without a great
risk to their life. Because there is this group of patients with adrenal cortical hyperplasia,
they will just virilize through life and then we do not know which way they are going to
go. By doing nothing, we have created so many situations we will not know how to
handle in the future. So we are doing something that we know less about than what we
are doing now?
Answer - Well, that strikes me as a condition which there might well be equipoise and
you would test non-intervention against various interventions that held promise based on
past experience. So it struck me that might be a good candidate for just the kind of
research protocol that I was proposing. There is an assumption in medicine that serious
conditions left unmanaged are always worse than serious conditions clinically managed.
But when you get enough experience that the morbidity, especially psycho-socially is
becoming non-trivial, then you have got to be willing to challenge whether every
increment of intervention is worth the iatrogenic biopsycho-social burden you are adding,
and I think we are at that stage. We have seen this in critical care. The assumption in
critical care was every reduction in mortality is worth whatever morbidity there is among
survivors and except in neonatal critical care nobody in critical care believes that
anymore, I think. They are willing now to question that. It is what I call the traditional
logic of beneficence. That is the effort to constantly improve medicine and benefit the
patients should always be undertaken regardless of the outcomes, especially if you are
reducing mortality. But we have learned from som{~ of the other areas of medicine that
there are times at which the tradition logic of beneficence needs to be challenged. And I
think the outcomes as we currently understand them, in areas like the one you have just
described where there is enough equipoise, then we should be willing to challenge it.
How do you manage that? With a clinical trial or with a strong consensus formation
process?
GENDER IDENTITY AND SEX ASSIGNMENT: A
REAPPRAISAL FOR THE 21 sT CENTURY
William G. Reiner
Division of Child and Adolescent Psychiatry, Department of Psychiatry, Division of
Pediatric Urology,Department of Urology, Johns Hopkins Medical Institutions,
Baltimore, MD
Of all relations the most universal is that of identity.

David Hume, A Treatise of Human Natun~, 1739
INTRODUCTION: EVOLUTION AND GENDER IDENTITY IN

HUMANS
How Did We Get Here and Where Are We Going?
Identity is the core of human existence. This existential reality manifests itself in the
human as a species-specific evolutionary focal point. It appears that no other earthly
species abstractly contemplates its very nature. This is as important for a discussion of
identity in general as it is for the concept of gender identity.
A brief review of evolutionary reductionism might be helpful at this time. First,
reductionism is the view that a process or organism as a whole can be broken down to its
individual components, or parts, and be understood as well or better than by examining
the whole. In the case of Homo sapiens sapiens such evolutionary processes are complex
and only partly decoded. In addition, the mind as a whole may appear to be greater than
the sum of its parts. Nevertheless, evolution is both apparent and data-based [Simon &
Schuster, 1996). That natural selection in the face of environmental vicissitudes has led
to present humans cannot be ignored.
How such seeming chaos leads inexorably to such order in life is, however, another
matter. For the sake of time and simplicity let me rder to three crucial principles of life
[Coffey, 1988]:
Self organization
Self maintenance
Self transcendancy.
Self organization is the process of naturally forming patterns, or organization, from

apparent chaos--whether we speak of molecules, cells, organs, or organisms. Self
maintenance is the process of sustaining existence. Self transcendancy is the potential
for changing the pattem--the central theme of evolution. These three principles imply
and create the central theme of life forms, namely, that a living system, although

Edited by Zderic et aI., Kluwer Academic/Plenum Publishers, 2002
176 W.C. REINER
dependent upon its environment, is not determined by it. Thus, humans have two arms
and ten fingers and a creative brain. The environment may relieve anyone person of a
finger or an arm as it may stimulate or inhibit creativity in the brain. Yet without that
potential, without the genetic patterns that define humans from all other species, no
environmental influence could affect any of us in those ways. Ten fingers as well as
language potential are determined by our evolutionary genetics, not by environmental
influences, yet maintaining all ten fingers or speaking a particular language may well be
dependent upon multiple environmental parameters [Simon & Schuster, 1996; Coffey,
1988; Gould, 1994].
Language, for example, is a self organizing evolutionary process that permitted further
self organization of Homo sapiens and accentuated self maintenance and self
transcendancy potentials. One's language potential is not determined by environment,
even though language itself may accelerate self transcendancy potential and influence
human evolution itself and, thus, environment [see, for example, Jusczyk, 1997 and
Lieberman, 1999].
These are concepts that are central to the idea of identity as well as gender identity.
For it is every bit as likely that gender identity is a self-organizing evolutionary reality as
language is. Yet we have had little hard data to argue the point-in any direction. [See,
for example, the contrasting views in the paper by Meyer-Bahlburg in this volume, for a
discussion of social construct theories and behaviorist theories of gender identity
formation.] Thus, at present the concept of gender identity itself is more a metaphysical
speculation than a physical data-based theory. Recognizing this fundamental reality, we
must prepare to move from speculation to observation, from observation to querying, and
from questions to designing research methodology to assess, answer, and elicit more
questions. We must be leery of providing prognoses before we understand diagnoses--we
must be prudent in our desire to do something for children with troubled genitalia by not
doing too much-because we are not cognizant of the implications and ramifications of
what we do. Ethically, we must recognize that we are not at all assured of the end result
of sex reassignment from the genetic sex in infants and children.
Perhaps, then, we should commence with the outcome and maneuver in reverse. The
children, it would seem, will tell us what their gender is; they will relate to us who they
are. Only we must be prepared to listen with divers ingenuity. Once we begin to
understand which children with which sets of clinical circumstances choose which
gender-i.e., the outcome-then we can begin to assign the likeliest gender-outcome to a
newborn with particular ambiguous clinical circumstances and maintain clinical ties for
years to come in order to adjust as necessary. In other words, we must be flexible.
Psychosocial and Psychosexual Implications of Sex Assignment
Where Are We and Where Should We Be?
Beginning with the pioneering work of John Money and the Hampsons in the 1950's
and 60's, an approach to children with ambiguous genitalia developed that evolved into a
GENDER IDENTITY AND SEX ASSIGNMENT 177
unifying medical-surgical paradigm by the late 1960's [Money et aI, 1955; Kessler,
1990]. The approach was founded in behaviorist theories steeped in a fluid premise that
much if not most of human behavior was environmentally induced or at least
conspicuously influenced. If environment is so crucial, the argument goes, then an
appropriately "engineered" environment could alter behavior in a desired pathway. This
notion of a sort of tabula rasa of gender identity allowed the creation of a medical and
surgical construction of gender identity for the many unfortunate neonates born with
genital ambiguity or -worse yet-complete absence of the phallus in a 46, XY genetic
male or complete virilization in a 46, XX congenital adrenal hyperplasia (CAH) female
[Kessler, 1990].
As experience with the idea broadened, the traditional paradigm intensified, with its
emphasis on early sex assignment, the importance of potential or lack of potential sexual
function with the extant genitalia, preservation of fertility in 46, XX genetic female
neonates with CAH, and the good cosmetic appearance of the surgically constructed new
genitalia. These have become the guiding factors in decision making in sex assignment
in the neonate with abnormal genitalia even to the present [Bradley et aI, 1998 and
Glassberg, 1998].
Unfortunately, cohesive and coherent data supporting such decision-making factors is
largely absent. The much heralded hallmark case [Money, 1975], now the so-called
"John/Joan Case", was refuted by a longitudinal follow-up into adulthood [Diamond et aI,
1997]. Other supporting data [Money et aI, 1986] or contradictory data [for example,
Reiner, 1996 and Imperato-McGinley et aI, 1979] themselves are reports of cases with
unclear or wildly divergent prenatal hormonal milieux or are individual case reports-with
comparable weaknesses to the single case "John/Joan" report.
On the other hand, the complexity of sexual differentiation and genital development
during embryological and fetal periods provides ample opportunities for the assessment
of internal and external errors. Recorded at least since ancient Athenian times, genital
anomalies occur in nearly one percent of the population. Etiologies vary yet commonly
include genetic errors, embryonic hormonal mistakes, or pelvic field defects, such as
cloacal exstrophy. Hormonal errors commonly emanate from mutations or other genetic
errors [Migeon et aI, 1998]. Pelvic field defects may proceed from serious early genetic
errors as well as anomalous tissue induction. The distinctions are important. Gender
identity is likely at least in part to be determined prior to birth, during critical periods in
brain development related to hormonal imprinting. The existence of normal or abnormal
genitalia or the existence of genitalia at all may play less of a role in gender identity
formation than before realized.
The interface of genetics with human evolution is undeniable. Observations of sexual
dimorphism have been recorded at least as early as Aristotle [Haqq et aI, 1998].
Nevertheless, conceptions of the relationship between genetics and human behavior have
been far more controversial during the 1960s through the 1980s probably than at any
other time in history [Kessler, 1990]. Recently, genetic and other biological determinants
have again appeared at the forefront, beginning with the discovery in 1971 by Raisman
and Field of structural sexual dimorphism of the brain [Raisman et aI, 1971]. Subsequent
brain research has recognized other sexually dimorphic structures or phenomena; such
research remains active [Gorski, 1985; DiPietro, 1996; Giedd et aI, 1997; and Swaab et
aI, 1995].
178 W.C.REINER
Human evolutionary history includes a "home-base concept" as a foundation for

families [Roberts, 1997]. These evolutionary origins of rooted behaviors may have
played an important part in the development of gender roles, although their relationship
to gender identity is largely speculative and beyond the scope of this paper. The reader is
referred to experts in evolution for further discussions of the evolution of gender identity
and gender role [Simon & Schuster, 1996; Gould, 1994; Roberts, 1997; and Potts, 1996].
To state that we comprehend the evolutionary mechanisms or pressures resulting in
given traits or attributes such as gender identity is probably naive at best. The evolution
of the human brain and the attributes of behavioral traits, drives, or potential are
inextricably interconnected and interwoven. Reductionism as a scientific focus of
comprehending gender identity, as an example--that is, looking at neuronal systems or
nuclei, or cellular and molecular biology for the etiology of such sexual dimorphism--
may conceivably be as accurate for the dimorphism of the brain as it is for the
dimorphism of the genitalia [Simon & Schuster, 1996; Imperato-McGinley et aI, 1979;
Migeon, 1998; Gorski, 1985; and DiPietro, 1996]. In other words, we recognize that
such dimorphism exists. It is our goal to understand the etiology of dimorphism both for
general somatic features as well as for behavioral and identity features. After all, the
mind and the body are not disconnected.
Scientific basis, Scientific Bias, and Scientific Faith
Mechanistic Versus Systems Approaches
We are products of our environment even if not determined by it. Thus, our own
views and perceptions focus on the culmination of 3 or 4 centuries of scientific thought
and about twenty- four hundred years of western philosophy. But if we can engender a
perception of those three organizing principles of life-self organization, self maintenance,
and self transcendency-we should be able to recognize that the pattern of organization
remains the same and that the potential for change-for evolution-is the central theme.
It was, in fact, an historical crisis of perception that has led to the methodology upon
which we have based our approach to gender identity these last several decades. The
mechanistic approaches from the Great Clock of Descartes (that is, that all the universe
works like a great clock) have held sway from the 17th or 18 th centuries into the twentieth
in much of scientific thought. More recently-and interestingly, in business and
technology-has systems analysis (simply, that the whole is made up not of parts but
rather the parts exist because there is a whole) become attractive to the natural and
behavioral sciences. The Scientific Method can be mechanistic, Cartesian, with the Great
Clock as the central metaphor. And the Cartesian and the Newtonian revolutions were
certainly valuable adjuncts to Renaissance philosophy. Yet novel-non-mechanistic-
conceptions have discerned special relativity, chaos theory, and abstractions of order-
from-chaos [Coffey, 1988].
Indeed, we have had great difficulty making the paradigm shift from Cartesian
dualism and the Newtonian revolution of the 17th and 18 th centuries. We appear to be
more nearly facile with a "parts approach." OUf present-day perceptions of how to
approach questions or problems are somewhat akin to the mechanistic approach that most
of us utilize in our day-to-day lives. We do not fully integrate the space-time continuum,
as it were, into our concepts of life. We observe the troubled (clinical) parts and perceive
a requisite repair or reconstruction of the part, rather than grasping the system as a whole
and the reality that the parts exist only because the system exists.
When we successfully correct a physical malfonmation or treat a mental illness, when
we intervene for a problem of one part of the body, we congratulate ourselves not only
for a job well done but for an intervention that benefits the patient. It seems obvious to
state, but nevertheless we need always remind ourselves that the reconstructed arm is a
part of a body and the body is a part of the person, and that in fact the person remains-
even if the arm is lost. An individual's existence does not depend upon the existence of
her eyesight Of his hearing, nor his penis or her vagina.
It is not particularly natural for us as clinicians, as mental health professionals, nor
even as patients for that matter to observe a person or ourselves as a system, as a whole.
Yet when we conceive of the good of the system we must understand the existential
whole itself rather than the summation of the parts. It is true that losing an arm may
change the way the system-the person -responds to her environment. It is true that the
environment has affected the system. Yet it is most important to realize that the essence
of the system has not changed. Mary remains Mary., Bob is still Bob - even though either
or both of them may feel less happy or less capable of coping with the world. But that is
the whole point. The essence of the system is unchanged and will adapt.
Our perception of gender-and gender identity and gender role- is a perspective of
viewing the whole in its parts. We see gender identity as something to dissect, something
to observe from environmental perspectives, an entity to comprehend in and of itself. I
would argue that the brain itself - that is, even though parts of the brain may be more
important to the formation of gender identity than other parts - the brain itself effects this
notion of gender identity as a part of a greater identity system, a personality system, an
adaptive system. This is "The being as such" as Aristotle conceived, or "the actuality"of
a being, as it is often translated. Gender identity as a potentiality or, as is more likely, as
an actuality, is an admixture of intricately interwoven internal concepts, genetic forces,
realizations, internal and external observations, hormonal influences, environmental
influences, and so on. At any given time any or all of these potentialities might be
conscious or quiescent or perhaps somewhere in between.
Gender identity is like the electron in quantum physics, the electron that is a
"schmeer." At any given time, the electron has a probability of being somewhere in some
pattern - but when we pinpoint it we can observe it only as it is a part of that probability
pattern. That is, the electron is part of a series of probability patterns of interconnections
that make up the atom itself. It is these patterns of interconnections that create the whole
atom. It is these patterns and interconnections within the human body - and especially
within the human brain - that make up identity, gender identity, gender role, sexual
orientation, temperament, and so forth.
Within the brain what we say are the parts are really a set of interconnections reaching
out to interact, to relate to, other interconnections. The essence of the existence of the
brain, the essence of the being, is within these interconnecting and interdigitating neural
networks. The brain is a systems organ. To speak of Alex having autism is to observe
180 W. G. REINER
Alex's brain, Alex's syndrome. No two people with autism are alike. To treat Alex's
deficits in language, in social skills, in perseveration is to deal with individual symptoms
or sets of symptoms. But it is not to deal with Alex's brain as a whole. It does not
change the essence of Alex or of Alex's syndrome, if you will.
The same can be said of gender identity, "normal" or "abnormal", "typical" or
"atypical". If we truly grasp this concept of a system of interconnections each relating to
other interconnections, then we are face to face with the futility of assigning a sex of
rearing and expecting a child to grow and develop and be happy with our choice.
The pattern of organization of the brain that leads to gender identity formation remains
the same in spite of us. Once the brain has been organized under the influence of specific
developmental pressures, be it female development or male development, and once the
critical periods of that development are passed, the pattern of gender identity is unlikely
to change-and we have no data that it does, transsexuals and gender dysphoric
individuals notwithstanding. That environment influences its expression, or that social
norms playa part in gender roles, does not alter the pattern of gender identity.
I am suggesting that there is a language that we have not yet developed, a language
about the systems aspects of the brain, a language about the formation of human
attributes and personality, a language about gender identity, about the formation of
gender identity. The apparent chaos of the masses of neurons is in fact an ordered set of
patterns, patterns of interconnections, patterns determined by self organization - self
organization of the neurons themselves. It is no accident that Bob is Bob or Mary is
Mary. Would Mary be Mary had she been reared by parents other than her own? Can
the essence of Mary truly be altered (other than by injury to the brain)?
A living system, although dependent upon its environment, is not determined by it.
The pattern of organization remains the same. The interconnections of the brain reach
out to interact with and to relate to other interconnections within the brain and within the
environment and the essence of the child's existence lies in those relationships-the
relationships of the interconnectivity.
The Etiology of Gender Identity
The etiology of gender identity may be neither obvious nor easily conceptualized. Yet
what is obvious is that the presence of androgen is critical. It is the determining factor in
the development of specific cytoarchitectural as well as behavioral sexual dimorphism in
humans--genital structure, shape of the pelvic girdle, red cell production, male-typical
behaviors, masculinization of the brain, and enzyme activities, to name just a few. The
trendy notion that Homo sapiens must develop gender identity or any attribute in a
divergent mechanism from other primates or even other mammals is species-narcissistic.
That humans must develop gender identity at all-that is, under environmental influences
- is an unproved assumption validated by little data. In the human fetus, the neonate, and
even in the very young child, society may well playa lesser role than many social and
behavioral scientists would like to admit. Ethological similarities of gender role, at least,
across mammalian species in general seem rather striking [Simon & Schuster, 1996;
Coffey, 1988; Gould, 1994; and Potts, 1996].
Second, methodological weaknesses or biases can be very problematic in sociological-
behavioral studies of human attributes. Assumptions can be grounded in perception,
rather than rational or objective observation or even objective speculation. Sexual

orientations, for example, are no longer looked upon as social constructs or family or
socially induced conditions but rather as variations of the norm [APA: DSM-IV, 1994].
Third, the evolutionary happenstance and selection in arriving at the present status
cannot be denied. That we have incomplete comprehension of the mechanisms or
realities of the etiology of gender identity should not reduce us to circumstantial
deductions but lead us to rational and logical investigation. The mere presence of
obvious sexual dimorphism, such as genitalia or breast development, or even the less
obvious cognitive or cytoarchitectural differences might well direct us towards concepts
of fundamental brain dimorphism. In other words, our assumptions about gender identity
must be carefully analyzed to preclude false assumptions and clinical misadventures.
Psychosocial and Psychosexual Outcomes of Sex Assignment
Sex assignment, then, and surgical or medical approaches to such decisions, must not
be dogmatic. The rather embarrassing paucity of literature relating to sex assignment or
gender identity in recent years reflects a fundamental failure of research. As Kessler
accuses, we construct gender [Kessler, 1990]. Yet decisions can be based upon the data
that is available. Clinical decisions may have to be altered as future data become
apparent. Sex assignment in the newborn period is an important clinical, psychosocial,
and psychosexual task with implications that we must not underestimate [Diamond et aI,
1997; Reiner, 1996; Imperato-McGinley et aI, 1979; and Reiner, 1999]. The infant
becomes a child, the child an adolescent, and the adolescent an adult. Gender identity is
a cardinal human fabric interwoven throughout any life-stage. We must be distrustful of
sallying forth clinically into areas we poorly understand. Rather, we must be prudent in
how we broach the subject of gender assignment-with parents and with clinicians-and
we must offer all of the data available, all of the controversy, plus the notion that we may
have to change directions with a given child in the future. After all, psychosocial-and
psychosexual-outcomes are really the foundation of our interest in sex assignment at all
[Migeon et aI, 1998; Reiner, 1999; Wilson et ai, 1998].
Clinical Decisions - Tradition, Dogma and Data
Clinical decisions and even the standard of care about sex assignment have
traditionally been based on few cases and limited clinical or research data, with
remarkably little outcome data. Recent discussions, as well as most of those in preceding
years, tend to emphasize potential for reproduction, sexual function, and genital
appearance as the major determining factors of sex assignment in children born with
ambiguous genitalia [Bradley et ai, 1998; Glassberg 1998; Money, 1975]. Furthermore,
it is frequently asserted that the decision regarding sex assignment should be made early
and the parents should have minimal ambivalence or ambiguity about both sex
assignment and rearing of the child [Bradley et aI, 1998; Glassberg 1998; Money, 1975].
Yet clinicians' faith in these assertions is generally inexplicable-the assertions are, in
182 W.G. REINER
fact, assumptions. For example, fertility may be irrelevant to a 46, XX salt wasting girl
who is fully virilized at birth, who never has sexual intercourse with a male because she
is attracted only to females, and who is uninterested in motherhood. Similarly, a
neovagina in a 46, XY aphallic male will have no sensory usefulness for the patient-it is
the patient's partner who may enjoy sexual function.
The traditional paradigm for sex assignment and reassignment in neonates with
abnormal genitalia rests largely on the innovative but predominately theoretical or
philosophical works of Money and his associates from the 50's and 60's. Data is largely
based on single case reports [Money, 1975] or discussions of multiple individuals with
ambiguous genitalia whose diagnoses are not clear but whose clinical homogeneity is
strongly in doubt [Money et aI, 1986]. Refutation similarly is often based on single case
reports, although Diamond and Sigmundson's case report refuted Money's original
hallmark case [Diamond et aI, 1997]. Others would argue that there are more than two
genders [Dreger, 1998; Elliott, 1998].
How should clinicians decide on sex assignment? Recently, "watchful waiting" has
been advocated as a more appropriate approach [Reiner, 1997; Schobur; 1998(2)]. As an
example, whereas the standard of care in the past for boys born with micropenis has been
sex reassignment at birth, Woodhouse has each reported on 2 series of males with
micropenis whose male sexual function and gender identity have been judged to be fair to
good [Woodhouse, 1998]. In my longitudinal series of 16 hormonally normal 46, XY
males assigned to female sex-of-rearing at birth due to absence of a penis, 8 have
spontaneously declared themselves male and seven live as males after ultimate parental
acceptance (one set of parents rejected the declaration)-three for more than 4 years
[Reiner]. Fifteen fall very close to the male-typical spectrum of gender roles, games,
activities, and behavior; one (castrated at age six) falls in the male spectrum.
Additionally, neo-construction and reconstruction techniques for the penis are
increasingly advanced and technologically sophisticated [Ochoa, 1998].
The Sexually Dimorphic Brain
Even today, hormonal effects on gender identity are not entirely clear [Imperato-
McGinley et aI, 1979; Migeon et aI, 1998; Slijper et aI, 1998]. Although testosterone has
definite effects on gender identity and sexual dimorphism of the brain, the roles of
dihydrotestosterone, for example, or Mullerian inhibiting substance are unclear [Reiner,
1999]. Additionally, inadequate or late gestational secretion of testosterone may lead to
inadequate or incomplete somatic sexual dimorphism and unknown effects on the
masculinization and sexual dimorphism of the brain [Reinger, 1996; Imperato-McGinley
et aI, 1979; Reiner, 1999]. Estrogen may have late gestational effects on the female brain
as well [Fitch et aI, 1998].
New tools should prove useful in the assignment-of-sex issue. McFadden has
evaluated sexual dimorphism in the auditory system that appears to be reproducible
across the life span, including at birth. He has found that click-evoked otoacoustic
emissions (CEOAEs) are sexually dimorphic [McFadden, 1998]. These are echo-like
waveforms emitted by normal-hearing cochleas in response to a brief transient click
sound. CEOAEs have been found to be differentially stronger in females than in males.
Prenatal androgen exposure appears to induce the CEOAEs of males just as it appears to
induce male-typical behaviors; the degree of masculinization of the brain and of CEOAEs
appear to be directly related [McFadden, 1998]. The technology is easily performed at the
bedside. If CEOAEs prove to correlate with the degree of masculinization of the brain,
then such a simple and inexpensive tool may be invaluable indeed for sex assignment in
newborns with ambiguous genitalia. A moderate lOr strong male CEOAE pattern, at the
very least, might assist in neonatal assignment of male gender for future psychosexual
and psychosocial realities regardless of genital appearance or function.
Sex Assignment in the Neonate with Ambiguous Genitalia: A

Reappraisal
Ultimately, appropriate investigations of the neonate with ambiguous genitalia will

reveal genetic sex, steroid metabolism, internal ductal anatomy, gonadal histology, and
hopefully the degree of masculinization of the brain. Rational decision-making for sex of
rearing must be based on an integration of all of these data. The goal would be that the
decision will have the greatest chance of matching the child's ultimate sense of self.
Early reconstructive surgery may not be critical - there are no data characterizing the
psychological impact of early versus later surgery on the parents or on the child.
Prediction, assessment, and measurement of virilization, of male-typical versus
female-typical behaviors and attitudes, and ultimately of gender identity will determine
the future paradigm or paradigms for sex assignment in these children. In the meantime,
answers to the questions of what we know and how we know it are neither obvious nor
facile. To predict the psychosocial and psychosexual prognosis for a child with a given
diagnosis it will be necessary for us to understand how children with that diagnosis
develop, respond, and perceive their identity longitudinally as opposed to cross-
sectionally. It is increasingly clear that children can come to recognize their gender
identity when it differs from their newborn-assigned identity without major psychological
collapse-although supportive, educational, cognitive, and sometimes
psychopharmacological interventions may be critical.
Research methodology for assessing sexual dimorphism continues to be refined.
Newer techniques may help correlate research findings and gender identity outcomes,
outcomes consistent with what a child ultimately perceives [Reiner, 1996, 1999 & 1997;
McFadden, 1998; Berenbaum, 1999; Hines, 1994; Berenbaum, 1997; Meyer-Bahlburg et
ai, 1994 ]. Avoidance of gender confusion at any age should be the goal. In the
meantime clinical and research data are limited but must be utilized to design present
guidelines for decision-making.
General Sex Assignment Guidelines
General guidelines must reflect the myriad of clinical diagnoses and situations, social
demands, and research data. Deficient information requires nonspecific guidelines.
184 W.G.REINER
Ethical demands preclude specific recommendations when the outcome is unclear

regardless of the diagnosis. Diagnostic and therapeutic options must be carefully
delineated for the parents. Dogmatic proclamations or decisions are probably unwise and
imprudent. Guidelines will become more specific as clinical research outcome data
accrues [Shaneyfelt et aI, 1999]. Guidelines should be evidentiary in form [Cook et aI,
1999].
It is with these considerations that general guidelines can be provided for the
assignment of gender in neonates with anomalous genitalia or prenatal development. The
spirit of the guidelines must recognize the obvious and the abstract ambiguity of the
child's (and thus the parents') situation. The treatment team must accept this ambiguity-
because it is the reality. Flexibility in longitudinal approaches to these children and
limited surgical reconstructions until gender identity is clear are the rubrics of these
guidelines. Rather than emphasizing good reproductive function, sexual function, or
normal looking genitalia [Bradley et aI, 1998; Glassberg, 1998], present guidelines
should be predicated upon clinical outcome. The prognosis at present can be determined
only in retrospect-by the patient. Thus, without the luxury of prognosis the treatment
team must be wary of irreversible surgical or medical interventions early in treatment.
Gender identity will be assigned. Society and family demand it. Arguments about the
timing of surgery will continue. Yet interest in genital cosmetic appearance, as an
example, begs the question-cosmetic for whom? Abnormal psychosexual development
in these children leads to a nearly ubiquitous anxiety that generally precludes their own
genital self exploration and often even genital awareness, let alone undressing in front of
others, regardless of what surgery is performed or how the genitalia appear to the parents
or surgeon [Reiner, 1996, 1999 & 1997; & Reiner]. The child will desire good genitalia,
cosmetically and functionally-but genitalia in line with hislher recognized gender
identity.
We must ignore our need to feel that we can obviate emotional trauma with medical or
surgical approaches. Malformations in a newborn virtually always create psychological
vulnerabilities in the parents. It is specifically psychological interventions that are
necessary for the parents and cannot be ignored. Such interventions throughout the
child's development can foster "the best fit" psychosexual and psychosocial directions as
well. It is this flexibility, with regular surgical, endocrinological, pediatric, and
psychiatric assessments, that can provide the child and family with the best potential
clinical outcome. Surgical reconstruction will simply most often occur when the child is
older [Reiner, 1999; Wilson et ai, 1998].
No algorithm for sex assignment will at present be entirely satisfactory. Given that the
child's sense of gender identity-the most important data-may not be available until ages
6 or 8 years, guidelines, carefully delineated and discussed with the parents, can assist the
treatment team to arrive with the parents at the most likely gender outcome based on
available data.
General Guidelines
The parents must comprehend the ambiguity-physical ambiguity first and, gradually,
the larger sense of clinical ambiguity. The clinical investigation will occupy their initial
time. They should be advised that a sex assignment decision will be based upon the
appropriate data. The child psychiatrist can initiate an assessment of the parents and
appropriate interventions for them. Interventions will be supportive, educational, and
cognitive.
Androgen imprinting implies a likelihood that the quantity, timing, and duration of
androgen exposure will all playa role in determining the degree of masculinization of the
brain. Such variables may also be pivotal for external virilization. Therefore, traditional
questions of potential sexual function, cosmetic appearance of reconstructed genitalia, or
potential fertility may be subsumed by the intensity of any androgen exposure in the
brain. Aspects of virilization- including, for example, CEOAEs-could be markers for
masculine gender identity. Neonatal androgen levels and perhaps even MIS levels may
be useful. All data should be recorded and reported in any published case series.
Once urgent medical management issues are corrected, the treatment team should
educate parents about normal genital variations, frequency of variations and anomalies,
and possible implications of the particular anomalies involved. Although the clinical
situation is ambiguous, it is likely that the child will ultimately recognize a specific sense
of gender identity. Thus, flexibility in the treatment team encourages flexibility in the
parents. Early supportive approaches must be consummated with long-term
psychological, medical, and surgical support. The parents will require interventions and
support for their sense of loss, grief, or guilt. They will need to be intimately involved in
decision-making but with appropriate guidance based on data. Referral to a support
group may be helpful.
Gender identity will be assigned. The parents must recognize the importance of
flexibility for the future. They will recognize that surgical reconstruction will be
indicated, but most likely in the future. They can understand the need for medical
interventions now (in CAR, for example) or for surgical interventions (malignant gonadal
potential, for example).
Truly comprehending that the child is very likely to sense or "know" a specific gender
identity, the parents can be taught effective child rearing approaches to gender and sexual
subjects that can decrease the possibility of future gender confusion and offer their child a
sense of participating in hislher medical treatment. In an open, ethical society, the
treatment team should advise open communication between parent and child and
treatment team and child regarding the child's condition. Within the framework of the
child's overall psychosocial and psychosexual development, the child's questions should
be answered simply and honestly. Assistance can always be found with the appropriate
member(s) of the treatment team for any given area of questions. Experience with
complex gender identity issues in children with a myriad of diagnoses has shown that
gender assessments may be fairly accurate after the age of five and sometimes even
younger [Diamond et aI, 1997; Reiner, 1996, 1999 & 1997; Wilson et aI, 1998]. Indeed,
children can often define their gender as young as six or seven years of age [Reiner,
1999; Wilson et aI, 1998], well before the development of abstract reasoning. Children
186 W. G. REINER
seem to tolerate their own gender recognition better than their parents-yet both will need
continuing psychological connection and intervention.
Specific Guidelines
This transitional approach to neonates with ambiguous genitalia can be capsulized as

follows:
Children who have significant or moderate virilization of the external genitalia:
regardless of the genotype, serious consideration should be given to a sex assignment of
male.
46, XY or 46, XY/4S, XO mosaic genotype-Gender identity will most likely be male.
Often these 46, XY children demonstrate male gender identity by ages three or four and
can be surgically reconstructed at a fairly young age.
46, XX genotype (CAH)-In 46, XX females with CAH and significant androgen
exposure the ovaries can be ignored well into the prepubertal years while careful
assessments slowly delineate the unfolding of the child's gender identity. The parents
must be educated that their child's behaviors will be more male-typical than female-
typical. Female gender identity may be associated with homosexual or bisexual
behaviors. Pregnancy after adolescence may be avoided by the patient. Children with
mild virilization of the external genitalia: the genotype may be only a fair predictor of
future gender identity.
46, XY or 46, XY/4S, XO mosaic genotype-The testes have most likely failed to
develop properly. If serum androgen levels at birth are very low or if MIS is low or
absent, female sex assignment must be seriously considered. However, other than for
prevention of gonadal malignant degeneration surgical reconstruction should await the
child's determination of gender identity.
Patients with micropenis and scrotal fusion generally will have male gender identity.
46, XX genotype-Simple virilizing CAH, for example, probably leads to female
gender identity, sometimes perhaps a mixed picture. Fertility issues may therefore be
very important. With low neonatal serum androgen levels and absent MIS, gender
identity will most likely be female. Female gender identity may be associated with
homosexual or bisexual behaviors sometimes. Appropriate surgical reconstruction based
on a child=s gender identity should be performed prior to the onset of puberty.
Children with female external genitalia or clitoral hypertrophy only-the gender
identity is very likely to be female. Any requisite vaginal reconstruction should be
conducted at an appropriate age for surgical success. Gonads can be treated based on
medical or developmental considerations (malignancy potential, breast development).
Clitoral reconstruction seriously risks partial or complete denervation [see Baskin et aI,
1998].
Androgen responsiveness or activity may vary within some diagnoses, and the
significance of the timing and intensity of exposure may differ for brain and genitalia.
Thus, for example, children with 46, XY genotypes and Sa-reductase deficiency are best
assigned male. The brain is probably already imprinted. Incomplete androgen
insensitivity syndrome children should be assigned according to the above guidelines but
with the caveat that brain imprinting will be very difficult to predict. These children may
have a female gender identity or a male gender identity, and the children may even
virilize further at puberty due to residual receptor or hormonal activity. True
hermaphrodites with their variable chromosomal and phenotypic pictures also fall within
the above guidelines, with gender assignment based on predicted gender identity.
Similarly, sex-chromosome anomaly syndromes-Klinefelter Syndrome and Turner
Syndrome, for example-fall within the guidelines and should be sex assigned male and
female respectively.
Conclusion
The paradigm for assignment of sex in the neonate with ambiguous genitalia is in
transition. Longitudinal outcome data are only now beginning to become available. Yet
the traditional paradigm does not appear to satisfy most of the various clinical dilemmas.
The brain is the most important sexual organ. If the outcome research from longitudinal
studies of children with ambiguous or absent genitalia identifies sex-reassignment as
developmentally perilous, then it will be critical to make the most appropriate gender
assignment depending on the brain-gender identity at birth. Utilizing new avenues of
research based upon genetic errors or new tools to provide strong evidence about the
degree of masculinization of the brain, we should be able to accumulate and inteI]Jret the
data at birth necessary to give us powerful clinical information in justifying sex
assignment. Genetic research tools and other molecular biological innovations have
allowed us to step into the realm beyond mere karyotype and genital appearance in
determining neonatal gender. We must recognize, however, that an understanding of the
development of gender identity may require additional new biological tools or
psychological tools or both. We must not confuse forms of social Darwinism with brain
evolution. Rather, we must attempt to comprehend what evolution has provided and to
track where it can lead us clinically.
Gender identity is a system within a set of systems that we call the mind or brain or
mind-brain. It is the whole that allows us to look at the brain as though it has parts, and
to see those parts as genetic, environment, structural, and perceptual. It is the realization
of gender identity as an actuality that we need to seek, to comprehend through the
individual child as that child contemplates identity through its (his or her) development.
We must avoid manipulations of that child based on our perceptions or biases. We must,
rather, await the outcome of the child's growth and development. This is the real
initiation of the paradigm shift.
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Questions for Dr. Reiner
Question - The concept about flexibility is something that bothers me. There are some
disorders out there, such as mixed gonadal dysgenesis or true hermaphrodism where we
are not sure what gender is best selected, because we do not know what the sexual
imprinting in the brain really is. What do you do in this situation of flexibility when a
child say to you at age 6 or 7, "Well, Mommie am I a boy or a girl? What am I?" How
do I deal with that? The other question is, how does that child's peers deal with the fact
that when they go swimming - do they go swimming - do they go into a male's locker
room or do they go into a female's locker room? I mean, these are real problems that a
child is going to have to deal with and how do we prepare them to deal with those
situations?
Answer - Those are very good questions. The children in my experience, and I have dealt
with - counting the ones by telephone (21) - but counting the ones in person, 18 kids
now, about half of whom have declared themselves the gender other than what they
assigned. They do not ask what their gender is; they state it. They sometimes will toy
with Mom and Dad when they are 6 and 7 and they will just kind of throw it out - kind of
like a little side line or a little side comment, but eventually usually by 8 they just flat out
tell you what their gender is. If Mom and Dad take them off alone and then talk to them
about it, they will often be very open. If Mom and Dad are very rigid about it, they
generally at that point will go underground. If one in my case - I have dealt with both
sets of parents, all kinds of approaches and the kids are very, very open and again, they
190 W.G. REINER
do not ask me what their gender is. I never get a question from the kids. It is always a
statement. They tell me what their gender is and they tell me what they think they are.
They tell me what they are going to grow up to be and that sort of thing.
In terms of the other issues, what do you do with the peers and all - again, privacy and
confidentiality is extremely important. I am not talking about secrecy, but privacy. And
what you find in these kids is that they do not go swimming with other kids. They do not
go into the locker room rather with other kids. They do not take their clothes off in front
of anybody else, including their own family, most of the time. You have assigned them a
sex, so they are going to go into the area - if they have to go to the bathroom they are
going to go into the area or the bathroom that is based on their sex. But if at age 7 or 8 or
9 they change their sex, or they tell you that they are really the other sex, than obviously
things are going to have to change. They are not a risk to anybody and as long, again, as
if they are dealing with the identity issues with their child psychiatrist and with their
surgeon and with their pediatrician and especially with Mom and Dad, presumably
nobody else is going to know. So that the issues in terms of peers and other people, are
only issues if Mom and Dad tell people something.
Response - You have a child who is identified as a true hermaphrodite or mixed gonadal
dysgenesis which is the real problem problems, at least in my experience, too, as they go
through - because some of them - and we have made wrong decisions at an early age and
that is obvious when they go through puberty. What do you tell the parents? Do you tell
the parents well we are not sure whether this is going to be a boy or girl and we need to
counsel you and counsel the child as they are growing up and then after puberty or as
they get older, they will declare what they want to be? How do you deal with that in a
newborn nursery, when they want to know whether they have a boy or girl?
Answer - Of course a child who is presenting a complex clinical problem, and obviously
you have to go through many, many different aspects of that problem and clearly it is not
going to be done all at one time or in most cases it won't, it will take 2 or 3 sittings
simply because the parents will not be able to absorb fast enough. But what I would do is
I would say that we have a problem. We are not exactly sure what the gender of our child
is, the way you would do with any child who is born. We have to do some testing. You
do the tests that you are going to do and you come up with your best guess, if you will, I
mean for lack of a better term right now I am going to use that. And that best guess is
based on what really would appear is the likeliest outcome of that child's psycho-sexual
outcome of that child's brain. I am not at all convinced by the way, as many people have
commented, that gender is something that evolves over time. It may or may not, but
there is no data to show that, or the data is extremely biased and poor. It may well be that
gender identity is relatively solidly imprinted at birth or after the 3 months of postnatal
androgens or what. We don't really know. But what we can say is that we also do not
really know exactly what that child is going to perceive itself as, at age 6 or at age 12 and
what we can say is our best guess and it is based on all of this data and we will give all
the parents the data. We think this child is going to be a girl - is going to identify as a
girl and that is what we would advise. But because there is a question, we need to wait
until this child is old enough that the child can tell us exactly how they perceive
themselves. As it turns out, the children handle this situation very, very well. They
really have remarkably little trouble, except for the first 2 or 3 months after they have
come to the point of saying, "I am a", whatever - the other/opposite sex of what I was
listed as. They do have some problems that need some care. They usually get a
tremendous sense of relief and happiness for the first week or two after they have
declared - sometimes for the first month and then they have a period of 2 or 3 months
where they have a great deal of difficulty and then they seem to come out of that. During
which time, by the way, they need to be evaluated because they very commonly have a
depressed episode. And then they come out of that and then they handle it well. The
parents who are relatively flexible and open to begin with also handle it well. It is the
parents who have been brought into the situation that this is the way the child is going to
be - we have to raise the child this way and have a very rigid view, those parents do not
handle it well at all and those can be psycho-social disasters. So I think the flexibility,
the importance of expressing the flexibility is to the parents.
Question - Have you had experience with kids in the newborn nursery that you followed
along because you are not sure what they are going to be and the parents have cooperated
with you?
Response - Again it is a lot of personality involved in this and you have to deal with the
parent's personality. In general, adults have a lot of coping mechanisms and kids do not,
by the way. So that in general, if you provide adequate rationale and reasoning of what
the possibilities are and why we need to be flexible, Mom and Dad can, most of the time,
live with that ambiguity. That is not as difficult as living with the rigidity and being
terrified that the child is going to find out there is a lie, which is very hard on them.
Question - Excellent talk. I just had a quick question about the cloacal exstrophy
patients. Did the patients expressing gender identity disorder undergo delayed
orchiectomy?
Answer - No, they were all castrated within 5 days of life. Most of them by 3 days.
Response - Does that suggest to you that a lot of this imprinting is going on prenatally
then?
Answer - Well yes and let me even say something further. The 6 year old who had a
pelvic field defect, but no cloacal exstrophy, he is the only one who fell in the male
range. He was castrated at age 6 and he fell in the male range on the gender assessment
schedules. The cloacal exstrophy kids who were castrated by 5 days of age fall very
close to the male range, but not in the male range.
Question - I think it is important when we are talking about intersex to remember that
the cloacal exstrophy population, are really not intersex per say. They are actually true
males,46xy's. They have an anatomical defect to their lower urinary tract and genitalia.
So it is important to not confuse the cloacal exstrophy population with the true
hermaphrodites or cases of mixed gonadal dysgenesis when we present this data.
192 W. G. REINER
Response - That is absolutely correct. These are not Intersex kids except in so far as they
loose their postnatal androgen. Even the true intersex patients have a myriad of
diagnoses even with any given diagnosis. For example, congenital adrenal hyperplasia is
not one diagnosis either, and the degree of virilization may be very different and
therefore the degree of masculinization of the brain may be very different. It may be hard
to predict or maybe it is not hard to predict. We don't know because nobody has sat
down and explored that. So what I am really advocating is that we need to understand
this. The trial I am running right now is considering what happens when the brain has
achieved most of it's androgen exposure and masculinization, and what happens to
subsequent behavior. But what I am also interested in is what happens in each of the
various diagnostic categories. So that we need to have long-term outcome studies done
where you take in all ages. In other words, you do not just start at birth, but you take in
every person with that diagnostic category and follow them longitudinally and come up
with an answer for each category. So that the paradigm - there is never going to be an
algorithm and I think one of the nice things about the original traditional paradigm was
that it provided almost an algorithmic approach that you just take the child and based on
A, B & C, here is what you do. And it was very nice and very handy because it covered
this huge myriad of diagnostic problem cases, problem kids. The catch is it doesn't really
work very well, so if it probably is never going to be one algorithm, there is going to need
to be a number of algorithms, based on what the real diagnosis is.
Question - Why not consider some of the children who have been allowed to be reared in
the sex of choice by the parents? I've got 4 patients in my practice who have reached
adulthood, who have had that opportunity and if you want to see anger in children and in
families, consider these situations where the parents were allowed to make the decision.
We have 2 older gentlemen with 46XX congenital adrenal hyperplasia who were raised
as males and they are both inadequate males, each has attempted suicide, yet both have
marvelous phalluses. But the bottom line, these are angry human beings who have never
functioned in their assigned roles. We have two patients with mixed gonadal dysgenesis,
who were allowed to go through life and make their decision later on and now that we
have reconstructed them as males, both of them have been in psychiatric care. There
have been experiences on the other side, too, to allow children who have reached young
adulthood or adulthood to go through an unbiased situation and they do not do well
either. So any way you look at it, there is going to be problems and it gets us back to the
original argument, what do you do at birth and when you offer patients the choice, which
many of us have been doing since the John/Joan thing came out, 100% of families, at
least in our experience, continue to make the decision to have a sex of rearing at that
time, that goes along with surgical reconstruction.
Response - You are bringing up anecdotal cases, again and what I am trying to say is that
what we need to do is have longitudinal outcome research and starting with every age
that is available in any given diagnostic category. I do not know what the answers are to
your 4 people and why they are that angry is interesting to me, too because I would say
the one thing about the kids I deal with is not angry. They are dealt with closely. They
do have a very good relationship with Dr. Gearhart and his team. If somebody is that
angry and that angry for that length of time, I would say they need some help. Something
is wrong someplace. And it may not be with their gender or with their assignment of
gender. It may be many, many other aspects of their life so something is going on there
far greater than just what their anatomy and physiology is dealing with. But regardless,
what I am saying is that we do not know the answers to these questions so that the
anecdotes are only helpful in so far as they give us some ideas on how to set up studies.
Once we really sit down and look, we need to set up the studies based on the kinds of
patients that are available and look at these patients longitudinally and have a protocol
that is going to give us some answers at the end that are useful answers. Hopefully, not
even at the end maybe even part way through.
Question - John Money and the Pediatric Endocrine group really had formulated their
policy based on a very large series of intersex youngsters in the 1950's and Money's first
work was published in 1955. My question to you is could you say more specifically what
is your current thinking in terms of how it is different from the traditional Hopkins policy
in terms of management? Where do you depart company from the earlier policies?
Answer - In terms of how does my paradigm vary from the paradigm that Money
formulated; I think what I was trying to imply was that I am not sure I have a paradigm
yet. The one thing that I think and I do not think John Money would argue this point, is
that the psycho-social, psycho-sexual and psychiatric aspects of these conditions are very,
very important and need to be looked at, followed and evaluated
Question - First of all, I enjoyed your lecture. I think while we would totally agree that
we have a problem ... the surgeons have a problem in making major surgical
recommendations based on the anatomical aspect and the surgical reconstruction point of
view with little consideration of how much sexual imprinting to the brain and the
psychological aspect, I mean in later life, would affect the child's future. But I think it
would be just as difficult for us to justify or to say that it is ethical or even practical for
the clinicians to tell the parents of a child with intersex problem that we do not really
know what is going on and let's wait until 15-16 years later and let the child determine
their own future. I do not think that is practical. Also, I think we need to set the record
straight. The John/Joan Case which has been repeatedly quoted, as well as the cloacal
exstrophy patients as already been pointed out very correctly, are not strictly speaking
intersex patients. I would like to share from my experience back in Hong Kong, China.
For some unknown reasons we have got a large population of patients with severe
hypospadias and a tiny penis. These boys with a hopelessly small micropenis with severe
hypospadias are, assigned a male gender because in Chinese culture, the male gender
always has been given some superiority. So I have a lot of problems in counseling the
parents for gender reassignment. And in fact, at least I can quote you two cases falling
into this category which I submitted to parental pressures and reared them as males and
then proceed with penile reconstructions. Both children developed severe urethral
strictures and then their early childhood was made miserable. In one of these cases, the
parents initially actually threatened to kill the child, if we were to raise the child as a girl.
They subsequently, came back to us and requested that the child be reassigned at the age
of 6 or 7 to a girl. The other child again has been brought up as a boy, and now at age 7
194 W.G. REINER
has himself indicated that he should be a girl. Now of course these are all anecdotal
cases.
I would like to ask, how much evidence or data have we got about patients with partial
androgen insensitivity which fall into the category I described, with severe hypospadias
and micropenis?
Response - There is more anecdotal evidence and every time somebody presents
anecdotes there is a limited amount of evidence that is presented and I am sure we all
present our anecdotes in our own way. We have our biases and we have to deal with our
biases. Clearly the cultural biases in Hong Kong are going to be very different than the
cultural biases in the United States which may be different still from other nations.
Clearly in the American society going into the 21 st Century individualism is held in an
extraordinarily high esteem not just socially, but legally. And the legal aspects are not a
minor aspect of all of this. So that the whole aspect of getting a truly informed consent
and getting a truly informed assent as Dr. McCullough was talking about, I think is
critical. And to argue that we do not have enough data to do that is absurd. We do not
have enough data period. That is my point. We do not have enough data to be doing
what we are doing. And yet we are doing it. So what I am saying is that we are - we
have protocols that we use now as surgeons and we have very little data to argue for. So
that to say that it is unethical to change what we are doing is sort of begging the question.
Is it ethical to continue doing what we are doing. I think what is ethical is setting up
longitudinal studies and examining these questions diagnosis by diagnosis. And what I
am hoping to be able to do with the cloacal exstrophy population is to be able to say here
are the psycho-social, the biopsycho-social outcomes of children who are 46XY
hormonally normal until the time of birth when they are castrated. Here are the
biopsycho-social outcomes; therefore this implies the following. That is going to be very
important because this is a hormonally almost normal group of people and they are a very
homogeneous population in terms of their hormonal imprinting. Then we can then look
and compare that group to a typical group of no surgical problems - no ambiguity and
compare that group to the salt wasting CAH kids - compare that group to the partial
androgen insensitivities and so on. So you have to set up the studies in order to answer
the very questions that you are asking and you what I am saying is that we already start
with the whole idea - my whole point is that we start with a prognosis and haven't got the
diagnosis. We have it backwards. So what we need to do is get the diagnoses, which
means the biopsycho-social outcome. So we need to do the outcome research in order to
be able to answer the very questions you are asking. I think in this society I do not think
it is difficult at all. I think that if people think that it is going to be hard to leave the
parents with ambiguity - I am not leaving the parents with a question mark on top of their
heads, you leave the parents with a plan. There is a definite plan. You still have a
protocol, but the protocol does not involve decision making or surgical approaches that
are going to be regretted by the patient and subsequently by the parents at a later date.
Question - I enjoyed your talk and specifically your plea for continued input from a Child
Psychiatrist and I wanted to bring up an issue that has not been addressed so far in this
meeting. Weare now having to fight managed care organizations and governmental
agencies on our patient's behalves. I remember vividly a patient who had ended up had
undergone a bilateral orchiectomy and came to me as an adolescent wanting testicular
prostheses and try as I might, the State Medicaid system would not approve this
procedure because it was "cosmetic". Perhaps one of the things that maybe should come
out of this meeting is that we recognize that we have an individual and perhaps a
collective obligation to lobby, not only state, but federal agencies and managed care
organizations to delineate what types of care these complex patients are going to need on
an ongoing basis and to develop some position papers so that we can show that this is
what really should be the standard of care.
Reponse - I agree.
Question - In Dr. Money's work conforming genitalia was obviously a very critical
aspect in the newborn period in order for gender identity to be established and we are
negating that now with this new philosophy. I would like you to answer a specific
question - when you counsel patients, do you talk to them about surgery if you do not
believe that that is true? And if they want surgery can the parents then give consent for
their child to have such in a medical-legal sense?
Response - I counsel the patients about everything. I try to present all the data and
present a brief history of what has happened and why it has happened. And I try to help
the parents along with the surgeon endocrinologist come up with the best guess about
what the likeliest outcome is going to be from a gender perception standpoint - from the
child's gender perception - assign that gender. I make no decisions and I do not advise
the parents to make decision. They make the decisions. I just give them data, they ask
questions and I answer them. If they ask questions about what I would do if it were my
child I'd tell them that is not an appropriate question for this particular circumstance,
because I do not know. I cannot put myself in that position.
Questioner - So then you give them no help in making a decision?
Bill Reiner - Well, I give them all the help I possibly can. I am available to them all of
the time. I am available to them in person, by phone, by email. They reflect off of me.
They talk to Dr. Gearhart. They'll talk to Dr. Migeon They will talk to whomever - they
will reflect off of me again and then they end up making the decision.
Questioner - So they make the decision then to have the gonads removed in the genitalia
conformed - what do you do then? If you do not think there is an answer to the question?
Response - By that time I have already told them what I think will happen. Or what
likeliest thing is that will happen. But is it medically-legally wrong for us to do that? I
don't know the answer to that question. I think there is no answer to that question.
Question - What were the instruments - what were the measures that people have used
for outcome? And a lot of the studies that I have read have consisted of interviews and
discussions and not necessarily standardized measures. I think anecdotal information that
196 W. G. REINER
people give is very helpful and I think what would be really useful would be designing
mixed method kind of studies that use standardized measures to look at the various things
that you want to get outcomes on, but also to use a real good qualitative approach as well
with a semi-structured interview that would really then be able to analyze the data and
you would still be able to get a lot of the information that one gets out of the kind of
anecdotal information.
Just a couple other things that I would like to say - the one thing I take issue with or have
some difficulty with as well is - and you mentioned yesterday about the anxiety disorder
and some pretty grim sounding outcomes for a lot of patients, which isn't necessarily the
experience that you read about or that I have necessarily had with the clinical population
I work with. And I really think people need to take a look at the resiliency literature that
is out there. I think kids do have marvelous coping mechanisms. They can be taught, as
can parents. And I think it goes again to the need to develop really good
multidisciplinary teams that cover a lot of areas, including the psychiatrist, the
psychologist, child life. You know you mentioned that anxiety was centered around a lot
of procedures. Most hospitals have a really good child life program. I think you really
need to look at what the resources are that you have to be able to pull them in and train
them so that they can work with this population.
Response - In terms of standardized testing I will just say that Dr. Meyer-Bahlberg is
going to refer to some of that in the next talk. But the testing that I do is relatively
standardized. The psycho-sexual history I do is drawn from the testing that Meyer-
Bahlberg has written out for adults. It is not really appropriate for kids but I have
modified it and covered basically everything. I also evaluate anxiety and all the anxiety
disorders, mood disorders, ADHD and so on, whatever you are going to be looking for. I
do use a semi-structured psychiatric interview that takes about 2- Y:z hours on an average
for each patient depending on the age of the patient. Longer for the younger patients
which is a fairly semi-structured and somewhat open-ended. And I also use open-ended
written materials for the parents to write down their perceptions, their feelings about the
interview, and so on. It takes me about 8-9 hours to fully evaluate a child. It is a pretty
broad array, and the instruments that I use are written up in the literature.
Question - Weare in the process of completing two studies. One involves five
individuals in their 30's. These are XY women, 3 with partial androgen sensitivity, I
with Kalman's syndrome, 1 with testicular dysgenesis, who have all lived as women, and
were assigned at birth as females. There is no gender dysphoria in those five individuals.
Four out of the five individuals are in long-term relationships. The sexual orientation of
one individual is homosexual.
The other study we were trying to extend the work of Drs Schober Woodhouse in
collecting a group of patients with small penis, including micropenis with and without
hypospadias and the outcome on that looks pretty good. We have also been able to
interview separately, again using structured instruments, the partners of these individuals
to confirm what they were telling us about their sexual lives and that looks pretty good.
Hopefully we will be able to get that in print shortly.
If I understood you correctly, in some of these young children declared to you that they
changed their sex differently than what they have been assigned and then you helped
them with that change. Is that correct?
Response - I have not affirmed any of those changes because in order to do that I would
have to - I think ethically deal with the parents in those counts in the first place. Now
once the parents affirm it, then I am quite willing to affirm it with the children. But no, I
do not affirm their changes. I accept them. I listen to them.
Questioner - You are just listening to what they are saying at this point?
Answer - Yes. I amjust collecting data and when the entire study is over I then go over-
you know if the child tells me that their sex is wrong I certainly discuss all that with the
parents.
Comment - I just have a comment about the lack of effect of the neonatal testosterone
surge. In analogous it is really not that surprising that the male patients with cloacal
exstrophy and those with aphalia have a male gender identity because an analogous
situation would be a boy who is born with anorchia where there is testicular torsion
bilaterally and utero. And those patients just, at least in my experience, are clearly male
in identity and their, of course, in their chromosomes, their sex chromosomes and their
hormonal status is essentially identical to these aforementioned conditions.
GENDER ASSIGNMENT AND REASSIGNMENT IN
INTERSEXUALITY: CONTROVERSIES, DATA, AND
GUIDELINES FOR RESEARCH
Heino F. L. Meyer-Bahlburg
New York State Psychiatric Institute, and Department of Psychiatry, Columbia
University, New York, NY
INTRODUCTION
In recent years, gender has become a hotly debated issue in regard to two clinical
syndrome categories: Intersexuality, i.e., individuals with ambiguities of the genitalia,
and Gender Identity Disorder (GID), i.e., individuals who have normal genitalia but
desire gender change. The intersex controversy focuses on the assignment of gender and
related issues of psychosocial and medical manag(:ment. The GID debate centers on the
question whether GID should be considered a mental disorder or be removed from the
Diagnostic and Statistical Manual (DSM) of the American Psychiatric Association (AP A,
DSM-IV, 1994) and declared a normal variant in analogy to the 1973 decision of the
AP A on homosexuality (Bayer, 1987». The GID debate extends to intersexuality,
because if intersex patients have significant gender identity problems, DSM-IV classifies
them as GID Not Otherwise Specified (GIDNOS) which implies a mental-disorder status.
In the current paper, however, we will leave the GID part of the debate aside and
concentrate on the question of gender assignment and related issues in intersexuality.
Intersexuality is usually defined endocrinologic ally and includes prenatal hypo-
androgenization in 46,XY males or prenatal hyper-androgenization in 46,XX females.
However, there are a number of conditions where anomalies of the genitalia are not
caused by endocrine disorders. This applies particularly to 46,XY individuals with penile
agenesis, cloacal exstrophy of the bladder, and traumatic loss of the penis (ablatio penis)
in infancy. When such cases are assigned to the female gender, gonadectomized, and later
treated with estrogens, the management considerations are very similar to those in
classical intersexuality. In the context of this chapter, I will therefore expand the term
'intersex' to cover both endocrine and non-endocrine categories of genital ambiguity.
Past and Present Policies of Gender Assignment
In regard to gender assignment and re-assignm(:nt of intersex patients, we distinguish

two major policies (Meyer-Bahlburg, 1998). The traditional one has been to determine
the true sex (since the 19th century usually understood as the true biological sex), to
assign gender accordingly, and to expect that everything else will fall in line, more or
less, with societal expectations-everything else meaning gender identity, gender role

200 H.FL MEYER-BAHLBlJRG
behavior, sexual orientation, courtship and love, sexual functioning, and psychological
health in general. The true-sex policy underlies the ubiquitous question at birth: "Is it a
boy or a girl?", implying that the infant is already a gendered person. Yet, what is the
definitive criterion of the "true sex"? Traditionally, it had been the external genitalia, in
the later 19 th and beginning 20 th century the gonadal histology (Dreger, 1998), and later
the sex chromosomes. Each of those criteria can be ambiguous, and all three may be
discrepant from one another.
The true-sex policy led some physicians to assign to the male gender patients with
complete androgen insensitivity (CAIS), because the gonads had a male histology,
although the defect in the androgen receptor permanently prevented the development of
any physical masculinization. It was the policy that drove some intersex patients to
suicide after their marriage plans were legally blocked because they had the wrong
gonads. (It also was the policy that led some clinicians to declare Turners' syndrome
girls 'neuters' because they had XO sex chromosomes and were therefore not deemed
eligible for marriage.)
The second policy is to determine the optimal gender (Meyer-Bahlburg, 1998; 1993) in
terms of future functioning, i.e., to assign the gender that for a given child carries the
best prognosis: for reproductive function (if attainable at all), sexual function, minimal
medical procedures, an overall gender-appropriate appearance, a stable gender identity,
and a reasonably happy life. The question the optimal-gender policy asks at birth is not:
"~ this a boy or a girl?", but rather: "Will this child have a better chance for a reasonable
life as a male or a female?" Thus, the basis for the gender-assignment decision is what
one can predict in infancy, given the child's particular syndrome and its severity, and
given all that is known about the natural history of the condition and its treatment
options. Under this policy, early surgery of the external genitalia is recommended to
avoid discrepancies between the child's assigned gender and genital appearance and,
thereby, to facilitate consistent sex-typing by the parents and others. With other words,
the rationale for early genital surgery includes gender-confirmation. Additional genital
surgery may be necessary later to facilitate intercourse. After infancy, when gendered
role behavior and identity become apparent, no gender re-assignment decisions are made
without careful evaluation of the child's behavioral development and self-concept.
This is the policy proposed by John Money and the Johns Hopkins school of pediatric
endocrinology in the mid-1950s (e.g., Money et ai, 1955; Money, 1968;1994). It was
formulated after literature reviews and direct evaluation of intersex patients had led to the
conclusion that gender of assignment predicts long-term gender outcome better than the
traditional biological criteria of true sex. Until recently, this policy was widely accepted
in the U.s. and other countries, although there certainly was never a universal consensus.
But even when there is a local consensus to follow the optimal-gender policy, decision
making in the individual case can be difficult, because the prognostic criteria are not
necessarily more definitive than the sex-diagnostic ones.
GENDER ASSIGNMENT AND REASSIGNMENT 201
Critical Perspectives on Current Policies
The Critics
It is this optimal-gender policy, or rather certain aspects of it, that has increasingly
come under attack in the past 5 years. Much of the current critique represents three
perspectives. The biological determinists point to selected cases with 46,XY
intersexuality who were raised as girls but changed gender to male in adolescence or
adulthood. The latter include, for instance, the observations by Imperato-McGinley et al.
(Imperato-McGinley et ai, 1979; Imperato-McGinley, 1999) on 5-alpha-reductase
deficiency (5-alpha-RD), the reports by Diamond (e.g., Diamond, 1997; Diamond et aI,
1997) on scattered cases of other XV-intersex conditions such as the John IJoan case with
a history of traumatic loss of the penis, and Reiner's work on 46,XY patients with cloacal
exstrophy of the bladder or with penile agenesis (Reiner et aI, 1995; Reiner, 1999). Given
the extensive animal literature on the role of the sex hormones in the sexual
differentiation of brain and behavior, it is entirely plausible that such biological
explanations are also invoked for gender change in humans (Hines et aI, 1993).Thus, it is
assumed that the prenatal endocrine milieu does not only determine the sexual
differentiation of the genitalia but also of the brain, and the status of the genitalia at birth
is interpreted as an indicator of the degree of prenatal masculinization of either organ
(except for the syndrome of 5 -RD where a metabolite of testosterone,
dihydrotestosterone, is needed specifically for the masculinization of the external
genitalia, and except for the conditions of non-hormonal genital abnormalities).
One has to keep in mind, of course, that the 1955 concept of the gender-
undifferentiated neonate has long been discarded by John Money himself, after he
began in 1964, together with Anke Ehrhardt and others, to investigate the prenatal
hormone theory in humans. Already their first publications in 1967-1968 (Ehrhardt et aI,
1967; 1968(2); Money et aI, 1972) showed masculinization of gender-role behavior in
girls and women with a history of prenatal androgen excess, and these findings were
confirmed in many subsequent studies (Ehrhardt et aI, 1981; Dittmann et aI, 1990; Zucker
et aI, 1996; Berenbaum, 1999). Thus, to some extent, the attacks of the biological
determinists are historically outdated.
In contrast to the biological determinists, the social constructionists usually come with
a perspective that is decidedly anti-essentialist, i.e., anti-biological. Thus, we have a
curious constellation here: John Money, the non-essentialist of 1955 who saw the
newborn as gender-undifferentiated and emphasized the importance of social factors in
gender development, and then spearheaded the research that demonstrated the influence
of prenatal hormones on human psychosexual differentiation, is now attacked jointly by
biological determinists who vouch for the predominance of biological factors in gender
development, and by social constructionists such as Kessler (Kessler, 1998) who object to
the practice of making the status of the external genitalia the basis for sex assignment
decisions in intersex newborns. However, the basic thrust of the social constructionists'
argument is not the question of the etiology of gender in the specific psychobiological
sense, but a critical examination - often from specific feminist, queer-theory, andlor
202 H.F.L. MEYER-BAHLBURG
trans gender perspectives-of the gender-assignment decisions and its underlying

assumptions and of the related question of decision power (e.g., Dreger, 1998).
In regard to these questions, the social constructionists have common ground with the
intersex activists, i.e., individuals who are living with an intersex condition and have
personally experienced medical management and its consequences. Best known in the
U.S. are the activists from the Intersex Society of North America (ISNA) (Chase, 1998).
ISNA is a peer support group which provides an urgently needed meeting ground for
intersex persons who are often desperately isolated. Moreover, it constitutes a forum
particularly for those who feel wronged by medical management. Beyond these
functions, ISNA has become, under the determined leadership of its founder, Cheryl
Chase, a major public voice and promoter of policy change. The most outspoken
members of ISNA belong to a generation of intersex patients who, when assigned and
raised female, underwent excision of the clitoris ("clitorectomy"), a procedure which
tends to markedly diminish erotic sensitivity and orgasmic capacity. As a consequence,
ISNA's primary focus is genital surgery and its risks to sexual function, and ISNA's
1995 Recommendations (ISNA, 1995) advocate a delay of all elective genital surgery
until the patient herselflhimself can give informed consent. In addition, many from the
now middle-aged generation of intersex patients have experienced marked difficulties in
finding out about their medical condition and gaining access to their medical records.
The Major Questions
As the intersex debate is conducted in many diverse media such as pamphlets,

newsletters, websites, internet lists, videos, newspaper reports, meeting presentations, and
scientific publications, it focuses on three major clusters of issues: gender assignment,
genital surgery, and information management. In regard to gender assignment, the key
questions are:
1. How strong are the hormone effects on brain development; do they really determine
the outcome in terms of gender identity?
2. For which condition and to what extent can penile size at birth be interpreted as an
indicator of prenatal androgenization of the brain?
3. Dependent on the answer to the first two questions: Which XY individuals should be
assigned female, and which XX should be assigned male?
4. Is it appropriate to insist on a two-gender classification, and should our society not
make room for a third gender or for an even more flexible arrangement of genders?
The primary questions concerning genital surgery are:
A. Which outcome criteria should be the critical ones in making surgical
decisions?
B. Should we in all cases of elective genital surgery hold off until the patient
reaches the age of legal consent?
C. If genital surgery is planned, which techniques of clitoral, vaginal and penile
surgery yield the outcome that is seen as most important?
A third major area of controversy has to do with the management of medical

information such as the patient's history of sex assignment and re-assignment, including
the history of genital surgery, the histology of the gonads, or his/her sex-chromosome
complement, with the following main questions:
I. About what, when, and how does one inform the parent, and about what, when, and
how does one educate the intersex child?
2. Are there any grounds to withhold any information from any adult intersex patient, for
instance those with a major psychiatric disorder?
3. And what role can patient and parent support groups assume in information
management?
Essentially, all of these questions are based on adverse outcomes that have been
observed in individual cases. The answers should be formulated for the goal of
minimizing adverse outcomes and be based on systematically researched empirical
evidence. But here we face a major dilemma, namely, a very limited database because of
the relative rarity of patients with an intersex condition. Only long-term follow-up can
definitively demonstrate whether decisions on gender assignment and genital surgery
were successful, or what the range of consequences of variations in information
management are. Ideally, we would have prospective studies following sizable numbers
of intersex individuals from infancy to late adulthood. But given the low incidence of
such children and the diversity of syndromes and conditions, the logistical difficulties in
conducting prospective follow-up studies are daunting. Also, the small number of
investigators working on psychosocial intersex issues has slowed the progress in the
methodology of assessment and other aspects of research design. It is therefore not
surprising that the overall status of long-term outcome research in this area leaves much
to be desired. We do not have the space here to deal in detail with all the questions listed
above, but will focus the discussion on psychosocial aspects that are particularly relevant
to the urologist.
Long-term Outcomes: Goals/Criteria
General Considerations
Psychosocial goals of clinical intersex management that most patients and

professionals can agree on are a good overall quality of life, including a stable and
uncontlicted gender identity, attainment of a level of education commensurate with
intellectual potential, appropriate functioning in work, the capacity for long-term
partnering (with child-rearing if desired), satisfying sexual functioning, a reasonable
social life, and freedom from significant psychopathology. These goals can be
operationalized for the assessment of long-term outcomes. Ideally, the clinical
management would serve to diminish the putative differences in long-term outcome
between intersex patients and non-intersex control groups. The underlying assumption is
that without medical and psychosocial intervention, intersex patients will show increased
rates of impairment in the various outcome domains listed. To date, impairment of

intersex patients who did not undergo a professional intervention is mostly inferred from
clinicians' experiences with individual cases some of which were published over the past
150 years, but we still lack epidemiologically sound studies that could serve as a
scientific framework for the evaluation of clinical management. (Incidentally, strictly
speaking, there is hardly any intersex patient who does not experience some form of an
intervention, because in most cases the social environment responds to the condition at
birth or whenever it is recognized or diagnosed later, and these responses themselves may
foster and/or hinder the development of psychological health.) When the long-term
outcome of a patient is less than desirable, the reasons may lie in biological factors
associated with the condition itself, in secondary biological/medical side effects of the
condition over time, in the spontaneous reactions of the social environment, in the side
effects of the professional interventions, or in an interaction of such factors. Only careful
investigations of patient samples of sufficient size can give us reasonably definitive
answers. One needs to withstand the temptation of attributing the cause of impaired
outcomes to a particular medical intervention or some of the other potential factors on the
basis of mere suspicion.
OukomeCrikriafurGffiH~Su~ery
For the urological surgeon, the psychosocial goals of genital surgery are of particular
interest. As mentioned earlier, the major focus of ISNA's 1995 Recommendations
(ISNA, 1995) is the avoidance of genital surgery unless required for reasons of medical
health. Presumably, no one would argue against genital surgery if it were always
successful, but quite a few intersex patients feel that genital surgery did them more harm
than good, mostly because of impairment or loss of sexual function, in some cases also
because of the loss of the distinctive 'intersex' characteristic (e.g., ISNA, 1998). Thus,
one has to consider a variety of potential goals of genital surgery and their corresponding
outcome criteria, and these have to be operationalized and reported in detail. The current
surgical literature is replete with surgical outcome assessments where investigators label
surgical results "satisfactory" or "excellent" without specifying the outcome criteria, the
rating procedure, or the raters (Kessler, 1998). Such reports do little to advance our
knowledge. Editors of medical journals are in a particularly powerful position to effect
rapid improvements of standards in this area.
Potential goals and outcomes of genital surgery include the following.
1. There are purely medical criteria, e.g., the absence of acute surgical complications or
of marked scarring, or later complications such as the development of vaginal
stenosis, penile fistulas, and clitoral loss, or the attainment of appropriate urinary
function (form, direction, and quality of the urinary stream), etc., but they will not be
further considered here because they are dealt with in greater detail elsewhere in this
volume.
2. The most frequently quoted criterion in the surgical literature is cosmetic: The
external genitalia look normal in size, shape, and location and do not raise any
question of abnormality.
3. Looking normal also means looking typical for the assigned gender which constitutes
the gender-confirming goal of the surgery of the external genitalia. If the genitalia
look markedly gender-!!typical, consistency of rearing in the assigned gender may be
threatened.
4. Also normal urinary function may serve to confirm the assigned gender: the children
can assume the gender-typical position for urination. Otherwise, intersex children
may have to employ gender-atypical urination procedures. For example, in the
John/Joan case (Diamond et aI, 1997), Joan's urinary opening after the feminizing
surgery was still located so high that she had to direct her urinary stream downward
with her fingers. Or such children may experience teasing by peers and, thereby,
become highly sensitized, or may need special bathroom privileges in school which
constitutes another opportunity for stigmatization, etc.
5. From a long-term perspective, one hopes that, during puberty, the external genitalia
can develop in size and shape as is typical of the adolescent's gender. If the genital
appearance in older adolescents and adults is gender-!!typical, the patient may
develop anticipatory anxiety about exposing his/her nude body and what a potential
sex partner's reaction might be, and the anxiety, in tum, may become a barrier to
courtship and sexual involvement.
6. A major outcome, of course, is sexual functioning. It is often interpreted as the
anatomic fit of a penis and vagina, which is what the simplistic 'pole' and 'hole'
statements of the past referred to. There are certainly many examples of patients for
whom a minuscule or non-erectile penis or a stenotic vagina presented a major
impediment in their sexual lives. But the traditional pole/hole consideration
represented a rather crude and incomplete understanding of sexual functioning. It
also disregarded the fact that there is a substantial minority of intersex patients to
whom the heterosexual paradigm does not apply.
7. At least as important as the aspect of anatomical fit, and for many patients more
important, are erotic sensitivity and orgasmic capacity, be it during sex with a partner
or by self-stimulation. Both aspects of sexual function have major implications for
overall sexual satisfaction. (In reviewing the literature, one gets the impression, that,
in the context of the post-industrial Western society, medicine tends to underrate the
importance of erotic pleasure and overrate the importance of reproduction.) Both
male and female genitalia are complex systems relying on an intricate interplay of
muscular, vascular, neural, and biochemical elements. The recognition of the role of
the neurovascular bundle in clitoral functioning has led to significant progress in
clitoral surgery (Schober, 1998), but as a recent report (O'Connell et aI, 1998) has
shown, even the anatomic features of the clitoris are not yet fully understood. There
is now a consensus that clitorectomy is likely to variably reduce or even eliminate
erotic sensitivity and orgasmic capacity (Meyer-Bahlburg, 1999). But the data
available on erotic function after clitoral resection and clitoral recession are quite
unsatisfactory, and recent reports (e.g., Alizai et aI, 1999) raise doubts as to how
universal the frequently claimed successes of these procedures really are, particularly
from a long-term perspective.
206 H.F.L. MEYER-BAHLBlJRG
8. In question is not only the patient's own erotic sensItlvity. The appearance and
function of the patient's genitals may also have implications for the erotic
functioning of the sexual partner, and the couple's sexual and emotional relationship
overall. For instance, when working clinically with heterosexual couples including
intersex patients and their partners, one hears that quite a few women find the intra-
vaginal sensation of a full-sized erect penis and of its pre-ejaculatory volume
increase arousing, and some may need the experience of deep thrusting for obtaining
orgasm. Many men consider peno-vaginal intercourse the only 'real sex' which can
become problematic when the woman partner has vaginal stenosis. It is likely that
sexual problems of intersex patients can be somewhat ameliorated by sex
counseling/therapy, but to what extent is not known.
9. Last, but not least, there is the outcome criterion of reproductive potential. For
instance, considerations of reproductive potential playa major role in the gender
assignment of infants with true hermaphroditism as practiced in France (Fekete,
1999), while the presence of functional internal female reproductive structures and
their preservation along with the preservation of fertility has always been a major
argument for the assignment of 46,XX patients with CAH to the female gender, even
in the presence of male-like external genitalia.
Overall, we need substantial studies on the quality of life - also to include the quality
of sexual life - for a full evaluation of all aspects of outcome of genital surgery. We need
to learn from the intersex patients themselves what they see as their priority goals for
genital surgery. We also may need to take into account that patients vary considerably in
the importance they themselves attribute to various outcomes.
Who Makes Management Decisions?
If we reconsider and diversify the goals of genital surgery, we also have to revisit the
question of who makes the decision on genital surgery. If the outcomes of genital surgery
essentially belong to the domain "quality of life," the patient ought to be the one to weigh
the beneficial outcomes against the risks. But in the intersex situation, the patient is
usually a child. ISNA (ISNA, 1995) recommends, therefore, to wait with all elective
surgery until the child is old enough to give informed consent. But can one wait that
long? Those aspects of genital surgery that serve to confirm the assigned gender start
having their impact, if any, much earlier already. Also, a child may experience
significant teasing by the peer group because of urination problems, or an adolescent may
develop marked anxieties and related romantic and sexual inhibitions because of genital
inadequacies. And how is a sexually inexperienced young adult going to weigh the
benefits of sexual pleasure? In this context, it is important to note that in a recent written
survey of ours on adult 46,XY patients (Meyer-Bahlburg et ai, 1999) the majority of
respondents did not agree that genital surgery should be postponed to adulthood, when
the individual can legally provide fully informed consent. More survey data of this
kind-including data from parents-will be needed for a comprehensive reconsideration
of such policy issues.
ISNA (lSNA, 1995) demands that, instead of elective genital surgery, counseling by
professionals experienced in psychosocial intersex issues be provided to all patients to
facilitate gender-consistent rearing. On the basis of my clinical experience, I certainly
consider counseling a very important part of psychosocial management, although a
systematic documentation of its effectiveness in the intersex area has not been attempted,
not only because of logistical problems but also because of the ethical problems the
establishment of a non-counseled control group would pose. I doubt, however, and we
certainly do not have the data to demonstrate, that preventive counseling can sufficiently
take care of all genital-status-related problems and make elective genital surgery in
childhood totally obsolete. Also, it is quite unlikely that the needed counseling services
will be available in the foreseeable future, given the current HMO climate in the
provision of medical services.
Thus, I expect that for the foreseeable future considerable decision-making on genital
surgery will remain with the parents, in consultation with their child's physicians, during
the intersex child's early years. If the parents are to give truly informed consent,
however, the full range of outcomes needs to be discussed with them, and the information
on outcomes should be based on data, or at least the parents ought to know what is data
based, and what represents opinion. Here again, we run up against the difficulty that the
database is so limited.
Long-term Outcomes: Data on Gender and Sexual Functioning
What is known about long-term outcomes of intersex patients? What we have for most
syndromes, is data from individual case reports or from small series of cases that vary
tremendously in what is investigated and how it is assessed. Only for classical CAH do
we have several studies from independent investigators who evaluated modest-sized
clinical samples of adult patients. My own team is currently conducting follow-up studies
in collaboration with several clinics, covering both female and male
pseudohermaphroditism. From everything that is available in the literature and in our
own data, we can summarize some preliminary conclusions concerning gender
development and sexual functioning of patients in these two categories. (For reasons of
space, the literature on true hermaphroditism is not reviewed here.)
46,XX Pseudohermaphroditism
Patients with 46,XX pseudohermaphroditism are mostly represented by classical CAH

with 21-hyrodxylase deficiency (21-0HD). One can visualize best the variability in
genital appearance at birth in terms of the Prader (Prader, 1954) stages of genital
differentiation which range from mild clitoral enlargement (Prader 1) to extreme male-
like masculinization with a penile urethra (Prader 5). (If normal females and males were
added to the Prader scale, they would be classified as 0 and 6, respectively.) The CAH
condition can be associated with any of these stages. Of the two major subtypes of
classical 2l-0HD, Simple Virilizers (SV) and Salt Wasters (SW), the SW subtype, on
average, shows more severe masculinization of the: genitalia (Therrell et aI, 1998), but
both subtypes range across all Prader stages. (Note that classical Prader staging may not
be appropriate for CAH patients who have undergone prenatal dexamethasone treatment,
because the latter may differentially affect the development of the external and internal
genitalia, depending on the timing of the treatment.) Major psychological findings on
CAH females are as follows.
In comparison to control groups, CAH females are behaviorally masculinized, but
with much interindividual variability (Ehrhardt et aI, 1968(2);1981; Money et aI, 1972;
Dittmann et aI, 1990; Zucker et aI, 1996; Berenbaum 1999). The psychological
masculinization is much more pronounced in SW than in SV women (e.g., Dittmann et aI,
1990; Meyer-Bahlburg et aI, 1999). There is great variability of gender-role behavior
within each CAH subtype. Some of this variability is probably accounted for by
variations in prenatal masculinization as represented by the Prader stages. Thus there is
some degree of a dose-response relationship between prenatal androgen excess and
gender-role behavior (Meyer-Bahlburg et aI, 1999).
In spite of this variability in childhood gender-role behavior, almost all CAH females
maintain a female gender identity. This illustrates that a core gender identity can
accommodate much variation in gender role behavior. Only very rarely do women with
CAH change to a male gender in adulthood (Meyer-Bahlburg et ai, 1996). If gender
change occurs, it seems to happen among those who had prolonged genital ambiguity in
the early years and/or lack of consistent androgen suppression by glucocorticoid
treatment during childhood. On the other hand, some 46,XX CAH infants with high
genital Prader stages are mistaken for cryptorchid 46,XY and assigned to the male
gender. When their condition is finally diagnosed in adolescence, most elect to stay male
and apparently do reasonably well in all spheres oflife except fertility (for references, see
Meyer-Bahlburg et aI, 1996), but good systematic follow-up data on even a moderate-
sized adult sample are lacking.
Diamond and Sigmundson (Diamond et aI, 1997) have suggested to assign all Prader-
stage 5 females with CAH as males. This recommendation is presumably based on the
belief that the penile masculinization indicates the same degree of (male-typical) brain
masculinization in the 46,XX infant as in the 46,XY infant, but at this time we do not
have the comparative data to support this assumption. In addition, such an assignment
would involve ovarectomy and hysterectomy and, thereby, iatrogenic infertility.
However, we currently do not have the data to show that the psychological and sexual
functioning and the subjective quality of life are better one way or the other. Most
physicians would be reluctant to deliberately deprive a person of his or her reproductive
capacity, unless there is a demonstrable gain in the function and quality of life in the
selected gender. We need systematic comparative follow-up data to support a policy
change with empirical evidence.
Studies of sexuality show in CAH women as a group delayed and reduced sexual
activity and libido (Meyer-Bahlburg, 1999; Meyer-Bahlburg et ai, [in press]). They also
show less heterosexual activity and imagery, and (in a minority) increased bi- and
homosexuality.
Again, the differences from control groups are more pronounced in SW than SV
women, and there is much within-group variability.
One factor in the relatively reduced sexual activity of women with CAH is the genital
status in later adolescence and adulthood. Quite a few CAH women - especially those
with the SW subtype-find intercourse painful or even impossible or stay away from
heterosexual involvement altogether because of their awareness of having an inadequate
vagina (Meyer-Bahlburg, 1999; Meyer-Bahlburg et aI, [in press]; Mulaikal et aI, 1987).
When clitorectomy is used, diminution or loss of erotic sensitivity and orgasmic capacity,
and with it, of sexual satisfaction, are probably quite frequent, although some women
appear to retain both to varying degrees. To what extent the more recent techniques of
clitoral resection and recession improve the picture remains to be studied. Published
functional outcomes of these techniques appear promising (for refs. see Meyer-Bahlburg,
1999), but a recent report (Alizai et aI, 1999; see also Passerini-Glazel, 1999) raises
troubling questions. Only detailed studies on sexual functioning in adult patients will
provide the answers we need.
46,XY Male Pseudohermaphroditism and Related Conditions
For many syndromes of 46,XY male pseudohermaphroditism, genital staging can be

assessed by means of the Quigley (Quigley et ai, 1995) scale, ranging from 1 (normal
male) to 7 (normal female). Originally, the scale was defined only for the spectrum of
androgen insensitivity (AIS) patients. Straightforward Quigley staging is not appropriate
for 5-alpha-RD, where the androgenization deficit is limited to the dihydrotestosterone-
dependent external genitalia, and to androgen-independent penis anomalies such as penile
agenesis, cloacal exstrophy of the bladder, and ablatio penis. Where Quigley staging can
be used, it is again assumed that the genital stage reflects the degree of prenatal
androgenization.
The extreme degree of undermasculinization, that is, Quigley stage 6, in combination
with estrogen exposure is represented by the syndrome of Complete AIS (CAIS) in which
both the genitalia and the brain appear to be underandrogenized. The other end of the
masculinization spectrum is represented by children who were born with normal male
internal and external genitalia (Quigley stage 1) but lost the penis later, and were then re-
assigned to the female gender. In such cases, we have to assume that both genitalia and
brain were fully prenatally androgenized. When the patients are gender re-assigned and
their external genitalia surgically feminized, their brains are not medically altered and
stay masculinized. In addition to children with penile ablatio, children born with normal
testes but with agenesis of the penis or cloacal exstrophy of the bladder also fall in this
category of masculinization. It is possible that also the syndrome of 5-alpha-RD belongs
here as far as brain masculinization is concerned, depending on whether testosterone or
its metabolite dihydrotestosterone play the major role in the prenatal sexual
differentiation of the brain (which is currently not known). All other types of male
pseudohermaphrodites fall in between the two poles of the Quigley scales.
The data available on long-term gender outcome in male pseudohermaphrodites are
very limited (Meyer-Bahlburg, 1999). By and large, the findings are in line with
expectations . Gender-role behavior is least masculinized when there was no effective
prenatal androgen effect in peripheral tissues as illustrated by patients with CAIS. By
contrast, there are strong indications of behavioral masculinization in patients with a
male-typical prenatal sex-hormone milieu, as is the case in the non-endocrine conditions
of genital ambiguity in 46,XY patients.
In the least masculinized syndrome, CAIS, no female to male gender change has been
reported. By contrast, patient-initiated female to male gender change in the most
masculinized syndromes (cloacal exstrophy, aphallia, penile agenesis) may perhaps
approach 50% ([Reiner), but these data are as yet unpublished and based on very small
samples. In the other syndromes, some gender change from female to male occurs,
particularly when there is prolonged visible genital ambiguity (Money, 1986), but it is
relatively uncommon.
In our ongoing collaborative follow-up study of adult 46,XY pseudohermaphrodite
patients at Johns Hopkins Hospital, now at a sample size of 58, the vast majority of male
pseudohermaphrodites are satisfied with their gender, regardless of having been raised as
males or females, although about one third had experienced times in their life when they
were uncertain (Meyer-Bahlburg et ai, 1999). Thus, these data appear to confirm the
studies by Ellis (Ellis, 1945) and Money et a!. (Money et ai, 1955) which led to the
conclusion that most intersex patients identify with the gender to which they have been
assigned.
Diamond and Sigmundson (Diamond et ai, 1997) have recommended that 46,XY
individuals with penile agenesis, cloacal exstrophy, traumatic loss of the penis, or 5-
alpha-RD, all of whom presumably have normal testes, be routinely assigned to the male
gender, and that the same decision be made for 46,XY individuals with other intersex
syndromes who have genitals of Quigley stages 2-3, as well as for patients with
micropenis. Again, only good comparative studies of patients in sufficient numbers who
have been raised in one gender or the other can provide definitive answers to the question
in which gender psychological and sexual functioning and the subjective quality of life
are really better for a given condition. There are individual case reports or small-sample
studies showing good and poor outcomes on both sides (for references, see Meyer-
Bahlburg, 1999), but no data that permit an estimate of the differences in rates of positive
or negative outcomes.
Concerning sexuality, much less is known about 46,XY pseudohermaphrodites than
about 46,XX classical 21-0HD. The spotty data available suggest increased rates of
homosexuality (gynecophilia) in female-raised 46,XY patients other than CAIS (Money
et ai, 1986) and especially in those syndromes where gender change to male is common
in adolescence and adulthood (Imperato-McGinley et aI, 1979; Imperato-McGinley, [ ];
Rosier et aI, 1983). Case reports suggest the dependence of sexual activity level and
libido on appropriate androgen replacement where indicated, but the evidence is
insufficient for more specific conclusions. The majority of the 46,XY
pseudohermaphrodites in our own ongoing collaborative project with the Johns Hopkins
Hospital clinic are 'mainly' or 'somewhat satisfied' with their sexual functioning,
independently of their gender (Meyer-Bahlburg et ai, 1999). A few reports have
specifically addressed the sexual functioning of men with a micropenis or microphallus
with a female partner and presented encouraging results (Money et aI, 1985; Reilly et aI,
1989), but we need more detailed assessments in unbiased samples of reasonable size to
have solid empirical evidence.
Summary
In summary, we have learned a great deal about syndrome-specific patterns of

behavioral development. For both female and male pseudohermaphrodites, the data seem
to be compatible with a prenatal-androgen model of human psychosexual differentiation.
Wherever we compare two conditions whose relative degrees of peripheral prenatal
androgenization are known to us, we find that the condition with the higher degree shows
more masculinization (Meyer-Bahlburg, 1999), although there is again much unexplained
within-group variability. The lack of studies that share identical assessment procedures
does not permit us a simultaneous comparison of multiple groups from the existing
publications and therefore no finer quantitative analysis. The data are also insufficient to
solve the question of penile size as an indicator of brain androgenization (apart from the
fact that in some conditions - such as 5-alpha reductase deficiency or penile agenesis - a
penis does not develop, although male-typical prenatal testosterone levels are present).
Thus, the evidence is insufficient for the answers to the most pressing questions that were
listed earlier.
Methodological Problems and Guidelines for Future Research
Conceptualization
In an earlier section, we identified the three major issues of the current debate: gender
assignment, genital surgery, and information management. For all three issues empirical
data are needed as a basis for decisions on management policy. From the psychosocial
perspective, long-term outcome studies have the highest priorities, because only they can
give us the necessary feedback on the long-term effects of specific management
decisions and techniques.
The age group of patients to be studied depends on the focus of investigation. For
instance, in regard to the outcome of gender assignment, we need data on adult patients
until at least midlife in order to get an appropriate overview of the frequency of patient-
initiated gender change. But if we want to understand the factors that contribute to gender
change - especially the psychosocial ones - WI~ also need to study our patients in
childhood and adolescence, preferably prospectively, because retrospective assessment of
child-rearing behavior is so difficult to do. In regard to genital surgery, we need to study
its gender-confirming aspects particularly in childhood, but investigation of the impact
of surgery and resulting genital status on courtship and sexual functioning obviously has
to wait until the individual is old enough for dating and sexual activity. Regarding the
outcomes of specific surgical techniques, we have to take into consideration the
difference between efficacy-the outcome of a newly developed specific technique as
performed in systematic trials by an expert, usually the inventor-and the efficiency-the
outcome when a technique is routinely adopted by a wider range of surgeons. In the area
of information management, acute and short-term follow-up studies of medical education
and counseling concerning issues of information transmission and affective response of
both patients and parents would be desirable. At present, however, we are also in need of
long-term follow-up surveys in regard to the patient's severe emotional reactions
(including suicidality) to the disclosure of medical information - also via the media --,
the impact of frequent genital examinations by diverse medical staff, patients'
experiences with access to medical charts, the benefits and disadvantages of patient
contacts with patient-support groups - both face to face and over the internet, and so on.
As to the question, which syndromes should have priority for long-term follow-up
investigation, one has to say that none have been studied sufficiently to provide a fully
satisfactory basis for the policy management decisions we are facing. Because of their
relative frequency and homogeneity, we know relatively more about 46,XX patients with
classical CAH than any of the male pseudohermaphrodite syndromes, but even here
major questions are poorly researched, for instance, the comparison of long-term
outcome of 46,XX CAH born with a penile urethra in male-assigned versus female-
assigned patients, or sexual functioning in dependence on the technique of genital
surgery. The diversity of syndromes and the low prevalence of each has hampered
follow-up research on male pseudohermaphrodites even more, and all of the various
46,XY syndromes and conditions are understudied in all psychosocial respects.
Sampling
To provide the data base needed for improving the psychosocial prognosis and thereby
the empirically based rationale for decisions on gender assignment and genital surgery,
we need to analyze outcome data by patient groups that are defined on the basis of both
definitive endocrine and molecular-genetic criteria where applicable and by the degree of
genital ambiguity at birth in terms of Prader (Prader, 1954) and Quigley (Quigley et aI,
1995) stages, rather than by current genital status alone. We also need to systematically
compare patients with the same condition who have been reared female to those who
have been reared male. Similarly, we should try to assess the natural history of a
condition, i.e., compare the outcome of surgically and hormonally untreated patients,
where available, to the outcome of treated patients.
The question of control data and control groups needs to be carefully considered. For
instance, gender change also occurs in non-intersex persons with a certain prevalence
(Meyer-Bahlburg, 1999). Problems in sexual functioning among intersex patients should
not be compared to an assumed standard of 100% perfection, but to community samples
where sexual dysfunctions such as anorgasmia in women are not uncommon. For
instance, Laumann et al. (Laumann et aI, 1999) found for a nationally representative
sample that about a quarter of sexually active women had periods of several months or
more in the 12 months prior to study when they were unable to achieve orgasm.
Ideally, we would conduct prospective follow-up studies of regional or national
cohorts formed by the systematic screening of newborns, both those with an intersex
diagnosis and non-intersex controls. In recent years, this has become possible for the
syndrome of CAH for which systematic newborn screening has been introduced in a
number of states. However, given the urgent need for outcome studies, we cannot wait
until such cohorts reach mid-adulthood, but must also conduct follow-up studies on
patients who are now in their early and middle adult years.
The eXlstmg literature relies mostly on sman clinical samples or individual case
reports. Even if reports concern groups of patients, the sample sizes are usually too small
for reasonably definitive conclusions, given the variability in diagnosis, genital status at
birth, surgical history, and sex-hormone treatment history. In order to obtain clinical
follow-up samples of sufficient size, we urgently nc~ed multisite collaborative studies with
uniform protocols across clinics. A centralized regional or national registry of intersex
patients could also be extremely valuable in this regard.
Case reports in particular are suspect of selection bias, since it is often not the average
patient, but the one with special characteristics who is considered worthy of publication.
Some single cases have been totally overvalued in their importance; the widely
publicized John I Joan case (46,XY with penile ablatio; Money, 1975;1999, Diamond et
aI, 1997) is a prime example. Originally, the case was overused as an illustration for the
power of social rearing. Currently, the case is overused as an example for the power of
biological factors in gender development (Meyer-Bahlburg, 1999), notwithstanding the
publication of another case with the same condition, but a different outcome (Bradley et
ai, 1998).
Selection bias is not limited to case reports. It may also seriously affect follow-up
samples collected by clinics. For instance, clinic populations of patients with a specific
genotype or endocrine phenotype are often not representative of people with the same
genotype or phenotype in the general population, and marked referral biases in terms of
severity of the medical condition, socioeconomic status of the family, and local
demographic factors such as race/ethnicity are quite typical. As a consequence, clinics
may differ in the demographic or other characteristics of patients seen.
In addition, there is the bias of differential attrition. Long-term follow-up studies
typically suffer from very substantial loss of patients over time. Who stays with a clinic
over many years and who participates in follow-up research ist determined by many
psychological factors. These include satisfaction with the doctor-patient relationship and
the medical management. Dissatisfied patients are likely to leave a clinic and to not
cooperate with research studies initiated by that clinic; the limitation of the study to the
remaining patients may create a bias towards positive outcomes.
Selection bias is not limited to clinic samples. For instance, when researchers like
Diamond specifically search for XY patients raised female who later change to male, they
deliberately introduce a patient-selection bias in the research process-which may be
heuristically useful, but does not provide an epidemiologically satisfactory sample.
Another example is ISNA: the outspoken people among its members appear to include
increased numbers of patients with dissatisfaction about their gender or genital status.
Perhaps, intersex organizations such as ISNA that are highly critical of medicine and
appear to have many members who are dissatisfied with their medical management may
be in a position to complement clinic studies by studying their membership, provided
such studies are conducted with appropriate methodology and protection against
assessment bias. Alternatively, patient support groups could be asked to collaborate with
clinics so that patient participation and representativeness are increased.
Sample selection biases often affect psychosocial outcome variables more strongly
than biological measures. Thus, careful planning of sampling procedures and the detailed
characterization of non-participants in order to gauge the generalizability of findings are
very important. How many of the original population have died, and does this number
indicate just medical risks or also suicide risks? How many prospective participants could
not be traced, and does this imply a socioeconomic bias or a selection for personality
characteristics? Who are the refusers, and have their refusal reasons to do with
unsatisfactory management and inadequate outcome? The participation in such studies of
social or psychiatric epidemiologists would be highly desirable.
Assessment
The outcome assessments employed in the available literature are often not systematic
and sometimes highly impressionistic. In view of the small sample sizes typical of most
studies in this area, there is an urgent need for the employment of standardized outcome
measures across studies so that results can be combined for greater statistical power. The
problem is the paucity of standard measures available for behavioral intersex research.
There is a need for the development of additional systematic assessment tools in this area.
As I have argued elsewhere (Meyer-Bahlburg, 1999), research on health-related
quality oflife (HRQL) constitutes a useful guideline for long-term outcome assessments
of intersex patients. Formal HRQL assessment needs to take place on three levels: (a)
generic broad-band assessments; (b) intersex-specific assessments of gender role/identity
and sexuality that vary with gender and developmental stage; and (c) syndrome-specific
assessments, e.g., of specific cognitive deficits or specific techniques of genital surgery
used (for available methods see refs. Meyer-Bahlburg, 1999; McDowell et aI, 1996). A
good example of an HRQL approach to intersex research is the study by Kuhnle and co-
workers (Kuhnle et ai, 1995).
Quantitative assessments, e.g., systematic ratings or multi-item scales, should be
sufficiently fine-graded so that we can achieve a good differentiation of syndromes and
individual cases along psychological continua. For instance, the employment of finely
differentiating gender scales shows that subtypes of patients with classical CAH differ
markedly in gender-role behavior (Meyer-Bahlburg et aI, 1999), which has implications
for both etiological research and clinical management.
Particularly on the syndrome-specific level, systematic qualitative interviews will be
an important additional tool, either in preparation for the development of new
quantitative or structured assessment methods or as a complement to the latter.
'Qualitative' in this context does not mean 'impressionistic' or 'unsystematic'. Rather,
qualitative techniques as used in anthropology and other social sciences constitute a
rigorous approach to the inductive development of pertinent constructs and their
operationalizations from the narratives that interviewees provide to open-ended question
(Krueger, 1988; Berg, 1998). An important source of qualitative material can be
autobiographic accounts of intersex patients (Dreger, 1999). Money's Biographies of
Gender (Money, 1991) is an outstanding illustration of the qualitative approach at the
case-report level. Qualitative assessment is also a good starting point for the studies of
parents' attitudes to specific aspects of intersex genitalia such as clitoral size or
hypospadias, of parents' - and, later, patients' - primary concerns and worries regarding
an intersex condition, of patients' reasons for satisfaction and dissatisfaction with post-
surgical genital appearance and functioning, and of patients' reasons for refusing genital
surgery, dilatation procedures, and wearing intravaginal forms, or for avoiding going to
physicians altogether.
As this chapter is not suited for a detailed and comprehensive review of the
assessment methods available, we will list below the instruments we have used andlor
are using in our own work in this area.
Gender
Gender identity, i.e., the sense of being male or female (or some form of trans gender),
can be assessed by asking a person directly although there is no standard formulation in
use. Often, the presence of a gender identity as male or female is derived from the
absence of frank gender change or of gender doubts and other signs of gender
dysphoria.For the latter, Zucker and co-workers (Zucker, 1993) have developed a
Gender Identity Interview for Children which seems applicable from preschool to the end
of childhood. It was developed for the the assessment of GID in non-intersex children,
but we are using it clinically and in an ongoing research project for intersex children and
find it very useful (unpublished data). Two analogous scales for adults, which, however,
also include items on gender-role behavior and sexual orientation, are the Gender Identity
Scale for Males (Freund et aI, 1977) and the equivalent for females (Blanchard et aI,
1983). Both were originally developed for non-intersex adults with GID, but are used by
my team also for intersex patients (e.g., Meyer-Bahlburg et aI, 1996).
Gender-role identity, i.e., the degree to which a person experiences himlherself as
masculine and as feminine, can be assessed in adolescents and adults by a simple
numerical 5- or 7-point rating scale with the extremes labeled "not at all masculine" and
"very masculine" (or feminine), respectively.
For gender-role behavior in childhood, our unit uses several measures. Two of these
are parent-administered questionnaires, the Child Game Participation Questionnaire
(CGPQ; Meyer-Bahlburg et aI, 1994) and the Child Behavior and Attitude Questionnaire
(CBAQ; Meyer-Bahlburg et aI, 1994) which are modified, re-scaled and re-normed
versions of earlier forms originally developed by Bates and Bentler. Their most
comprehensive subscales, 'Gender' of the CGPQ and 'FEM' of the CBAQ were found to
yield very large effect sizes for gender of 3.9 and 5.7, respectively, in the norm
population of 6-10 year olds. The Gender-Role Assessment Schedule - Child (GRAS-C;
Meyer-Bahlburg et ai, 1988; Cosentina et aI, 1993) is a comprehensive semi-structured
interview for children; a very similar version, the M-GRAS-C (Meyer-Bahlburg et aI,
1988), can be administered to the mothers to report on their children. Both interviews
take 1-2 hours to administer, and statistical analysis is done by individual item or on the
basis of item groupings to be determined for each study. For adolescents and adults we
use analogous interviews, the GRAS-A and M-GRAS-A (Ehrhardt et aI, 1984(2»; they
combine recalled and concurrent information. More recently, Zucker's team has
developed a brief self-administered questionnaire, the Recalled Childhood Gender
Identity Scale (Mitchell et aI, 1991; Zucker et aI, 1996) which combines retrospective
items of gender-role behavior and gender-role identity.
A recall questionnaire for adults that focuses on play behavior is the Child Play
Activities Questionnaire from Bencler's team (Grellert et aI, 1982). The closest to a
comprehensive adulthood questionnaire concerning concurrent gender-role behavior is

the Sex-Role Behavior Scale-2 by Orlofsky et al. (Orlofsky et ai, 1982), but it is only
now being applied by us in a study of intersex patients.
Recently, Berenbaum has developed specific measures for selected sub domains of
gender-role behavior, for instance, a scale for assessing interest in infants in childhood
(Leveroni et aI, 1998) and a new self-report scale for assessing sex-typed activities and
interests, including vocational interests, in adolescence (Berenbaum, 1999). To what
extent individual component variables of human sex-dimorphic behavior are
differentially affected by the dosage and timing of prenatal hormone exposure - as we
know it for specific variables of sex-dimorphic behavior in lower mammals - is an
unsolved issue, and the development of such more specific measures for humans may
allow us to address that question.
There are many other psychological scales for the measurement of gender-role
behavior and gender attitudes for the general population (Beere, 1990), but most of these
are not specific enough for the needs of intersex research, and their effect sizes for gender
are usually rather modest. The psychological literature on gender often discusses the
question whether a bipolar (male- female) continuum is an appropriate construct for the
operationalization of sex-dimorphic behavior, or whether two independent (masculine
versus feminine behavior) or more dimensions are more appropriate. As I have
demonstrated elsewhere (Meyer-Bahlburg et ai, 1994), the answer to this question
depends to a large extent on one's investigative goal and on the procedure chosen for the
development of such measures. For research designed to prepare a policy on gender-
assignment decisions in a two-gender culture, a bipolar composite scale seems to be an
adequate instrument to start with.
Sexuality
Many instruments are available for the assessment of sexual behavior in the general
population (the best sourcebook is Davis, 1998), but they lack specificity for the intersex
population. Both clinically and in our own research we use a comprehensive sexual-
history interview for adolescents and adults, the Sexual Behavior Assessment Schedule
(SEBAS-A) (Meyer-Bahlburg et ai, 1983), which captures psychosexual milestones,
sexual orientation, and current sexual activity, but requires trained interviewers.
The SEBAS-A also includes a detailed section on sexual dysfunctions. Two good
general screening questionnaires for sexual dysfunctions in the general population are the
Brief Sexual Function Questionnaire for men by Reynolds et al. (Reynolds et ai, 1988)
and the Brief Index of Sexual Functioning for Women by Rosen et al. (Rosen et aI,
1998). However, given the particular genital status and surgical history of many intersex
patients, such standard instruments must be complemented by more specific inquiry, at
this stage of our knowledge preferably by qualitative interviews. In addition, specifically
tailored rating scales such as the ones used by Mureau (Mureau et ai, 1995;1996;1997)
can be used for patients' ratings of satisfaction with cosmetic and functional results of
genital surgery. Ideally, studies of sexual functioning would utilize both physiological
and psychological assessments. For instance, if it can be shown that pudendal nerve
conductance is preserved at the end of genital surgery (Gearhart et ai, 1995), we know
that a basic component of the genital system is functional at that time, but we still need
confirmation that erotic sensitivity and orgasmic capacity are retained in adulthood
(Chase, 1996).
General Assessment Procedures
The utilization of multiple informants - indepf:ndently assessed - can strengthen

interview- and self-report-based findings considerably. This is obvious if one deals with
sexual functioning where former and current spouses and lovers can validate crucial
aspects of sexual activity and function. It also applies to gender role behavior and gender
identity change where observations and impressions of family members and peers can be
a valuable adjunct to the patient's own account.
As in rigorous treatment studies generally, assessments of outcome for systematic
research purposes should not be conducted personally by those who provided the patient
care, be it by way of genital surgery, hormone treatment, or intersex counseling. The risk
of inadvertent biases in favor of the intervention is too high. In behavioral studies, the use
of written self-report questionnaires or audio-computer-assisted interviews (Turner, et aI,
1998) administered by research assistants may help in this regard. Analogous
considerations apply to the evaluation of somatic outcomes of medical treatment.
Behavioral outcome studies should be done under blind conditions whenever possible.
This may require that the initial sections of a follow-up interview protocol are conducted
by interviewers who are unaware of the medical diagnosis and genital condition of the
research participant, while the latter is instructed not to disclose his/her medical condition
to the interviewer. Other sections are conducted later in the protocol after the interviewer
has been informed about the medical situation.
Assessment topics such as gender identity, sexual functioning, genital status, and
reaction to medical management are of central importance to outcome research on
intersex patients, but often highly emotionally charged because of societal taboos or
personal experiences. The results may be self-presentation bias including underreporting
and distortions. A number of techniques have been developed to facilitate disclosure and
diminish distortions by the interviewee (Money, 1991, ch. 1; Catania et aI, 1995; Money,
1994, chs. 6 and 7).
Research interviewing is a social process (Catania, 1997) in which considerable
demands for social skills and research discipline are put on the interviewer. Great care
must be taken that research interviewers in this sensitive area of inquiry are well selected
and well trained in research interviewing , both in regard to general research
interviewing technique and coding and in regard to rapport building and the facilitation
of self-disclosure and accurate reporting on part of the study participants. In my
experience, clinical training and experience in either medicine or mental health does not
automatically qualify someone to conduct good research interviews. The opposite is often
the case: Medical professionals tend to lack patience for psychological complexities;
mental-health clinicians tend to get over-involved in them; and neither may be free of
judgmentalism in matters of gender and sexuality. Some systematic manuals are available
for other areas of sex research interviewing (e.g., Gruen et aI, 1991; Dugan et aI, 1997)
that can serve as models for interviewer selection and training in the intersex area.
Conclusions
In the mid-1950s, the optimal gender policy constituted a significant step forward in
planning intersex management. In its general approach, this policy appears still valid. It is
no accident that most of the practical clinical guidelines of Diamond and Sigmundson
(Diamond et aI, 1997) are essentially the same as those that can be found in Money's
many summaries on the subject (e.g., Money, 1968;1994). Moneys guidelines are based
on his own immense clinical experience in the psychosocial management of such patients
while most of his critics are either not clinicians or at least not specialized in this area.
On the other hand, not only has society changed, but our scientific knowledge of
intersexuality has greatly increased since 1955. Thereby, the accuracy of our prognosis of
the psychosocial outcome has improved for at least some patients with specific
syndromes, and this continuing development will gradually increase our ability to make
more syndrome- and severity-specific decisions regarding gender assignment and
surgery. Planned policy changes should be informed by empirical data and followed by
assessments of the long-term outcome of the new management approaches. This demand
is in line with the recent increase in requirements for changing diagnostic criteria in the
preparation of DSM-IV (APA, 1994) and the grounding of psychological therapies in
systematic studies of outcome (e.g., Heiman, 1997). As illustrated by these two examples,
such guidelines should not be left to individuals, but should be arrived at by
multidisciplinary committees of appropriate specialists with opportunities for input from
others working in this area and the patients themselves. The work of such Committees is
not going to be easy. It is highly unlikely that follow-up studies will result in simple
clear-cut findings. Even if we should find ways of integrating outcomes over different
syndromes and conditions, we will probably end up with mixed patterns of more or less
probable advantages and disadvantages of gender assignments, surgical procedures, etc.,
and it will require a gradual process of consensus development to arrive at management
guidelines that can be endorsed by at least a clear majority of committee members.
The history of science teaches us that most scientists - and others - see only a limited
piece of the legendary elephant. Many grasp a kernel of truth, but the entire elephant is
much more than the parts we have our hands on. Thus, skepsis is advisable regarding any
individual viewpoint. This also applies to intersex research, particularly when it comes to
psychosocial matters. A constructive approach to this dilemma is to begin with the
assumption that everyone has something to contribute and to refrain from
oversimplifying the positions of others with whose views we disagree. Respect for
different perspectives is in order, along with an attempt to understand in which context
various individuals try to provide answers to which questions. Then, wherever applicable,
let sound empirical research evidence be the arbiter. Sound psychosocial research also
needs to take into account the historical changes societies undergo, and how these
changes affect outcome evaluation. In Western post-industrial societies, for example,
societies' definition of gender roles are changing, transgender individuals encounter more
tolerance, and patients have achieved greater autonomy in medical decision making. Such
societal changes influence the life experiences of the afflicted patients and, thereby, the
evaluation of long-term outcome, and need to be considered in any revision of the
psychosocial and medical management of intersexuality.
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PRENATAL DIAGNOSIS AND TREATMENT OF
INTERSEX STATES
Natalie E. Rintoul, M.D. and Timothy M. Crombleholme, M.D.

Department of General and Thoracic Surgery and the Center for Fetal Therapy, The
Children's Hospital of Philadelphia
INTRODUCTION
Congenital adrenal hyperplasia (CAH) is the most common etiology of ambiguous

genitalia in the neonate [Miller et aI, 1987]. Furthermore, it is the only cause of
ambiguous genitalia currently amenable to prenatal intervention. In over 95% of cases,
CAH results from a genetically determined deficiency in 21-hydroxylase activity.
Prevalence of the carrier state for this disease in the general population is estimated to be
approximately I in 60 and the incidence of clinical 21-hydroxylase deficiency in
caucasians has been found to be I in 14,000 [Pang et aI, 1988]. There is no difference in
the incidence of CAH based on sex. Due to the autosomal recessive manner of
transmission, congenital adrenal hyperplasia carries a 25% recurrence risk for subsequent
siblings.
During adrenocortical steroid hormone biosynthesis, hydroxylation of the steroid
nucleus at carbon 21 is a prerequisite for the eventual production of both glucocorticoids
(cortisol) and mineralocorticoids (aldosterone). In the absence of this hydroxylation step,
the immediate precursors (ie. progesterone and 17-hydroxyprogesterone) accumulate and
are eventually processed via the sex-steroid pathway. As a result, excess androgens
including androstenedione and testosterone are produced. This excess of androgens leads
in turn to virilization of the developing external genitalia. The internal reproductive
organs remain unaffected and undergo normal differentiation to form the female gonads
and reproductive tract due to the absence of Mullerian inhibiting substance. As a result of
deficient cortisol production, the pituitary gland secretes excessive ACTH which results
in adrenal gland hyperplasia. The deficient 21-hydroxylase enzyme also impairs the
aldosterone pathway and results in the salt wasting variety of congenital adrenal
hyperplasia.
Three potential phenotypes may result from 21-hydroxylase deficiency. The first is
simple virilization wherein adequate aldosterone synthesis is present and patients are
often first diagnosed between birth and five years of age. The second phenotype involves
not only virilization, but also salt wasting. These patients exhibit more complete
masculinization, inadequate aldosterone production, and if untreated may experience a
life threatening adrenal crisis in the neonatal period. Lastly, the mild or non-classical
phenotype is associated with a complete lack of prenatal abnormalities and represents the
late onset form. Patients with this phenotype initially present with hirsutism and
oligomenorrhea in adolescence. Therefore, presentation at birth with ambiguous genitalia

226 N. E. RINTOUL, ET AL.
occurs only in the classic forms of 21-hydroxylase deficiency (ie: simple virilizing and
salt- wasting).
Despite postnatal treatment for patients with congenital adrenal hyperplasia, adult
males are characteristically short in stature and females are short, obese and often
infertile [Migeon, 1996]. Infertility is more common amongst those females. with the
classical salt-losing form of the disease and occurs despite functional gonads and normal
development of the fallopian tubes, uterus and the upper one third of the vagina. Elevated
progesterone levels have been proposed as one possible cause of this infertility [Van Wyk
et aI, 1996]. Alternatively, some argue that exposure to excessive androgen levels in utero
may result in masculinization of the central nervous system and disruption of the cyclic
secretion of gonadotropins [Money et aI, 1984].
Embryology
The adrenal cortex begins to form around the seventh week of gestation. Growth of the
adrenal glandular tissue is regulated via a negative feedback loop encompassing several
intermediates including adrenocorticotrophic hormone (ACTH). This inhibitory feedback
is presumed to exist from very early in gestation. Therefore, treatment of congenital
adrenal hyperplasia may be achieved by the early administration of dexamethasone which
is felt to inhibit ACTH release from the fetal pituitary gland.
Sexual differentiation in humans occurs between 9 and 13 weeks gestation. In the
undifferentiated state the fetus has a genital tubercle, genital folds, and genital swellings.
The undifferentiated genital tubercle is sensitive to androgen action at 8-9 weeks
gestation. In males, testosterone is converted to dihydrotestosterone by 5 a-reductase. It
is the presence of dihydrotestosterone that is responsible for the formation of the scrotum
and penis at the end of the first trimester. Genital fusion is nearly complete by 12 weeks
gestation under the influence of androgens.
Females affected with congenital adrenal hyperplasia produce increased androgens
and as a consequence may have a urogenital sinus (fused urethra and vagina), an enlarged
clitoris, and fused and rugated labia majora. The formation of the urogenital sinus begins
as early as 6 weeks gestation. There is a wide spectrum of virilized female phenotypes.
Surgical correction involves clitoroplasty and/or vaginoplasty. Males affected with
congenital adrenal hyperplasia appear normal, often with hyperpigmented scrotal skin.
Prenatal Diagnosis
Prenatal diagnosis of ambiguous genitalia poses particular difficulties that are not an
issue in the newborn nursery. The diagnosis can not always be made on the basis of the
sonographic appearance of the genitalia. However, a prenatal diagnosis of ambiguous
genitalia can be suggested by a variety of clinical circumstances.
PRENATAL DIAGNOSIS AND TREATMENT 227
In some cases, a problem is first suspected when there is discordance between prenatal
ultrasound findings and fetal karyotype. Advances in ultrasound technology now provide
detailed resolution of fetal genital anatomy. Nonetheless, differentiation of a small penis
from a large clitoris remains difficult. The only ultrasound finding considered
pathognomonic during fetal sex determination is visualization of testes in the scrotum
[Smith et aI, 1996]. However, the testes do not appear in the scrotum during normal
development until after 28 weeks gestation. During a typical ultrasound examination the
scrotal contents are not well visualized in 30% of cases. In addition, even when scrotal
contents are well visualized, erroneous gender assignment will occur in up to 3% of
cases. Notwithstanding these limitations, it is possible to clearly image the genitalia in a
significant number of patients [Figures 1,2]. Discordance between the ultrasound and the
karyotype can occur in the instance of overandrogenized females (eg. congenital adrenal
hyperplasia), underandrogenized males (eg. testosterone deficiency, androgen receptor
deficiency, 5-0. reductase deficiency) or in certain chromosomal abnormalities (eg. pure
gonadal dysgenesis, mixed gonadal dysgenesis, true hermaphrodite). In some situations,
ultrasound will detect ambiguity of the genitalia without the benefit of genetic
amniocentesis [Figures 3,4A and B]. Secondly, from a purely gonographic perspective,
ambiguous genitalia may be included in the diagnosis of a larger complex structural
anomaly such as cloacal exstrophy or bladder exstrophy [Figure 5]. Thirdly, ambiguous
genitalia are known to be associated with a number of genetic syndromes. Smiths'
textbook lists a total of 21 syndromes in which ambiguous genitalia are frequent and an
additional 46 in which it is an occasional finding (Smith, 1997). Finally, a prenatal
diagnosis of ambiguous genitalia may be pursued more aggressively in those cases with a
family history, as in the case of congenital adrenal hyperplasia.
Figure 1. Normal male genitalia. Figure 2. Normal female genitalia demonstrating labia.
Figure:3. The s(}nographk l1ppcaran':e nf a !:lUlu scnmm\ A del<likd giil1itlll UhraS(Aiild wrvey in combinnti6n
wilh::l high index ofsuspiduf! arc 1l<~~dcd to oded the nlkn suNk ::lpptilfllnce nf tlmj,igUC>(ls genitalia.
Figure 4 A and B. This is the sonographic appearance of a virilized genotypic female (Al and the appearance
of an undervirilized male with an underdeveloped phallus (8). This demonstrates the difficulty of making the
diagnosis of ambiguous genitalia in utero.
Figure 5 A & B. Ultrasound views of a case of bladder exstrophy in which there is no bladder in the abdomen,
a mass is seen in the lower abdomen with the umbilical cord at its superior margin and the epispadic penis is
seen below (A). The clinical appearance of cloacal exstrophy in the neonate (B).
Prenatal diagnosis of congenital adrenal hyperplasia has undergone an evolution

during the last two decades. The first technique was established in 1975 with the
demonstration of increased 17-*hydroxyprogesterone (17-0HP) in second trimester
amniotic fluid samples [Frasier et aI, 1975]. Since 1984, prenatal diagnostic testing has
been available at as early as 10 weeks gestation by chorionic villus sampling (CVS).
DNA obtained from the chorion can be assessed for HLA genetic linkage marker analysis
and direct molecular analysis of the 21-hydroxylase locus CYP21 [Forest et aI, 1993]. At
present, prenatal diagnosis of congenital adrenal hyperplasia involves a combination of
these studies.
Measurement of amniotic fluid hormone concentrations in second trimester fetuses
affected with congenital adrenal hyperplasia reveals elevated 17-0HP and deJta-4
androstenedione. Normal 17-0HP levels range from 18-269 ng/dl. Levels >300ng/dl are
diagnostic of 21-hydroxylase deficiency [Mercado et aI, 1995]. However, hormone levels
are only reliable with the salt-losing classical congenital adrenal hyperplasia [Pang et aI,
1985]. 17-0HP may be normal in non-classical and simple virilizing forms of congenital
adrenal hyperplasia. In addition, the hormone levels are uninterpretable in the face of
maternal dexamethasone treatment.
HLA genetic linkage marker analysis involves DNA analysis of the HLA-DR locus.
HLA typing determines the fetal genotype: homozygous affected, heterozygote (carrier),
or homozygous unaffected. It is a particularly useful tool in the diagnosis of a fetus with a
previously affected offspring. HLA typing is useful in the diagnosis of both the classical
and non-classical forms of congenital adrenal hyperplasia. This technique does have
limitations in that there may be intra-HLA recombination or the parents may have the
same HLA antigens. Advances in molecular biology have allowed identification and
cloning of the gene for 21-hydroxylase, known as CYP21. The CYP21 genes are located
on chromosome 6 [Dupont et aI, 1977]. Direct molecular analysis of CYP21 can be
performed on fetal DNA obtained from CVS. The C4-CYP21B gene locus can be studied
by Southern blotting. Additionally, point mutations in the CYP21B gene can be studied
by PCR method. Overall, 20% of patients have a significant deletion, while 80% have
only a point mutation in which there is an adenine to guanine conversion in the second
intron. This type of point mutation is called gene conversion [Speiser et aI, 1992). In this
type of mutation an active portion of the normal gene is converted into an inactive gene
or a pseudogene by the transfer of a base. Congenital adrenal hyperplasia results from
inherited mutations of both 21-hydroxylase B genes. These genes are nestled between the
HLA-B and the HLA-DR genes of the major histocompatibility complex and next to the
genes encoding the fourth component of complement (C4B genes) [White et aI, 1985].
HLA genetic linkage marker analysis is also subject to limitations in that there is a 2%
de novo mutation rate in affected families [Speiser et aI, 1994]. It is important in both
counseling and caring for these patients to be aware of diagnostic errors: each of the
techniques described is only 89-95% accurate. Finally, it is important to karyotype the
fetal cells obtained either from the amniotic fluid or the chorion villi.
Congenital Adrenal Hyperplasia: Diagnosis and Treatment In Utero
The first reported treatments of congenital adrenal hyperplasia were in the 1950's with
postnatal administration of cortisone which suppressed androgen production [Wilkins et
ai, 1950]. The first prenatal treatment of congenital adrenal hyperplasia involved the
injection of hydrocortisone directly into the amniotic fluid [Nichols, 1969). However, the
first successful treatment of a fetus affected with congenital adrenal hyperplasia in utero
was not reported until David and Forest in 1984 [David et aI, 1984]. This was a
pioneering study of fetal therapy which reported suppression of fetal adrenal glands
through maternal oral dexamethasone treatment. The investigators initiated treatment of 6
mothers at high risk for congenital adrenal hyperplasia. Of the six at risk, only two
pregnancies were subsequently found to have affected female fetuses and were treated to
term. Prenatal therapy was stopped in the other cases in which the fetus was determined
to be either male, heterozygous or a homozygous unaffected female. In the two affected
female fetuses, treatment was started at 9.4 weeks gestation in one mother and at 5 weeks
in the other. The first infant had moderate virilization and the second infant had normal
genitalia. These two infants were each felt to have severe forms of congenital adrenal
hyperplasia based on high levels of 17-hydroxyprogesterone prior to initiating postnatal
treatment. Each infant was markedly less virilized than their respective older affected
sibling.
Evans et al reported their experience with the prenatal treatment of a fetus at 9 weeks
gestation [Evans et aI, 1985]. This infant was mildly growth retarded with weight and
length in the 5th percentile, but head circumference in the 25th percentile. Further
postnatal testing of the infant revealed that she was only heterozygous deficient and was
subsequently weaned from the hydrocortisone. This case demonstrates the need for
accurate prenatal diagnosis of affected infants.
The report from Forest et al in 1989 was a landmark paper. They treated 43 at risk
pregnancies in a large multicenter trial [Forest et aI, 1989]. Treatment started at 7 weeks
gestation. Dexamethasone doses of 0.5 mg (14-22 mcg/kg/d) orally every 12 hours were
administered in 2/3 of the pregnancies and 0.5 mg (20-36 mcg/kg/d) orally every 8 hours
were given in the other 1/3 of pregnancies. Four mothers aborted 2-5 weeks after
initiation of treatment. In utero fetal demise occurred in one fetus near term, 18 weeks
after cessation of treatment. The fetal loss rate in this study was 9%. Intrauterine growth
retardation occurred in one pregnancy, however the mother involved had had a previous
similarly affected infant in an untreated pregnancy. In 25 of 43 pregnancies, treatment
was stopped following amniocentesis demonstrating an unaffected fetus. In 6
pregnancies, prenatal diagnosis predicted an affected fetus. A lower maternal estriol level
was indirect evidence of fetal adrenal suppression [Tulchinsky et aI, 1972]. There were
no significant maternal complaints or complications other than excessive weight gain
managed by salt restriction. Some of the mothers receiving the higher dose steroids
reported irritability and nervousness. In those treated to term, treatment was stopped at
delivery and none had any clinical symptoms of steroid withdrawal. Two babies treated
with the higher dose steroids were small for gestational age. Follow-up at 6 months to 8
years of age of the prenatally treated affected children revealed normal growth, normal
psychological development and normal lenses on opthalmological examination. In
summary, this was the first large, multicenter study to report the successful in utero
treatment of 6 fetuses affected with CAH resulting in 4 normal and 2 mildly virilized
females.
Speiser et al reported their experience with two successful prenatal treatments of
affected females [Speiser et aI, 1990]. Treatment was started at 8 and 9 weeks gestation
respectively. In both situations the index cases had required genital surgery, but the
treated siblings did not.
An update of the original French multicenter study was reported in 1993 by Forest et
al [Forest et aI, 1993]. The update reported an additional 20 pregnancies. Three girls were
found to be affected with CAH. Treatment was unsuccessful in one case where the
diagnosis was made at 14 weeks gestation and fetal adrenal suppression was not achieved
until after that time. In this group of pregnancies there were two early spontaneous
abortions not thought to be a complication of prenatal treatment.
Karaviti et al reported their experience with the treatment of a series of pregnancies at
risk for congenital adrenal hyperplasia in 1992. As in other studies, at risk pregnancies
included those in which the couple had had a previous child with 21-hydroxylase
deficiency [Karaviti et ai, 1992]. Fifty-five fetuses in 54 pregnancies were identified and
41 pregnancies were treated with maternal oral dexamethasone 20 iJ.g/kg/d in three
divided doses. Of these, twelve fetuses were predicted to have classical congenital
adrenal hyperplasia and seven were prenatally treated. The initiation of treatment ranged
from the 4th to the 21 st weeks of gestation. Genitalia were normal in affected females
treated prenatally prior to the 8th week of gestation. There was no difference between
treated and untreated groups with respect to head circumference, weight, and length at
birth.
Mercado et al reported their 8 year experience with prenatal therapy in 1995 [Mercado
et ai, 1995]. This represents the largest experience reported to date from a center in the
United States. Dexamethasone was initiated in 101 fetuses after I3-HCG confirmation of
pregnancy. In one group of mothers, CVS was performed at 9-11 weeks with karyotype
and 21-hydroxylase deficiency testing. If the fetus was male or an unaffected female,
therapy was stopped. In a second group of mothers, amniocentesis was performed at 15-
18 weeks with karyotyping and 21-hydroxylase deficiency testing. Again, if the fetus was
male or an unaffected female, therapy was stopped. Subsequently 22 fetuses were
confirmed to have classical congenital adrenal hyperplasia and of those, 13 were female
232 N. Eo RINTOUL, ET AL.
and 9 were male. Overall, the prenatal diagnosis was accurate in 95% of the cases with
amniotic fluid 17-0HP levels, 89% accurate with the HLA-DR typing and 91 % with
CYP 1 analysis. Treatment was started prior to 10 weeks gestation in 9 of the 13 affected
females. In all 9 females treated prior to 10 weeks gestation there was significantly
reduced virilization and normal genitalia in four of these cases. In contrast, treatment
starting after the 10th week was not effective in preventing virilization. Six women of the
101 reported complications of prenatal dexamethasone therapy including excessive
weight gain, hypertension and striae. With regard to fetal complications, five fetuses
spontaneously aborted. Four of the five were in the treatment group, three of which were
prior to amniocentesis or CVS and one was related to an incompetent cervix. This
resulted in a fetal loss rate of 3.8%. The treated neonates had normal birth weight, head
circumference and length consistent with other reports. There were no documented birth
defects.
Dexamethasone is the steroid of choice in prenatal treatment as it is only partially
degraded by the placenta and easily crosses this barrier to reach the fetus [Pang et aI,
1990]. Dexamethasone does not bind well to transcortin (corticosteroid-binding globulin)
and has a relatively long half life of four to six hours. Cortisol is less effective for fetal
therapy as only a small amount crosses the placenta due to its high affinity for transcortin.
In addition, cortisol is quickly degraded by the placenta to inactive cortisone.
When a pregnancy is known to be at risk for congenital adrenal hyperplasia, maternal
treatment with dexamethasone should begin in the fifth week of gestation prior to
definitive diagnosis. The usual starting dose is 20 /Jg/kg/day in three divided doses
[Forest et aI, 1989]. Others have proposed a higher dose of 0.5 mg every 8 hours to lessen
the number of partially virilized females [Loeuille et aI, 1990]. Maternal compliance and
efficacy should be assessed by frequent determinations of serum cortisol and estriol
levels. Maternal estriol levels reflect the conversion of placental pregnenolone to
dehydroepiandrosterone sulphate by the fetal adrenal gland. Success is related to maternal
levels of estriol. In general, low levels correlate with adequate fetal adrenal suppression
and better outcome. Estriol levels should be monitored throughout the second and third
trimesters. A normal cortisol level indicates a need for increased dexamethasone dosing.
Adequate fetal adrenal suppression is obtained with low amniotic fluid steroid levels and
low maternal serum estriol levels at rnidgestation [David et aI, 1984; Forest et aI, 1989].
Treatment failures have been hypothesized to be secondary to late onset of treatment,
poor compliance, treatment interruption, ineffective dosage, or differences in
dexamethasone metabolism [Forest et aI, 1993].
In one series of prenatal therapy patients, a stillbirth at 33 weeks occurred [Karaviti et
aI, 1992]. The authors did not attribute this to the prenatal dexamethasone treatment
which had been stopped at 19 weeks gestation. Forest et al. also reported a fetal demise in
their series, but did not attribute the event to dexamethasone [Forest et aI, 1989]. A large
study looking at the effects of multiple courses of betamethasone therapy for preterrn
labor found an increased mortality in the lower gestational age neonates [Banks et aI,
1999].
From the experience reported in the literature, there has been no association between
prenatal dexamethasone therapy in humans and teratogenicity. In contrast, it is well
established that treatment of pregnant animals with high doses of glucocorticoids is
associated with teratogenic effects. Multiple courses of maternal glucocorticoid therapy
have been associated with growth retardation in fi~tal sheep [Jobe et aI, 1998; Ikegami et
aI, 1997]. In a study of monkeys, fetal growth retardation was found only in those
monkeys that had received pharmacological doses of dexamethasone (40-160 mcg/kg/d)
[Novy et aI, 1983]. Interestingly, daily maternal treatment with dexamethasone has not
been found to cause growth retardation in human fetuses [Mercado et aI, 1995]. There is
no evidence of differences in birth weight, head circumference or length in affected
neonates treated prenatally versus those that were not treated.
In summary, there is no evidence to date of an increase in malformations, growth
restriction, intrauterine fetal demise, or spontaneous abortions following fetal therapy
with dexamethasone. Follow-up to 5 years of age of infants treated in utero has shown no
untoward effects [Forest et aI, 1987). Children followed up to 8.5 years of age appear
normal in terms of growth and cognition [Forest et aI, 1989). Based on the normal growth
and development found in these studies it is believed that prenatal dexamethasone
therapy is a relatively safe form of treatment at present. It is important to recognize that
even if dexamethasone is started prior to 8 weeks and virilization of the external genitalia
is averted, this may be too late to prevent the effect of androgens on the developing brain.
Further long term studies examining cognitive and psychosexual effects as adults are
needed.
Maternal sequelae of prenatal dexamethasone at 1 - 1.5 mg daily include varying
degrees of excessive weight gain, hypertension, glucose intolerance, hirsutism and
excessive striae [Pang et aI, 1992]. Overall, mothers of affected female fetuses treated for
the entire pregnancy had a 57% incidence of relatively minor complications. Despite this,
all mothers were willing to repeat the treatment with subsequent pregnancies. Stress
doses of hydrocortisone should be administered during labor. Maternal steroids should be
weaned following delivery.
It is important to note that prior to the advent of prenatal therapy, there had never been
a female neonate affected with the severe salt wasting form of congenital adrenal
hyperplasia delivered with normal genitalia at birth. Furthermore, female siblings with
the same form of 21-hydroxylase deficiency generally have had the same phenotype with
similar degrees of virilization. In one series of 50 affected infants, 49 required surgical
reconstruction [David et aI, 1984]. Therefore, the fetal adrenal gland does respond to
suppressive dexamethasone therapy. This provides evidence that fetal adrenal function is
regulated by ACTH, at least in part. ACTH is not the entire story though, as it is known
that anencephalic fetuses are not adrenally insufficient [Benirschke, 1956].
Speiser recently reviewed the literature on congenital adrenal hyperplasia and
recorded over 300 pregnancies treated worldwide from 1984 through 1994. From this
population it could be seen that the outcome of preventive therapy could be broken down
roughly into thirds: 113 of neonates were born with normal genitalia, 113 were partially
virilized, and 113 were significantly virilized [Speiser et aI, 1994). These results initially
appear disappointing. However, when the effect of gestational age is considered a more
encouraging picture emerges. Those treated before 8 weeks gestation (9/11 patients) were
normal or minimally virilized. In contrast, those treated after 9 weeks (3/5 patients) were
severely virilized.
The goals of in utero therapy is to ameliorate the masculinization of the female fetus
with congenital adrenal hyperplasia. This would not only avoid the difficulty in gender
identification at birth, but also avoid the need for subsequent corrective surgeries. Even
234 N. E. RINTOlJL, ET AL.
reducing the degree of virilization may be beneficial in reducing the magnitude of genital
reconstructive surgery needed postnatally.
Future Treatments
The imperfect nature of the treatment for congenital adrenal hyperplasia opens the
door to more innovative approaches. The future holds the promise of at least two
potential improvements: in utero gene therapy and in utero adrenal transplantation. Each
offers the potential for definitive cure, but remain theoretical possibilities. The mutations
responsible for CAH have been well characterized which may permit the development of
effective gene therapy strategies. Postnatal gene therapy could eliminate the need for the
current imperfect steroid replacement therapy. Perhaps one day even pre-implant embryo
gene therapy strategies could be developed for parents at risk for CAH.
For adrenal transplantation to be effective the dysfunctional adrenals must first be
removed surgically or suppressed medically. At present, adrenal transplantation has only
been performed in the laboratory setting in animals. In humans, surgical and medical
adrenalectomy has been performed in select cases to simplify management.
Van Wyk reported the use of adrenalectomy in two human patients less than 10 years
old with 21-hydroxylase deficiency [Van Wyk et aI, 1996]. Without adrenal glands these
patients required replacement therapy similar to patients with Addison's disease. It is felt
that adrenalectomy may benefit girls with the most severe form of congenital adrenal
hyperplasia and nonfunctional CYP21 genes. This approach is most effective if
adrenalectomy is performed before the secondary damage of postnatal glucocorticoid
over treatment occurs.
Other investigators have used medication to suppress the adrenal glands [Cutler,
1996]. Antiandrogens, aromatase inhibitors and ketoconazole have all been used to block
the deleterious effects of androgens. Merke reported the results of a pilot study on 12
children using flutamide, an antiandrogen, and tesolactone, an inhibitor of the conversion
of estrogens to androgens [Merke et aI, 1997]. Flutamide and testolactone were used in
combination with a lower daily dose of hydrocortisone. Results suggested that the
combination can reduce the weight gain and rapid bone maturation with early closure of
the epiphyses and resultant short stature that generally occur with the traditional high
doses of hydrocortisone necessary to suppress adrenal androgens.
Studies of adrenal transplantation in animals hold promise. In a syngeneic rat model,
fetal adrenal glands were transplanted into adult rats who had undergone surgical
adrenalectomy [Trammer et aI, 1994]. All of the animals survived and had serum sodium
levels within the normal range. Another animal model demonstrated that adrenal cells
alone could be transplanted into adrenalectomized mice and could survive. Clonal bovine
adrenocortical cells grown in tissue culture have been injected into adrenalectomized scid
mice [Thomas et aI, 1997]. The tissue formed by the injected cells assumed the functions
of adrenal glands and these animals also maintained normal serum sodium levels.
Studies such as these raise the possibility of in utero transplantation of adrenocortical
tissue in human fetuses.
Discussion
Congenital adrenal hyperplasia is the first inborn error of metabolism that has been
successfully treated in utero. To date, most in utero therapy has been surgical. However,
in the case of congenital adrenal hyperplasia, medical therapy in utero has the potential to
prevent the virilization ofthe external genitalia.
Fetal therapy for this disease is unusual in that all at-risk pregnancies are treated
initially, prior to establishing the diagnosis. As a result, eight pregnancies at risk for
congenital adrenal hyperplasia must be treated for everyone fetus that may benefit from
fetal suppressive therapy. Three of four female fetuses will be unaffected following the
laws of autosomal recessive inheritance and hence do not require fetal therapy and no
males require fetal therapy to prevent ambiguous genitalia.
In patients at-risk for congenital adrenal hyperplasia, the fetal adrenal gland is
pharmacologically suppressed by oral maternal dexamethasone starting as soon as the
pregnancy is detected. The importance of initiating treatment early must be stressed:
beginning treatment before confirming the status of the fetus is imperative for success.
Treatment must be initiated prior to 8 weeks gestation, and ideally prior to 5 weeks. This
early time point is particularly important because the urogenital sinus begins to form at 6
weeks. In addition, the sooner therapy is started, the less time the surplus androgens can
effect the developing brain. Fetal therapy is stopped in all males and unaffected females.
Affected females are treated in utero until delivery.
Considerable problems remain with the postnatal treatment of patients suffering from
congenital adrenal hyperplasia: adequacy of reconstruction, issues of gender identity,
overtreatment resulting in Cushing's syndrome, and undertreatment resulting in
precocious puberty and short stature.
The goals of treating congenital adrenal hyperplasia in utero are to prevent virilization,
avoid or minimize reconstructive surgery, and prevent the psychologic sequelae of gender
ambiguity. Despite the pitfalls in reaching a diagnosis and the variable results based on
gestational age, the benefits of in utero therapy for congenital adrenal hyperplasia
outweigh the risks.
Future endeavors involving gene therapy and adrenal transplant are on the horizon, but
at present dexamethasone therapy should be offered for all at-risk pregnancies.
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Questions for Dr. Crombleholme
Question - Your last line of your last slide indicates that it is uncertain what the treatment
might do after 10 weeks on the developing brain. But are you certain about the effect of
the treatment before 10 weeks? There is a whole evolving field which is called
functional teratology i.e. what are the effects of chemicals on the developing brain. It is
clear that you can't say at the moment of birth that the child does not have functional
defects. You have to follow them up until the school going age, so even until the
reproductive age in order to say so. So I wonder what all the children that I have treated
with dexamethasone which is known to interrupt the cell cycle of nerve cells during cell
division might do on those children?
Answer - You make a good point. They have already sort of moved out of the window
where you would affect virilization of the external genitalia. And in general, the answer
has been, well you are not going to affect that, why continue therapy. All I am saying is
that it may be worth considering continuing treatment in those patients. What affect has
happened on the developing brain of those children who were treated as early as 5 or 6
weeks of gestation really has not been looked at and so I think it is an open question.
Question - The red queen in Alice in Wonderland's being quoted before here already.
One of our other famous quotes is sentence first - trials later. And dexamethasone
treatment in a sense is falling into that category, to a degree. In the rat there is clear
evidence of their effect on glucocorticoid receptors withthe hippocampus, and
hippocampal function. And given the fact that in those same animals treated with
dexamethasone, by the time that those animals hit puberty they have clear glucose
intolerance type II diabetes and hypertension. What are we actually trying to achieve
with this treatment? It is not true to say, I do not think, based on that data - on those data
- say that this stuff is safe. We simply do not know what the long-term effects are. It
may be that you are rightly trading off the effect of virilization with all it's accompanying
problems. But substituting it - with developing the risk factors for cardiovascular disease
later. And the most important issue really comes from the fact that it is not just the trade
off that is happening in those individual females who are affected. They are actually
imposing this treatment on certain other individuals who would never be exposed to
dexamethasone in that stage of gestation.
Response - You are correct. That is something that the family needs to buy in to. But
they will be exposed for a very short time between 8 and at most 18 weeks gestation to
glucocorticoids. Now despite that, not just in the issue of the treatment of CAH, but in
other patients who are on glucocorticoids during gestation for other indications, there
really is not a lot of data to suggest that there are a lot of adverse fetal effects. Now the
long term effects that you were referring to, no. I do not think - I am not aware that
anyone has looked that far, but I think you can only extrapolate so far from a rat to a
human fetus.
Response - Maybe I can add something to this. We published 2 years ago, a pilot study
behavior follow-up on children treated prenatally with dexamethasone, and among other
things included several questionnaires for kids of preschool age. We found in
comparison to controls, increased emotionality and internalizing psychosomatic
symptoms in these kids, but that was a sample of about 40. Currently we have a major
study on the way of about 500 total. This study was driven by the primate data that
prenatal glucocorticords have effect on the hippocampus of primates also. And we are
doing a second study which has not got us far where we do a very intense face-to-face
interview doing a psychological test and behavior observation study on kids with this
condition, too. Our total will be about 128 that we will report next year.
ANATOMICAL STUDIES OF THE FETAL GENITALIA:
SURGICAL RECONSTRUCTIVE IMPLICATIONS
Laurence S. Baskin, MD,

Department of Urology, University o/California, San Francisco
INTRODUCTION
Under the influence of the SRY gene, testes determining factor, and androgens fetal
differentiation precedes to form the normal male genitalia. Genetic and environmental
abnormalities can disrupt normal development leading to an ambiguous state of the
genitalia. In most societies it is the accepted norm; that the genitalia either look male, or
female, with an ambiguous state not being socially acceptable. Based on societal norms,
the previous standard of care was to surgically recreate either the male or the female
physical appearance. Long-term data validating this approach, however, is lacking and
recently there has been an effort to document the optimal treatment for patients who do
not fit into either a classic male or female category. The new concept that is gradually
being embraced is that sex assignment is not an irrevocable phenomena. In other words,
it is not a social or medical emergency to assign a sex immediately after birth, and in fact,
the sex of the patient may actually change during life depending on genotypic,
phenotypic and environmental conditions. This awareness has been initiated by both
physicians as well as advocate groups with the hope that difficult but correct decisions
can be made for patients with ambiguous states in the future.
In an attempt to be able to understand the anatomy of both normal and abnormal
genital development, we have embarked on an extensive histologic analysis of fetal
specimens. These studies by their inherent nature are not complete but are in progress
and depend on the availability of adequate tissue for analysis. The present paper is based
on over 50 fetal specimen that have been carefully analyzed. The anatomy of the human
genitalia has been looked at from a structural, cellular, neural and vascular point of view.
The implications of the fetal anatomy has been applied to the strategic design of
reconstructive procedures.
Materials and Methods
These investigations have been approved by the Institution's Committee on Human

Research. Human fetal male and female specimens between 8weeks and the newborn
period were analyzed after being embedded in paraffin and serial sectioned (6/-1).
Specimens with hypospadias were also studied. Fetal sex was determined by
fluorescence in situ of the X and Y chromosome specific probes. The entire fetal
specimen was preserved with each slide containing 2 to 4 histologic sections. Specimens

Edited by Zderic et al., Kluwer Academic/Plenum Publishf~rs, 2002
240 L. S. BASKIN
were dated based on fetal femur length when possible, or fetal heal-toe length. Staining
was performed with hematoxylin and eosin and Masson's trichrome.
Immunohistochemical staining was performed selectively with antibodies raised against
smooth muscle a-actin, neural markers S 100 and PGP 9.5, epithelial cyto-keratin
markers and markers for blood vessels, such as von Willebrand's and factor 8. The
avidin-biotin peroxidase procedure was used with Vectastain ABC kits (Vector·
Laboratories, Burlingame, CA» and cobalt intensification. Antibodies against smooth
muscle a-actin and the neuronal markers S 100, as well as the cytokeratins were obtained
from Sigma (St. Louis, MI). Anti-neuronal cytoplasm protein gene product 9.5 was
obtained from ultra clone in the island Well's United Kingdom. Double immunostaining
for both smooth muscle, a-actin and the neuronal markers S100 and PGP 9.5 were
performed using biotinylated secondary antibody obtained, obtaining a brown color with
antimouse-IgG from sheep. A pink stain was obtained using biotinylated secondary
antibody antimouse IgG made from horse and labeled with alkaline phosphatase factor
AK-5002. The substrate was Vector Red (Vector SK-5100) (Vector Laboratories
Burlingame, CA).
Anatomical three-dimensional computer reconstruction was created of normal male,
female and specimens with hypospadias using an Olympus digital camera, Adobe
Photoshop, 5.1, NIH imaging and an Apple computer, as described in previous
publications (Baskin et aI, 1997,1998,1999). In short, every 10th to 20 th section was
imaged and then digitized. The corporal or erectile body, glans, foreskin, clitoral hood
and areas of interest were manually outlined. Nerves and vascular structures as well as
the corporal erectile tissue outlined were contrasted and enhanced with the respective
background. Accuracy of the enhanced images was checked against still photographs of
the original immunostained specimens. Three-dimensional reconstruction was performed
in the X and Y axis. Animated motion picture and views of interest were captured as
images. Coloring of the genital anatomical structures was artificially assigned.
RESULTS
Normal Human Fetal Penis
Localization of nerves with anti-PGP neuronal marker was noted in specimens of all
fetal ages--prominent dorsally at 11 and 1 o'clock, but also extending around the tunica
to the junction of the corpus spongiosum and corpora cavemosa suggesting that we may
be injuring these structures in penile straightening procedures (Baskin et aI, 1996,1997).
(Figure 1 & 2 ) The nerves continued into the glans on the dorsal aspect suggesting that
glans reduction in feminizing genitoplasties should be performed on the ventral aspect.
Smooth muscle was first noted at 10 weeks' gestation, with epithelial differentiation
occurring in the earliest specimens studied (8 weeks' gestation) (Baskin et aI, 1997).
Over time, smooth muscle density was highest in the corpus spongiosum, especially
between urethra and the corpora cavemosa. Smooth muscle also developed in close
proximity to the urethral epithelium. The tunica albuginea showed consistent variations
ANATOMICAL STUDIES OF THE FETAL GENITALIA 241
in thickness, with the mid dorsal 12 o'clock position being the thickest followed by the 5
and 7 o'clock periurethral positions,
Figure 1. Normal Human fctal penis, 25 weeks.
A. Transverse section distal penile shaft (25X) S I 00

B. Transverse section distal penile shaft (25X) S 100
C. Transverse section mid penile shaft (25X) SIOO
D. Transverse section mid penile shaft (25X) S I 00
E. Transverse section proximal penile shaft (25X) S I 00
F. Transverse section proximal penile shaft (25X) SIOO
G. Transverse section proximal penile shaft (25X) S I 00
H. Tra'nsverse section proximal penile shaft (25X) SIOO
Note localization of S-IOO nerve marker in brown completely surrounding the cavernous bodies up to the
junction with the urethral spongiosum along the penile shaft except at the 12 o'clock position (A-D). On the
proximal penis at the point where the corporal bodies split into two (E) and continue in a lateral fashion inferior
an adjacent to the pubic rami the nerves localize to an imaginary triangular area at the II and I o'clock position.
At this point (E) the nerves reach there furthest vertical distance from the corporeal body (- one half the
diameter of the corporeal body) and continue (F-G) in a tighter formation at the II and I o'clock position well
away from the urethra. (Used with permission [Baskin et aI, 1998)),
242 L. S. BASKIN
Figure 2. Normal Human fetal penis, 25 weeks' gestation. Four views of a computer-generated three-
dimensional reconstruction (A, Side; B, Front; C, Side; D, Back E, Front (without urethral); F, Side (without
urethral)). Note the nerves in red and their absence at the 12 o'clock position. The tunica is represented in
blue, the urethral lumen in yellow and the urethral spongiosum and prepuce in lime. (Used with permission
[Baskin et ai, 1998]).
Normal Human Fetal Clitoris
The normal fetal clitoris consists of two corporal bodies with a midline septum
(Baskin et aI, 1999). (Figure 3 & 4) The ultrastructure of the female corporal bodies is
similar to the male counterpart. The glans clitoris forms a cap on top of the distal end of
the narrowed corporal bodies. The glans clitoris has a midline septum starting on the
ventral aspect and extending ~half way into the glans. Large bundles of nerves course
along the corporal bodies with the highest density on the dorsal aspect (top). No nerves
are noted at the 12 o'clock position, however, nerves extend completely around the
tunica, in a fashion similar to that of the fetal penis. The innervation of the glans occurs
via multiple perforating branches that enter at the dorsal junction of the corporal body
and the glans. The lowest density of nerves is in the glans on the ventral aspect in
juxtaposition to the glans septum (Figure 3 and 4).
ANATOMICAL STUDIES OFTHE FETAL GENITALIA 243
Figure 3, Nonna! human tina! ditoris 24 weeks, t\·lL Serial coronal sections through the nurmal thnah:
ditorh $lailKd with SIOO r~,pre~cnljng nCTVC$ (arrows), Magnifkalkm 4(}X,A dit()nd hond; labiil minonl and
majora, B Nt'fVt£ on {(II' of thccfccti!c body and lOp of glans c!ilt)fis (asterisk) C-E glans ditonsand
er(,ctik bodies f-G, klwcr part of glans clitoris with midline ddi- H, end of glans dilf)ris and vllgin;ll
mtrOltus, (Used with pcmlission [Buskin et ai, 1999j),
Figure 4. Normal Human fetal clitoris, 24 weeks' gestation, Four views of a computer-generated three-
dimensional reconstruction (A, Top; B, Bottom; C, Back/Top; D, Bottom. Note the pathway of the nerves in
red with a paucity of nerves on the bottom of the clitoris as well as in the top midline. The tunica of the
corporal bodies is represented in purple with the inside of the corporal bodies being yellow. The glans clitoris
is represented in green with the clitoral hood represented in dark green and yellow. (Used with permission
[Baskin et ai, 1999)).
244 L. S. BASKIN
Anatomy of Hypospadias
A unique opportunity came about when fetal specimens with hypospadias were
obtained for anatomical analysis (Baskin et aI, 1998). The hypospadiac penis showed no
difference in neuro and vascular anatomy except at the region of the abnormal urethral
spongiosum and glans. (Figure 5 & 6) The corporal bodies, tunica albuginea and
vascular anatomy were similar. The nerves started proximally as two well defined
bundles under the pubic rami superior and slightly lateral to the urethra. As the two
crural bodies converged into the corpora cavernosa bodies the nerves diverged, spreading
around the cavernous bodies up to the junction with the urethral spongiosum not limiting
themselves to the 11 and 1 o'clock position. Along the entire shaft of the penis the 12
o'clock position did not have neuronal structures.
Figure 5. Hypospadiac Penis, 33 weeks' gestation
A. Transverse section glans ( 16X) SIOO

B. Transverse section distal penile shaft (25X) S 100
C. Transverse section distal penile shaft (25X) S 100
O. Transverse section mid penile shaft (l6X) S 100
E. Transverse section proximal penile shaft (l6X) S I 00
F. Transverse section proximal penile shaft (16X) SIOO
Note the anatomy of the hypospadias penis is the same as the normal penis except for the abnormal
formation of the distal urethra and glans (A-C). The nerves are black staining in Figures A-O and brown
staining in E and F. (Used with permission[Baskin et aI, 1998]).
The most striking difference between the normal penis and the hypospadiac penis is
the difference in vascularity. The hypospadiac penis has huge endothelial lined (factor
VIII immunostain) vascular channels filled with red blood cells (Figure 5). In contrast,
the normal penis has well defined small capillaries around the urethra and fanning into
the glans (Figure 1). The vascularity under the urethral plate was also extensive. The
nerve distribution in the abnormal glans is also less extensive when compared to the
normal penis.
Figure 6. Hypospadiac Penis, 33 weeks' gestation. Four views of a computer-generated three-dimensional

reconstruction. (A, Side; B, Back; C, Front; D, Side). Note the nerves in red and their absence at the 12 o'clock
position. The tunica of the corporal bodies is represented in blue in A and B and blue and yellow in C and D,
the urethral lumen in orange and the urethral spongiosum in lime. The hypospadiac penis has the same
innervation and anatomy as the normal penis except the abnonnal urethral spongiosum. (Used with permission
[Baskin et aI, 1998]).
Reconstructive Applications
Penile Curvature
Increased knowledge of the normal penile anatomy and the anatomy in the
hypospadiac penis with respect to the nerves, the corporal bodies, the glans and the
vascularity is useful in the strategic design of penile reconstructive procedures (Baskin et
aI, 1998). Based on these anatomical studies we have modified our approach to the
correction of penile curvature associated with hypospadias. Previously, we advocated the
TAP (Tunica albuginea plication) procedure (Baskin et aI, 1994,1995). We now feel that
it is impossible to "lift up out of harms way" the neurovascular bundle as previously
246 L. S. BASKIN
described without damaging some of the branches of the neurovascular bundle that
clearly wrap well around the penis. We now perform penile straightening by placing
rows of plication sutures at the 12 o'clock position in a nerve free area (Figure 7) (Baskin
et aI, 1998). The 12 o'clock position is also the thickest and hence the strongest part of the
tunica (Baskin et aI, 1998). This technique has been successfully applied to young men
with congenital penile curvature (Baskin and Lue, 1998). We have also used the 12
o'clock plication technique in patients with penile curvature secondary to hypospadias
with successful short term results (Baskin et ai, 1998).
Figure 7. Dorsal Plication Nerve Sparing Technique. Schematic of penile straightening techlllyue uSlIlg
plication sutures In the 12:00 o'clock position thereby avoiding the nerves of the penis. (Used with permiSSion
[Baskin et ai, 1998J).
Feminizing Genitoplasty
A clear understanding of normal female anatomy should benefit our design of

reconstructive surgery in patients with congenital adrenal hyperplasia. The goal of
feminizing genitoplasty is to recreate the normal female anatomy. Historically, the
enlarged clitoris/phallic structure has been dealt with by amputation (Gross and Crigler,
1966). Subsequently, more refined techniques such as recession clitoroplasty were
developed where the entire' clitoral organ is preserved by imbricating and burying the
proximal corporeal shaft and excess glans clitoris. The disadvantage of the clitoral
recession procedures may not become apparent until puberty when the recessed corporeal
bodies become enlarged and painful during sexual stimulation. To prevent this problem
Spence in 1973 developed a pwcedure involving subtotal resection of the shaft of the
clitoris with preservation of the glans (Spence and Allen, 1973). Further techniques to
preserve the neurovascular bundle via a ventral approach have been described by Mollard
and Goodwin (Rajfer et aI, 1982;Mollard et aI, 1981). Presently, more conservative
procedures have been employed to preserve both the sensory and cosmetic aspects of the
clitoris (Kogan et aI, 1983; Snyder, 1983; Duckett and Baskin, 1993). Nevertheless,
despite careful surgical technique nerves were still noted adjacent to the tunica (Figure 8).
Our approach to reduction clitoroplasty is based on the observations from our anatomical.
We propose that the tunica of the corporal body be preserved to spare as much of the
dorsal nerve as possible (Baskin, 1999). (Figure 9) The concept of lifting the dorsal nerve
off the tunica at the 11 and 1 o'clock seems inconsistent with the fact that the nerves fan
out extensively around the dorsal and lateral aspects of the clitoral body.
A second issue is the removal of erectile tissue. In severe cases of masculinization of
the genitalia it seems reasonable to reduce the amount of erectile tissue. Standard
treatment was to amputate the erectile body of the clitoris at the pubic arch leaving each
crural body and the neurovascular bundle with a strip of dorsal tunica. The long-term
effects of removing this erectile tissue on sexual function are unknown. In contrast,
leaving too much erectile tissue has been reported to cause pain in patients at the time of
puberty. A compromise, maybe, is to incise the corpora cavemosal body on the ventral
surface (bottom) at the 6 o'clock position remove erectile tissue within the tunica to
reduce the size of the erectile body thereby presenring some erectile tissue and all of the
nerves of the clitoris (Figure 9) (Baskin et aI, 1999).
Figure 8. Human specimen obtained at the time of feminizing genitoplasty. A-C. SIOO staining representing
nerves (arrows), transverse section. See high power of B. tunica of the corporal body with nerves and C.
erectile tissue. Note the thickness of the tunica of the erectile bodies. D-E. Serial transverse section stained
with smooth muscle a-actin (brown). Note the blood vessels (arrowheads) and the erectile tissue (asterisk)
and high power insert. (Used with permission [Baskin et ai, 1999]).
248 L. S. BASKIN
Figure 9. Schematic of Feminizing Genitoplasty based on Neuroanatomical Studies Clitoroplasty incision

B. Schematic representation of nerves based on anatomical studies C. Mobilization of urethral plate and outline
for ventral incision into corporal bodies D. Reduction of erectile tissue and incision for glans reduction E.
Corporal and Glans Reduction F. Reduced glans clitoris with erectile preservation. (Used with permission
[Baskin ct ai, 1998]).
The final issue is the reduction of the glans clitoris which may be the only surgical
procedure required for reconstruction if used in conjunction with recession of the clitoral
erectile tissue in less severe cases. Our anatomical studies would support reduction on
the ventral (bottom) part of the glans where the innervation is less dense than on the
dorsal aspect (Figures 3 & 4) (Baskin et ai, 1999). This should maximize sensation and
hence sexual function.
Conclusion
In conclusion, we have carefully defined the developmental anatomy of the normal

penis, the hypospadiac penis and the clitoris. Attention to anatomical detail will improve
surgical techniques for the reconstruction of patients with penile curvature and
masculinization of the external genitalia. The concept of sex assignment as a definitive
irrevocable psychological and surgical procedure is being revisited. Is it not
unreasonable to consider that sex assignment may be 'in progress" and not "definitive."
When indicated surgery should be as anatomically based as possible.
Supported by NIH Grants K08 DK02397-04, and R01 DKS7246-01

REFERENCES
Baskin LS, et at. Anatomical studies of Hypospadias. J UroI162:1108-1115,1998.

Baskin LS, et at. Anatomical Studies of the Human Clitoris. J Uro1162: I 015-1020, 1999.
Baskin LS, Lee YT, Cunha GR. Neuroanatomical ontogeny of the human fetal penis. Br J Uro/79(4):628-640,
1997.
Baskin U, Duckett lW, Lue T. Penile Curvature. Urology 48(3):347-356, 1996.
Baskin LS, Duckett JW. Dorsal tunica albuginea plication for hypospadias curvature. J Urol 151(6):1668-
1671,1994.
Baskin LS, Lue T. The correction of congenital penile curvature in young men. Br J Urol Inti 81(6):895-899,
1998.
Duckett lW, Baskin LS. Genitoplasty for intersex anomalies. Eur J Pediatr S80-84, 1993.
Gross R, Crigler R. Clitorectomy for sexual abnormalities, indications and techniques. J Surg 59:300-308,
1966.
Kogan S, Smey P, Levitt B. Subtunical total reduction clitoroplasty: A safe modification of existing techniques.
J Urol 130:746-748,1983.
Mollard P, luskiewenski S, Sarkissian 1. Clitoroplasty in intersex: a new technique. Br J Urol 53(4):371-373,
1981.
Rajfer J, Ehrlich RM, Goodwin WE. Reduction clitoroplasty via ventral approach. J Urol 128(2):341-343,
1982.
Snyder H, et at. Feminizing genitoplasty: A synthesis. J Uro1129: I 024-1 026, 1983.
Spence 1-1, Allen T. Genital reconstruction in the female with adrenogenital syndrome. Br J Urol 45: 126-130,
1973.
FEMINIZING GENITOPLASTY
Terry W. Hensle, MD & Jonathon Bingham, MD

Columbia University, College of Physicians & Physicians, Department of
Urology, Division of Pediatric Urology,Babies & Children's Hospital of New York, New
York, NY
INTRODUCTION
Feminizing genitoplasty is usually preformed in patients with genitalial ambiguity and

most commonly in patients with congenital adrenal hyperplasia. Congenital adrenal
hyperplasia (CAH) is the most common cause of ambiguous genitalia in newborn
children with an incidence of approximately 1: 14,000 live births (Hughes, 1988). CAH is
an autosomal recessive disorder in which the 46 XX female fetus is exposed to elevated
levels of adrenal androgens, resulting in varying degree of virilization of the external
genitalia and distal vagina. As there is no production of mullerian inhibiting substance
(MIS), the internal mullerian structures form normally.
The three structures that are commonly affected by the high androgen levels are the
clitoris, labioscrotal folds, and the urogenital sinus. A successful feminizing genitoplasty
must deal with all of these structural abnormalities in order to ensure a good cosmetic
result and adequate sexual function in individuals born with the virilizing form of CAH.
In the past, genitoplasty and vaginoplasty were preformed as separate, staged procedures.
Since the 1970's, however, many authors have shown that a one-stage operation is a more
efficient approach and that the cosmetic and functional outcome is usually superior. The
objectives of the one-stage procedure are to (1) reduce the size of the enlarged clitoris
while maintaining vascularity and sensory innervation, (2) reduce and feminize the
labioscrotal folds, and (3) most importantly, address the urogenital sinus, which usually
involves creating a separate vaginal introitus in the perineum.
Clitoroplasty
Over the years a number of techniques for clitoroplasty have been described, many of
which are now obsolete, such as total resection of the clitoris (Gross et aI, 1966) - (Fig. 1
A, B, C) and clitoroplication with recession of the corporal bodies without resection
(Lattimer, 1961) - (Fig. 2 A, B, C). All of the currently described surgical techniques for
clitoroplasty emphasize preservation of the glandular blood supply and sensation while
reducing corporal bulk.

252 T. W. HENSLE, ET AL.
®
CORPORA DISSECTED
OFf PUBIC RAMI
COMPLETED
t;L lTQRIO£eTOMY
Figure 1 A,B, & c. This diagram shows the now obsolete technique of complete clitoral resection (pan1es A
and B) , with a postoperative result shown panel C.
COIIPORt.
OISSECTED
FROIo! PIIelS
---
(~
H)~
SHAFT SUTURED
TO PUB, S
~ ~ q
A c
Figure 2 A,B,C. The technique of clitoroplication as described by Lattimer (L:attimer, 1961) in which the
corpora are dissec ted free of the pubis (panel A), and then are tacked back onto the pubis using interrupted
sutures (panel B and C).
Clitoral recession may be a valid possibility for children with mild clitoromegaly and
it is less involved than a reduction procedure (Ansell et aI, 1981; Randolph et aI, 1981).
When clitoral recession is preformed on a child with a large phallus, the result can be a
painful, conspicuous bulge during corporal engorgement (Fig. 3A). A modification of
clitoral recession was described by Hendren and Crawford (Hendren et aI, 1969), in
FEMINIZING GENITOPLASTY 253
which the glans is reduced by trimming the lateral margins, thus reducing the amount of
tissue to be concealed and in 1967, Stefan (Stefan, 1967) described a corporal plication of
the shaft. Using heavy longitudinal through-and-through sutures on each side of the
shaft, avoiding the neurovascular bundles, the shaft is shortened, partially obliterating the
erectile spaces. Another described technique is excision of multiple wedges of tunica
vaginalis as a way of shortening the shaft (Glassberg et aI, 1981). All of the techniques
for recession of the phallus are only possible in cases of mild-to-moderate clitoromegaly.
In severe cases, recession procedures are inadequate, and clitoral reduction is indicated.
The clitoral reduction technique originally described by Spence and Allen in 1973 is
now the most commonly performed (Spence et aI, 1973). As described, the corporal
bodies are completely resected, and the glandular tissue is debulked with meticulous
preservation of the dorsal neurovascular bundle (Fig. 3 B,C). The phallic skin is also
preserved and used as flaps in order to recreate the labia minora (Fig 4 A, B). Many
authors feel that this procedure was best performed in the first three to six months of life
(Hensle et aI, 1998; Passerini-Glazel, 1999).
Figure 3 A,B,C. In Figure 3A, clitroal recession alone has resulted in a painful and conspicuous bulge during
corporal enalrgement. As a consequence alternative procedures utilizing corporal reduction with or without
clitoral redcution evovloed. The method developed by Spence and Allen (1973) is shown in figure 38, where
the corpora) bodies are separated from the urethral spongiosum and the dorsal neurovascular bundles. This
allows for their remrnova) as shown in figure 3C.
The clitoral reduction technique originally described by Spence and Allen in 1973 is now
the most commonly performed (Spence et aI, 1973). As described, the corporal bodies
are completely resected, and the glandular tissue is debulked with meticulous
preservation of the dorsal neurovascular bundle (Fig. 4 A, B). The phallic skin is also
preserved and used as flaps in order to recreate the labia minora (Fig 5 A, B). Many
authors feel that this procedure was best performed in the first three to six months of life
(Hensle et aI, 1998; Passerini-Glazel, 1999).
Figure 4A,B. The excess phallic skin shown in fgiure 4A was used to recreate the labia minora shown shown
in figure 48.
Correction of the Urogenital Sinus/Vaginoplasty
Urogenital sinus abnormalities occur in a wide spectrum. In some instances the

vagina communicates with the urethra close to the perineum and in others, the vagina
joins the urethra more proximally, resulting in a long common channel or urogenital
sinus. In patients with a distal urethrovaginal connection, surgical repair can be
performed as a simple perineal procedure. There is little controversy and over the
techniques used in repair for these patients.
There is, however, significant controversy surrounding the repair of the patient with a
high vaginal-urethral connection. The anatomical distinction of high versus low
urogenital sinus is based on its relationship to the urethral sphincter. If the connection is
at the urethral sphincter or greater than two centimeters from the perineum, it is
considered to be a high urogenital sinus (Fig. 5A). Ifthe vaginal connection is lower and
closer to the perineum, it is considered to be a low urogenital sinus (Fig. 5B).
I
,(
Figure SA,B. Figures showing the distinction between a high CAl versus low (8) urogemtal sinus. If the
distance tram the perinuem to the urethrovaginal confluence exceeds 2 cm, the classifications scheme defines
this as a high urogenital sinus.
Low Vaginal Repair
When the vagina joins the urethra to form the urogenital sinus close to the perineum,
the surgical correction is relatively simple. The vaginal opening can be widened and
brought onto the perineum with or without the use of an inverted U skin flap. Dissection
for this procedure is routinely done via a perineal approach. Low vaginoplasty is best
performed using a flap of perineal skin (Fig. 6 A, B).
Figure 6 A,B. Preoperative views of the patients perineum shoing the outline of the U-shaped skin nap and the
Foley catheter in position. Traction sutures in the glans and labia help to provide exposure (fig. 68).
The procedure is done with the patient in the lithotomy position, and endoscopy is
used at that time in order to fully understand the anatomy and define the surgical
landmarks. A catheter is placed into the bladder anteriorly, and an inverted U-flap of
perineal skin is outlined and developed using electrocautery. The perineal skin flap is
held with traction sutures, and the length of the flap should roughly correspond to the
length of urogenital sinus to be opened. Once the perineal U-flap has been developed,
the urogenital sinus is opened in the midline posteriorly until the vagina is identified and
entered. The vagina can then be gently separated from the rectum posteriorly. It is often
helpful to have placed a rectal tube preoperatively or the nondorninant index finger
intraoperatively to help identify the anterior rectal wall. The inverted U-flap of perineal
skin is then turned in to join the apex of the posterior vagina and sutured in place using
interrupted long acting absorbable sutures (Fig. 7 A,B,C). The dorsal shaft skin of the
tJhallus, which is mobilized during the clitoral recession, is then divided in the midline
and brought inferiorly to join the newly exposed urogenital sinus, thereby creating labia
minora. The labioscrotal folds can then be mobilized, reduced in size and bulk, and
sutured inferiorly to create labia majora (Fig. 7 D). Postoperatively, the vaginoplasty is
stented for 3 days using either antibiotic impregnated gauze packing or a vaginal stent of
appropriate caliber. A Foley catheter is left in place for as long as the vaginal stent
remains (Fig. 7 D).
256 T. W. HENSLE, ET AL
o
Figure 7 A-E. Critical steps in carrying out a feminizing genitoplasty. The creation of a U shaped flap (Fig
7A) allows for the transfer of a vascularized flap of perineal skin into the urogenital sinus (Fig 78,C) which is
exposed via a midline approach. Dorsal shaft skin which is mobilized during the clitoral recession is incised in
its midline (Fig 78), and repositioned and reduced to create labia majora (Fig 7 D). Drains, a Foley catheter,
and a vaginal packing are left in place upon completion of the procedure.
High Vaginal Repair
In a relatively few children with severe virilization secondary to CAH, the vagina
enters the urogenital sinus at or above the urinary sphincter. For these patients,
vaginoplasty is often difficult and there is an increased risk of injury to the urinary
continence mechanism as well as the rectum due to the difficulty of dissection and
intricate reconstruction required. Many techniqu(~s have been used in an attempt to
restore normal appearing and functional anatomy, none are perfect.
Perineal Approach
Hendren and Crawford originally described the vaginal pull-down technique of

reconstruction in combination with both perineal and laterally based skin flaps (Hendren
et aI, 1969). In that procedure, after a small balloon (Fogarty) catheter is placed into the
vaginal introitus with the aid of an endoscope, the urogenital sinus is approached
perineally (Fig. 8A), just as one would do in a low-flap vaginoplasty (Hendren et ai,
1995). The dissection is carried out superiorly between the rectum and the urogenital
sinus until the catheter is encountered and the vagina is then divided just where it joins
the urethra (Fig. 8B). The proximal portion of the urogenital sinus can be tubularized for
a short distance and closed in two layers in order to create a longer functional urethra and
avoid the problem of female hypospadias.
The vagina is then mobilized anteriorly from the bladder and posteriorly from the
rectum (Fig. 8C). Because there is very little anterior vaginal tissue to bring to the
perineum, both lateral skin flaps and perineal skin V-flap have to be mobilized to bring
up into the high vagina (Fig. 8D). It is not unusual for patients to require mUltiple
vaginal dilations following this kind of procedure, and often, chronic home dilation is
necessary. In addition, the cosmetic appearance of the perineum is less than perfect.
Combined Approach
More recently, Passerini-Glazel (Passerini-Glazel, 1989) has described another

anterior approach to the high vagina. In this procedure the phallus is de gloved and
reduced in a fashion similar to that already described. The corporal bodies are freed and
resected, and the urogenital sinus is mobilized inferiorly. Once this has been
accomplished, the dorsal phallic shaft skin is mobilized both anteriorly and inferiorly and
divided in the midline (Fig. 9A). The urogenital sinus is then opened anteriorly with a Y
incision, and the previously mobilized anterior phallic skin flaps are brought inferiorly
and sutured to the open urogenital sinus and tubularized (Fig. 9B). This procedure allows
the urethral meatus to be in the normal sub clitoral position and allows the mobilized,
tubularized skin flaps to be free and redundant enough to move up into the perineum for
anastomosis to the divided vagina.
As originally described, the bladder can then be opened, and the posterior wall of the
bladder split in the midline (Fig. 9C). This dissection is carried to the junction of the
urogenital sinus and the vagina, with much of the mobilization carried out under the
bladder neck, which can be very difficult. It is often helpful to place a Fogarty catheter in
the vaginal opening preoperatively to allow better identification of the junction of the
urogenital sinus and vagina. Once the area has been identified and divided, the urogenital
sinus can be closed under direct vision to avoid postoperative fistulization (Fig 9C). The
mobilized phallic skin tube is then carried through the perineal opening and brought
upward sub trigonally for an anastomosis to the high vagina (Fig. 9C). Postoperatively,
packing and a urethral catheter usually remain in place for 4 to 5 days. The advantage of
this kind of procedure for the high vagina is that it can be performed in one stage, it
avoids postoperative vaginal stenosis, and it leaves the urethral meatus in a normal
position (Fig. 9D). In most instances the bladder does not have to be opened and the
procedure can be done through the perineum.
I
)
A
Figure 8 A-D. The Hendren Crawford technique for managing a high vaginal repair is performed
via a perineal approach (Hendren et aI, 1969). A Fogarty catheter is passed into the vagina under
endoscopic control (Fig. SA). The dissection is carried to the confluence of the urethra and vagina
(Fig. A, 8), and the vagina is dissected free of its attachment to the sinus (Fig S 8). The site where
the vagina was formerly attached to the sinus is closed with interrupted sutures (Fig Se) and the
vaginal cuff is then pulled inferiorly and anastomosed to the perineal skin flap. Lateral skin flaps
have to be added to compensate for the lack of anterior vaginal wall (Fig 8D).
)8
Figure 9 A-D. The combined perineal and transvesical exposure of the urethral vaginal confluence in the
management of a high urogenital sinus as described by Passerini-Glazel (Passerini-Glazel, 1989). The phallus
is de3gloced, the corporal bodies are resected, and the urogenital sinus is mobilized inferiorly as much as
possible,. l"e sinus is opened anteriorly with a Y incision (Fig. 9A) which allows for a plate of sinus to be
anastomosed to the shaft skin (Fig. 98). The three skin flaps shown in Fig. 98 are then rolled into a tube which
is then passed back to be anastamosed to the vaginal introitus which has been exposed through a transveslcal
and trans-trigonal approach (Fig. 9C). The urethral meatus ends up in a normal position (Fig. 9D).
Posterior Saggital Approach
Alternatively, Pena has described and popularized the posterior sagittal approach for
treatment of the high urogenital sinus (Pena et aI, 1992). He has described a similar
technique for repair of anorectal malformations, including cloaca (Pen a, 1989). The
transrectal posterior sagittal approach yields excellent anatomical exposure while
maintaining the important neurovascular structures of both the rectum and the urinary
continence mechanism. In the Pena procedure, it is safer to have the child undergo a
descending colostomy prior to the repair of the urogenital sinus. This is a protective
measure as infection can jeopardize not only the healing of the reconstruction, but also
the fecal continence mechanism.
For the reconstruction, the child is positioned prone with the pelvis elevated to give
adequate exposure to the perineum. A midline incision is made from a few centimeters
above the coccyx to the opening of the urogenital sinus (Fig. 10). Care is taken to ensure
that the incision is precisely in the midline, thus leaving an equal quantity of muscles on
both sides of the incision. In Pena's view, a midline incision through the rectum avoids
the need for possible circumferential dissection of the rectum which can lead to
de nervation and possible fecal incontinence.
Figure 10. This figure shows the view of the perineum and the midline incision made running from the tip of
the coccyx down to opening of the urogenital sinus. It is essential that this incision be made precisely in the
midline, thus leaving an equal amount musclde mass on either side and minimizing any denervation.
Dissection is continued in the midline, through the pectinate line. Once the urogenital
sinus has been opened, the anterior vaginal wall will come into view and a Foley catheter,
having been placed in the bladder can be visualized through the urogenital sinus (Fig. 11
A, B). The vagina can be separated from the urethra, creating a plane between the two,
this, however, is a very tedious and difficult dissection. Errors should be made on the
vaginal side as opposed to the urethral side, as holes in the vagina can be easily closed
and the entire vagina reattached to move the vagina away from the urethral closure.
Once the vagina has been completely mobilized, a circumferential dissection of the
vagina is then continued and the urethra is then reconstructed in two layers over the Foley
catheter (Fig. llC). The urethra and vagina are then brought out and sutured in place on
the perineum, with the vagina directly posterior to the urethra. The perineal body is then
reapproximated from the anterior to the posterior. The rectum is reconstructed in two
layers, both on the anterior and posterior rectal walls, thus reapproximating the
parasagittal fibers, the levator muscles, as well as the rectum.
Coccyx
Figure 11 A-C. Critical steps in the Penna approach to a high urogential sinus. The rectal wall has been split
at the 12 and 6 o'clock positions allowing for exposure of the high urogenial sinus which has been opened (Fig.
ItA,B). The vagina is completely mobilized (i.e. dissection above the dashed line in Fig IIA), a process that
is facilitated by the traction sutures on the vaginal walls (Fig II B). The urethra is reconstructed via
tubulurization over a Foley catheter (Fig IIC), and the vagina and urethra are then anastomosed to the perineum
(Fig II C).
More recently, Pena has advocated mobilization of the entire urogenital sinus complex
as a unit (Pena, 1989). This allows the whole complex to be placed more easily in the
perineum with the need for extensive separation of the vagina and urethra. In this
method, the urogenital sinus is mobilized together with the vagina and the two are
brought down as a unit and sutured to the perineum (Fig. 12 A, B).
There is a natural plane of separation between the urogenital sinus and the posterior
aspect of the pubis. After dissecting the urogenital sinus away from the pubis, one can
feel that the avascular suspensory ligaments are the only remaining structures preventing
advancement of the urogenital sinus and bladder. Once these ligaments are divided, a 2-4
cm of length is gained. The dissection continues in a circumferential manner, trying not
to damage the wall of the urogenital sinus or bladder. When the length of the common
channel is between 2-4 em., this maneuver is enough to repair the entire malformation.
The blood supply of both vagina and urethra remain intact and excellent and the chance
of urethrovaginal fistula is greatly decreased as compared to separating the two structures
(Fig. 12 C,D) and giving rise to an excellent functional and cosmetic result.
'-_.,...
""""
Figure 12A-D. These figures illustrate the principle advocated by Pena of mobilization of the cntire
urogenital sinus as one contiguous structure. As shown in Figure 12 A, the dissection proceeds without a
division of the urogenital sinus or the vagina, and it is possible to divide these structures away from the pubis
(Fig. 12 B). With diviSion of some avascular suspensory ligaments, the entire unit will reach the perineum (Fig
12B). The final postion of these structures is shown in Fig. 12C, with the final caosmetic result shown in Fig.
12 D. The excellent bloody supply that is preserved by this type of approach makes ischemic atrophy of the
introitus and fistula formation far less likely.
Rink and his colleagues have advocated a different prone procedure for repair of the
high urogenital sinus (Rink et aI, 1997). As described, the patient is placed prone, and a
posterior U shaped incision is then made with its ends on either side of the anus and its
apex at the position of the posterior aspect of a normally positioned vagina (Fig. 13). This
flap is then retracted posteriorly, while continuing the dissection in the midline between
the rectum and the urogenital sinus (Fig. 14A).
As the dissection progresses, the rectum is not divided but is held out of the way with
a retractor (Fig. 14 B). The dissection is continued until the urogenital sinus is reached.
The U-G sinus is then opened in the posterior midline and much like in the Pena
procedure, the vagina is then dissected away from the common U-G sinus (Fig. 14 C).
The urethra is then tubularized over the Foley catheter in two layers (Fig. 14 D), if
available, adipose tissue is incorporated into the midline to separate the urethra from the
vagina. For anastomosis of the vagina with the perineum, extra tissue from labial flaps is
helpful in some cases. In these cases, a preputial flap can be sutured to the opening of the
anterior vagina and if a preputial flap is not available, labial flaps can be constructed to
reach the anterior vagina, thus connecting it to the perineum in the appropriate anatomical
position. Just as with Pena' s modification of total urogenital sinus mobilization to attain
greater length and avoid separation of urethra and vagina, the same principle can be
easily applied to the Rink procedure.
Another modification of the perineal approach is the anterior sagittal transanorectal
approach (ASTRA) which has been described by Domini, et al (Domini et aI, 1997).
This procedure allows wide visualization of the U-G sinus just as with the Pena approach
but only involves opening the anterior rectal wall which the authors feel avoids any
potential compromise of sphincteric function and may avoid the need for a protective
colostomy.
Timing of Surgery
Timing of feminizing genitoplasty in patients with CAH has been a controversial topic
(Passerini-Glazel, 1999; Alizai et aI, 1999; Krege et aI, 2000). There are several reasons
for an early single-stage procedure. From a teehnical standpoint, the vaginal tissue is
thicker and more vascular in infancy due to the influence of the maternal hormones. In
addition, the vagina is also closer to the perineum in early infancy. Just as importantly,
parents often have enormous anxiety surrounding the condition of their child, as well as
concerns about the child's future development. Early repair reduces their anxiety sooner
and perhaps may enable the children to develop emotionally and sociallly with more ease
than they might with a later repair (Hensle et aI, 1998; Passerini-Glazel, 1999). The older
literature which suggests waiting until near puberty to proceed with the U-G sinus repair
is often based on failures associated with procedures not as technically sophisticated as
those described above.
Figure 13. Rink's approach to the high urogenital sinus begins with the patient in the prone position (Rink et aI,
1997). A small U shaped incison is made just above the anus as shown above allowing for a plane to be
developed between the rectum and the urogneital sinus.
.---~{ ~----.
---....,' i...------
I
c 0 1
Figure 14 A,B,C,D. Further dissection with the Rink approach continues in this plane until the sinus is reached
(Fig. 14A). The rectum is lifted superiorly with a band retractor (Fig. 14B). The bifurcation of the urgoential
sinus is reached, and the vagina is opened (Fig 14C). At this point, the urethra is tubularized over a Foley
catheter, and the vagina has traction sutures placed in its walls to allow for full mobilization (Fig. 14D).
REFERENCES
Alizai NK, Thomas DFM, Lilford RJ, Batchelor AGG, Johnson N. Feminizing genitoplasty for congenital
adrenal hyperplasia: What happens at puberty? } Uro1161: 1588, 1999,
Ansell JS, Rajfer 1. A new and simplified method for concealing the hypertrophied clitoris, } Pediatric Surg
16:681,1981.
Domini R, Rossi F, Ceccarelli PL DeCastro anterior sagittal transanorectal approach to the urogenital sinus in
adrenogenital syndrome: preliminary report. } Pediatr Surg 32:714, 1997.
Glassberg KI, Laungani G. Reduction clitoroplasty. Urology 17:604, 1981.
Gross RE, Randolph J, Crigler JF, Clitorestomy for sexual abnormalities, indications and technique. } Surg
59:300, 1966.
Hendren WH, Atala A. Repair of the high vagina in girls with several masculinized anatomy from the
adrenogenital syndrome. } Pediatr Surg 30:91, 1995.
Hendren WH, Crawford JD, Adrenogenital syndrome: The anatomy of the anomaly and it's repair. Some new
concepts, } Pediatr Surg 4:49, 1969,
Hensle TW, Kennedy WA. Surgical management of intersexuality, in: Campbells Urology, PC Walsh, AB
Retik, ED Vaughan, AJ Loein, eds" W,B. Saunders, Philaddphia, pp. 2155-2171, 1997,
Hughes lA, Management of congenital adrenal hyperplasia, Arch Dis Child 63: 1399, 1988,
Krege S, Walz KH, Haugga BP, Komer I, Rubben H, Long-term follow-up of female patients with congenital
adrenal hyperplasia from 21-hydroxylase deficiency, with special emphasis on the results of vaginoplasty.
Sr} UroI86:253, 2000,
Lattimer JK, Relocation and recession of the enlarged clitoris with preservation of the gland: an alternative to
amputation. }UroI86:113, 1961.
Passerini-Glazel G, A new one-staged procedure for c1itorovaginoplasty in severely masculinized female
pseudohermaphrodites, } UroI142:565, 1989,
Passerini-Glazel G, Editorial: feminizing genitoplasty, } Urol161 :1592, 1999.
Pena A, Filmer B, Bonilla E, Mendez M, Stolar C. Transanorectal approach for the treatment of urogenital
sinus: preliminary report. } Pediatric Surg 27:681, 1992.
Pena A, The surgical management of persistent cloaca: results in 54 patients treated with a posterior sagittal
approach. } Pediatr Surg 24:590, 1989,
Pena A. Total urogenital mobilization - an easier way to repair cloacas, } Pediatr Surg 32:263, 1997.
Randolph JG, Hung W. Reduction clitoroplasty in females with hypertrophied clitoris, } Pediatr Surg 16:681,
1981.
Rink RC, Pope JC, Kropp BP, Smith, Jr. ER, Keating MA, Adams Me. Reconstruction of the high urogenital
sinus: early perineal prone approach without division of the rectum. } Urol1 58:1293, 1997.
Spence HM, Allen TO, Genital reconstruction in the female with adrenogenital syndrome. Sr} UroI45:126,
1973.
Stefan H. Surgical reconstruction of the external genitalia in female pseudohermaphrodites, Sr} Urol 39:347,
1967.
Questions for Dr. Hensle
Question - Terry, I enjoyed your presentation and I have two comments. Number one, I
agree with you. We have had identical cases pretreated with dexthamethazone where you
have no clitoromegally because the treatment was initiated late and you prevent the
clitoral growth that occurs later in fetal life. Perhaps with very early treatment you
could change a urogenital sinus. This is in fact a disadvantage for the surgeon, because
you have less skin to do the repair. If you have a high bifurcation, this is something that
has to be considered. The second is that I think for the last two years I have repaired
most of these high sinuses with a total mobilization as described by Pena, but we do it in
the supine position entirely through their perineum without violating the rectum. And I
think it is very important to do a posterior u-flap to widen the opening of the vagina. One
virtue of that total mobilization is that you do not need to do the separation between the
urethra and the vagina.
Response - These are some very good points so let me deal with the last first. The total
mobilization concept is a wonderful concept and it does allow you to avoid that difficult
urogenital-sinus vaginal dissection, however, it only really works with medium to low
urogenital sinuses. With the very high ones like the in the line drawings I showed, you
just can't get - if you brought it all down everything would be out. You would have no
urethra, you would be right at the bladder neck. So there is some limitation to that.
Comment - The patient needs to be scoped to make sure that the urethra proximal to the
urogenital sinus is adequate. This is usually the case with congenital adrenal hyperplasia,
but not always with other urogenital sinus conditions.
Answer - I agree.
Question - Even in the hands of a skilled surgeon, what are you telling parents in terms
of what type of complications could be expected, how frequent they are, what is the
likelihood they will have to go back to surgery?
Answer - That is a very good question. I think that we stress very clearly that we are
doing early surgery in order to take advantage of blood supply, tissue planes, elasticity -
that sort of thing, but we are very clear, at least my group is very clear that we tell them
that vaginal stenosis is certainly part and partial to this and there may be the need for a
procedure at or around puberty. I do not give them a number because I do not know the
number. But I very clearly tell them that that is a potential.
Question - Could you comment on the use of the Hagar dilators postoperatively to
prevent stenosis? Is this something we should be doing or are we really creating a
problem?
Answer - Again, my opinion is just my opinion - but I think that Hagar dilators in little
girls are an anathema. I think that we should try and avoid that at all costs and one of the
benefits of doing early surgery is you do not have to do that. And there is nothing that
makes a family more unhappy then to have to go home from school or whatever and say
it is time for your dilation. It is just an awful thing to look forward to. I listen to family
after family tell me how bad it is and so I really would like to, at all cost, avoid Hagar
dilators or any other kind of dilator for that matter.
MANAGEMENT OF CLOACAL
EXSTROPHY
Michael E. Mitchell, M.D. and Chadwick Plaire, M.D.

Children's Hospital and Regional Medical Center, Seattle, WA
INTRODUCTION
The exstrophy complex represents a spectrum of congenital abnormalities that

includes the following conditions: epispadias, classic bladder exstrophy, and cloacal
exstrophy. These three deformations comprise a continuum with epispadias representing
the least severe, classic bladder exstrophy the most common, and cloacal exstrophy the
most severe. Cloacal exstrophy is extremely rare, occurring in approximately 1 in every
200,000 to 400,000 live births (Ziegler et aI, 1986). Although primarily a ventral
abdominal wall defect, cloacal exstrophy is associated with varying degrees of
abnormalities in the genitourinary tract, bowel, spine, and lower extremities. Until
recently, most children with cloacal exstrophy did not survive infancy because of the
complex multiple system abnormalities resulting in fluid and nutritional defects. In 1960,
Rickham reported the first survivor with cloacal exstrophy (Rickham, 1960). Primarily
due to advances in perinatal care and total parenteral nutrition, survival is now feasible in
greater than 90% of patients born with cloacal exstrophy. Thus, the focus is now directed
at quality oflife issues in these complex patients.
The anatomy of cloacal exstrophy is largely explained by an anterior herniation of the
bladder and hindgut in the fourth or fifth week of gestation, the cloacal phase of
development. This herniation prevents the midline fusion of the Mullerian ducts (hence
uterine didelphus) along with hindgut and bladder plate closure (hence two bladder plates
separated by a partially open hindgut plate). The normal curling of the tail bud is also
prevented resulting in spinal and renal malformations. The presence of the ventral
(anterior) bladder and bowel prevents the midline migration of the developing abdominal
wall resulting in a huge abdominal wall defect and pelvic flattening with symphyseal
diastasis. The typical anatomy of the patient born with cloacal exstrophy is an
exstrophied bladder separated into two halves by the exstrophied cecum. The distal
hindgut (large bowel) is usually underdeveloped and blind ending. Most patients have an
associated omphalocele of varying degrees. In one reported series of cloacal exstrophy
patients, spinal cord or vetebral abnormalities were identified in nearly all of the patients
(McLaughlin et aI, 1995). Orthopedic deformities in cloacal exstrophy patients include a
widely separated pubis symphysis and varying degrees of limb abnormalities. The
genital deformities found in cloacal exstrophy patients represent the most severe
deformities in the exstrophy complex. Patients with a 46XY genotype demonstrate a
spectrum of phallic abnormalities ranging from widely separated diminutive corporal
bodies with hemiglans to a small epispadiac phallus. The scrotum is typically bifid and

268 M. E. MITCHELL, ET AL.
the testicles are often located in an intrabdominal location. The 46XX genotype cloacal
exstrophy patients demonstrate varying degrees of abnormal mullerian duct development,
most commonly duplication of the uterus and/or vagina (Mitchell et aI, 1990).
Cloacal exstrophy is not in the strictest sense an intersex condition. Patients with
cloacal exstrophy have no ambiguity of their chromosomes or gonads. They,
furthermore, have a prenatal hormonal milieu consistent with their genetic sex.
Nevertheless, in the past, most children born with cloacal exstrophy and a 46XY
genotype were raised as females. These children were raised as females because the
diminutive, bifid phallus was thought to be inadequate for reconstruction. Until recently,
surgical procedures to reconstruct a functional phallus were lacking. Most surgeons
thought that it was easier to construct a vagina than to assemble a functional penis. Also,
data from the literature of that time would suggest that if reassigned at a young age,
children do well in the gender of raising regardless of their genetic sex (Money, 1975).
As a result, the majority of 46XY patients with cloacal exstrophy underwent orchiectomy
in the newborn period and were raised as females. Later in life, a vagina was surgically
created to allow for sexual intercourse.
However, a minority of the 46XY cloacal exstrophy patients were thought to have an
adequate phallus for reconstruction at birth and were raised as males. In 1989, Husman
et al reported extremely poor psychological outcomes in a small retrospective series of 8
cloacal exstrophy patients with a 46XY genotype that were raised as males (Husmann et
aI, 1989). Specific reasons for the poor psychological profiles were not clear and may
have been related to associated medical disabilities. Although this study raised concerns
about male gender assignment in these patients, no long-term studies adequately
document the biopsychosocial outcomes in a large group of 46XY cloacal exstrophy
patients raised as males.
The majority of children with cloacal exstrophy and a 46XY genotype have been
gender reassigned at birth. Solid data on the psychosocial result of this group is not
available. Anecdotal observations reveal that a large majority of these 46XY patients
raised as females exhibit male typical behaviors. One author reported that some of these
46XY cloacal exstrophy patients, initially raised as females, spontaneously declared
themselves to be male later in life (Reiner, 1999). It is unfortunately clear that long-term
data on the psychosocial outcomes in both groups of patients (46XY patients raised as
males and the 46XY patients raised as females) are deficient in the literature.
Currently, some of the newer surgical alternatives for repair of epispadias and
exstrophy allow for cloacal exstrophy patients to be reared in accordance with their
genetic sex. In 1994, Mitchell and Bagli described the complete penile disassembly
procedure (Mitchell and Bagli, 1996). In fact, this procedure evolved from attempts to
reconstruct the phallus of older 46XY cloacal exstrophy patients who were raised as
males. In the complete penile disassembly procedure, the penis is separated into its
component parts, two corpora cavernosa and the urethral plate. Then the urethral plate is
tubularized before being positioned in the proper anatomic position on the ventrum of the
phallus. Since the bladder is the proximal extension of the urethra, it became clear that it
was the repair of the epispadias which was the critical aspect of bladder closure in
exstrophy patients. Therefore, the complete primary repair of exstrophy as described by
Grady, Carr, and Mitchell, which is instrumental in reconstruction of the bladder and
creation of a functional phallus in the classic exstrophy patient, evolved from solving the
MANAGEMENT OF CLOACAL EXSTROPHY 269
problem of phallic reconstruction in the male cloacal exstrophy patient (Grady et aI,
1999). Our current approach is to perform complete primary reconstruction of these
children in the newborn period.
Technique
Our current approach to cloacal exstrophy patients is to first separate the exstrophied
cecum (with hindgut) from between the bladder halves completely. The cecum is then
tubularized and placed into continuity with the gastrointestinal tract as a colostomy. The
associated omphalocele is repaired, and the bladder halves are brought together
posteriorly. If possible, complete closure of the abdominal wall, bladder, and phallus is
also performed at birth. Sometimes it is not possible or advisable to perform complete
closure because of elevated intrabdominal pressure. In cases where complete closure is
not performed, the posterior wall of the bladder halves would be sutured together in the
midline posteriorly and the bladder left open. Thus, the closure would be staged, closing
the upper abdominal wall, but leaving the bladder open on the lower anterior abdominal
wall. In this staged approach, the bladder would act as a silo, which may allow it to
enlarge and permit the abdominal wall to stretch. Several weeks later, closure of the
abdominal wall, bladder, and phallus can be completed with the aid of osteotomies.
During this second stage closure, the penis is reconstructed using the complete penile
disassembly technique. This same strategy of closure may also be applied to girls
(46XX) with severe cloacal exstrophy.
Results
During the past 20 years, 50 patients with cloacal exstrophy have been treated by one
of the authors (MEM). Twenty patients were born with a 46XX genotype, whereas 30
patients were 46XY. Of these 30 patients with a 46XY genotype, 16 were raised as
males and 14 were raised as females. During the past 10 years, only 1 patient with a
46XY genotype has been assigned to a female gender. Of the patients contacted, despite
having early orchiectomies, greater than 50% of the 46XY patients raised as females
exhibit male type behavioral characteristics. Since no detailed psychological interviews
have been performed, information on these patients true gender identity and sexual
orientation is" yet to be evaluated. Our 46XY patients raised as males range in age from
1-24 years old with a mean of9.7 years. Two patients died in their twenties as a result of
sepsis and renal failure, respectively. Likewise, detailed psychosocial characteristics
have not been identified in this group. However, erections and orgasms are reported by
many of the older patients. Some of the males, also, report heterosexual relationships and
sexual activity. All of these patients appear to have male typical behavior. Complete
primary repair of exstrophy, without division of the urethral plate or use of para exstrophy
flaps was performed in the last 4 patients born with c:1oacal exstrophy. To date, these last
4 patients have not required bladder augmentation and their upper tracts remain stable.
Conclusions
As a response to this preliminary data, we have initiated a study of the biopsychosocial

outcomes of this important group of children. Even though cloacal exstrophy does not
represent an intersex condition, by studying these patients, we may gain a better
understanding of the complex issues relating gender to genetic sex. Our cloacal
exstrophy population is divided into subgroups: 1) 46XY patients raised as males, 2)
46XY patients raised as females, and 3) 46XX patients raised as females. It is anticipated
that a comparison study of these groups will provide insight into the optimal care for
these children. In our analysis, we will account for the fact that the older patients did not
benefit from the newer surgical techniques utilized in the younger patients. Our
preliminary data in the older patients and early clinical observations in the younger
cloacal exstrophy patients suggest that when possible it is better to reconstruct children
with cloacal exstrophy according to their genetic sex. This would assume that a 46XY
male would accept a less than perfect phallus. However, the broad and severe spectrum
of cloacal exstrophy demands individual consideration on a case by case basis such that a
46XY male should not have to accept no phallus at all.
REFERENCES
Grady RW, Can' MC, Mitchell ME. Complete primary closure of bladder exstrophy. Urol C/in N Amer
26(1 ):95, 1999.
Husmann DA, McLorie GA, Churchill BM. Phallic reconstruction in cloacal exstrophy. J UroI142:563, 1989.
Mclaughlin KP, Rink RC, Kalsbeck JE, Keating MA, Adams MC, King SJ, Luerssen TG. Cloacal exstrophy:
The neurological implications. J UroI154:782, 1995.
Mitchell ME, Brito CG, Rink RC. Cloacal exstrophy reconstruction for urinary continence. J Urol 144:554,
1990
Mitchell ME, 8agli OJ. Complete penile disassembly for epispadias repair: The Mitchell technique. J Urol
155:300,1996.
Money J. Ablatio Penis: Normal male infant sex-reassigned as a girl. Arch Sex Behav 4:65, 1975.
Reiner WG. Psychosocial concerns in Classical and Cloacal Exstrophy patients. Dial Ped UroI22:3, 1999.
Rickham PP. Vesico-intestinal fissure. Arch Dis Child 35:97, 1960.
Zeigler MM, Duckett JW, Howell CG. Cloacal Exstrophy, in: Pediatric Surgery, KJ Welch et aI, ed., Year
Book Medical Publishers, Chicago, 1986.
Questions for Dr. Mitchell
Question - Mike I was part of the Toronto group that reviewed all these patients and my
concern when we went over them in the paper I think we really alluded to a lot of the
problems with depression, etc., related to the phallus and as I have gotten farther and
farther along and look back at this I am afraid a lot of this was related to just the general
health problems with these kids. Do you have a sense of that?
Response - I do not know. They have so many variables that would need to be
controlled. The younger patients that we are seeing - and it is hard to pick up depression
in young kids - I do not think are going along that pathway. They seem to be much
healthier emotionally. The older patients, show that some are doing well and others are
experiencing some emotional difficulties. I do not think that the 46XY females were any
healthier than the boys, emotionally.
Question - One of the themes of your presentation clearly was that you have to sort of
individualize with these kids. There are a few older patients that we have seen at CHOP
that haven't had a complete reconstruction up front. How you would approach a patient
that was older that either moved to your town or was referred to you after a failure? They
come in all shapes and sizes and with all different types of problems, but one of the
common denominators that I have seen is that if they haven't had osteotomies or a good
closure early on there is not much that you can do with the vagina or the bladder. So
there is really two questions. One is how do you manage these patients if they come to
you at an older age, and what is the role of osteotomies in the cloacal exstrophy patient?
Response - We have used osteotomies quite commonly, even in the early closures
because I think it is very important to minimize the symphysial diastasis if you want to go
for continence. The older patients are such a mixed bag of problems and they really are
individualized. I usually work back from what does the patient want to achieve, and we
usually try to reconstruct them with that in mind.
Question - I didn't realized that John was at the microphone, too. I welcome his
comments. What has your approach been? Have you done osteotomies in patients that
are say 9,10, 11, 12 or even older?
Comment - Dr John Gearhart - I just want to make a comment about Doug's remark and
yours, too. I think if you are going to raise these 46XY cloacal exstrophy patients as
males, your osteotomy becomes even more important because it really allows you to get
those coporal bodies to the midline so you can join them. We do not use casts, we use an
external fixating device so they actually can go, after about 3 weeks, to the Ronald
McDonald House or somewhere with their fixating device and actually they do pretty
well with that. But we have seen some older kids who came in, girls especially, who had
such desperate looking lower abdominal walls and genitalias that really the only we
thought we could reconstruct them and make them look as normal as possible was with
an osteotomy. The trouble is in an older child, the 12 or 14 year old, it is a much more
morbid procedure than it is in a young newborn. But still at the end of the day I think it is
worth for them to have an osteotomy just to reconstruct their external genitalia and their
abdominal wall to make them look much more normal. But, certainly I would
recommend to you another trick that we have learned that the osteotomies is that we will
do the osteotomy and then we will put a fixating device on them and crank them together,
ever so slowly. Every day Dr. Sponsellor will do that for a period of 3 weeks or so and
you will be surprised how you will narrow the diastasis and then at 3 weeks when he
feels he has achieved the maximum he can get from his cranking the fixator together then
we will close the abdominal wall and do what we need to do and do the exstrophy, and I
think that is a nice little trick and I would recommend that to you and your orthopaedic
surgeons.
Answer - One of the problems with the diastasis and in my experiences I made the same
mistake, too - so I am just telling from what I have learned - is that you really need to cut
into the pelvic diaphragm very deeply in order to move everything back and if you do not
do that, then it does not make any difference what you do with the symphysis. You are
not going to get the results that you want to get. So you have to be very aggressive and
usually, for these redo kids, you just wind up taking everything apart and putting it back
together again.
It is important to remember that these children have a 30% shortage of bone in their
pelvis, so we have to make up that some way, too, with an osteotomy. I think that it pays
to be aggressive early on because you can take advantage of the fact that the pelvis will
grow if you give it the right framework in which to grow - so much better.
Question - Mike, with reference to the other issues that have been addressed over the last
two days, do you think with your vast experience in this, you will go back to Seattle with
any different approach in informing, getting consent, from your patients families? How
have these other issues we have talked about affect your approach to these patients with
cloacal exstrophy today?
Response - If you keep the genetic sex, many of the issues that we have discussed here
are non-issues. I do not worry about an imprinting because these patients have been
imprinted naturally. And I think that I can surgically create genitalia that are as good as
any genitalia I could have corrected made by converting these patients. So I do not have
any problems. The problems that we get into in my practice are the ones where patients
who referred in later in life like one patient who was incontinent and I made her
continent. But I was entering into her course of treatment late, and was one snapshot in
the history of her treatment. This is a much more difficult situation because there was so
much history that went just that they feel like boys. And then the other side - when you
were classifying the kids as being predominantly female, can you just say more what you
mean by predominantly?
Response - I think that these patients are not studied to the degree that we would like to
have them studied and that is why it was vague. And we are going to have to go because
I was not really very interested in going into them because I did not think that that was
where the real meat of this investigation was going to go. But as it turns out, it clearly is
where all the information is. These patients were clearly not going along the ideal track
where they were healthy, happy young ladies that we usually see. But we haven't done
the psychological testing and we are going to have to get into that just to have that
information. But, I have got enough sense I think from these early things that and
because we have developed techniques that I think we can use to prevent the need to
create any more patients that have these problems.
Question - Is your sense of the ones that you have cllassified as female or predominantly
female are unhappy living as girls?
Answer - As a group they seem to be healthy and basically happy people. But if you
observe what they do, they behave like the other sex that was not their assignment. And
they are smart, this is the way they compensate and for the most part they have good
family support systems. So they have been in on it their whole life and things are going
along well, but many of them were really kind of just getting into the point where they
are going into teenage and young adulthood, and here things can become difficult. before
she ever presented to us. As we have learned, that this is something that has to be
approached very gently, and continuity of care is essential.
Question - I think the advances you describe surgically are amazing. I want to ask you
about the slide of the 10 patients raised as girls. If you could just describe a little bit,
your classification where the heading was identity. It wasn't clear if you were saying
that the ones who you are saying are male have switched to living as boys or just that
they feel like boys. And then the other side - when you were classifying the kids as
being predominantly female, can you just say more what you mean by predominantly?
Response - I think that these patients are not studied to the degree that we dould like to
have them studied and that is why it was vague. And we are going to have to go because
I was not really very interested in going into them because I did not think that that was
where the real meat of this investigation was going Ito go. But as it turns out, it clearly is
where all the information is. These patients were clearly not going along the ideal track
where they were healthy, happy young ladies that we usually see. But we haven't done
the psychological testing and we are going to have to get into that just to have that
information. But I have got enough sense, I think, from these early things and that
because we have developed technique that I think we can use to prevent the need to
create any more patients that have these problems.
Question - Is your sense of the ones that you have classified as female or predominantly
female are unhappy living as girls?
Answer - As a group they seem to be healthy and basically happy people. But if you
observe what they do, they behave like the other sex that was not their assignment. And
they are smart; this is the way they compensate and for the most part, they have good
family support systems. So they have been in on it their whole life and things are going
along well, but many of them were really kind of just getting into the point where they
are going into teenage and young adulthood, and here things can become difficult.
TOTAL PHALLIC CONSTRUCTION, OPTION TO
GENDER REASSIGNMENT
Gerald H. Jordan, M.D.

Eastern Virginia School of Medicine. Norfolk. VA
INTRODUCTION
Total phallic construction was first accomplished by Borgoraz in Russia in 1936

(Borgoraz, 1936). He described a technique of tubed abdominal flap which was
transposed to the area of the penis by techniques of flap delay. A number of authors used
this technique with acceptable results. The phallus, so created, was insensible baculum
dependent for rigidity, and often did not create a urethra to the tip of the phallus. Thus,
patients were precluded from standing to void. The groin flap, as well as the gracilis
musculocutaneous flap, were also used for phallic construction. The cosmetic results of
phallic construction were greatly improved by Puckett (Puckett and Montie, 1978) using
the groin flap, and eventually the groin flap was used as a microvascular free transfer.
Again, prior to that, transposition of the phallus to the area of the penis was accomplished
by methods of delay or in multiple stages. The same short comings, as already mentioned
for the tubed abdominal flap, apply to these flaps.
A quantum jump was achieved with the description by Chang in 1984 of the radial
forearm microvascular free transfer flap for phallic construction. Varying designs of that
flap (Fig. 1) incorporated the innovations in cosmetics described by Puckett (Puckett and
Montie, 1978) and others, provided for erogenous and protective sensibility, allowed for
subsequent placement of prostheses for rigidity for intercourse, and allowed for the
urethra to be brought to the tip of the phallus, allowing the patient to stand to void. A
number of modifications have been proposed to the original "Chinese flap" design.
Many of these modifications were undertaken to center the urethral portion of the flap
over the cuticular vascular territory, thus improving the reliability of those tissues. Other
modifications were undertaken in order to incorporate vascularized bone under the
concept that these bony segments would provide sufficient rigidity to allow for
intercourse.
The fibular oseocutaneous flap was proposed as an option to forearm flaps by Sadove
and McRoberts (Sadove et aI, 1993, and chapter 18). This flap had a 100% urethral
complication rate, and unclear results with regards to rigidity and the inclusion of bone
being able to accomplish that. Lovie (Lovie et aI, 1984) described the ulnar forearm flap
primarily for use in head and neck reconstruction.. This center has adopted that flap as
the flap of choice for total phallic construction but has incorporated a number of the
modifications originally designed for the radial forearm flap, both for cosmetic and
urethral reliability (Fig. 2) We have not become enthusiastic about the inclusion of

276 G.H.JORDAN
vascularized bone to date, as we are not convinced that the literature supports the fact that
it works.
The goals of total phallic construction are as follows: 1. The phallus must be
cosmetically acceptable. 2. The patient must be allowed to eventually stand to void. 3.
The flap must allow for both erogenous and protective sensibility. 4. The flap dimensions
and configuration must be suitable for intercourse, and thus in correct time allow for
prosthetic placement. 5. The flap must be associated with limited donor site morbidity
and preferentially all could be accomplished in a single stage. Needless to say, these
goals have alluded reconstructive surgeons for decades, and I am not aware of any center,
in the pediatric population or otherwise, that has yet met all of these goals.
During the years, 1981 to 1999, at our center, 31 pediatric patients underwent total
phallic construction. Their age range was from 5 years to 15 years of age. Fourteen
patients were reconstructed following trauma; 8 patients underwent total phallic
construction for congenital anomalies such as exstrophy or cloacal exstrophy; 2 patients
underwent phallic construction for re-gender reassignment; 3 patients underwent phallic
construction for micropenis; 2 patients underwent phallic construction as a consequence
of circumcision accidents; and 2 patients were born with aphallia. Phallic construction in
the pediatric patient has not been undertaken without complication. Early in our series,
the urethral complication rate was 60%, and was comprised of either postoperative
strictures, fistulas, or both. With modifications in urethral anastomotic suture technique,
and in select patients, with the use of adjuvant gracilis muscle transposition or tunica
dartos flap transposition, we currently enjoy a 25% urethral complication rate. Out of 32
flaps used, we have lost 4 for a 13% flap loss rate. We have lost a substantial portion of
additional flap for a 3% partial flap loss rate. Cosmetic results were drastically improved
after 1986, when we moved to the exclusive use of microvascular free transfer for phallic
construction.
We began our experience in 1981 using composite flaps; these flaps used rectus
abdominous muscle, gracilis muscle, sometimes along with their respective skin islands,
along with graft techniques. Aware of the experience in China, in 1983 we moved to
microvascular free transfer as the procedure of choice, and in 1987 adopted the use of the
ulnar forearm microvascular transfer as the procedure of choice. Our currently preferred
flap design is illustrated in Fig. 2.
In our population, 7 patients have reached an age where sexual activity would be
appropriate, and of those 7, 3 are sexually active with prostheses. Two of those 3 patients
are married. In that we were plowing new ground in the pediatric population, we
obviously made mistakes and have learned some very significant lessons. The flap loss
rate is higher in children. Children thus seem to be at higher risk for vascular problems.
It is currently unclear to us whether this increased risk of vascular problems is inherent to
children because of either size, age, or both, or due to the fact that our pediatric
population is one that most have had considerable prior other surgery, IVs, A-lines, etc.
In other words, there is history to suggest that the forearm venous systems have been
potentially "ravished" because of the need for support of their prior surgical procedures.
Because of that, we now vigorously evaluate all of our pediatric patients with upper
extremity angiography, abdominal and lower extremity angiography, and as much as
possible try to evaluate the forearm venous anatomy with delayed films at the time of
angiography. We have come to the opinion that we require a resident expert
TOTAL PHALLIC CONSTRUCTION 277
angiographer for these evaluations and preferentially always schedule these children with
the same angiographer.
In the exstrophy and cloacal exstrophy patients, many were intermittent catheter
dependent. We originally felt that intermittent catheterization was reasonable and
possible through the phallic constructed urethra. We have now learned that these phallic
defunctionalized urethras become multiply colonized with difficult organisms. Our
children soon became chronically infected and symptomatic. Additionally, the constant
trauma of catheter passage through the constructed phallic urethra eventually attenuated
the suture lines and virtually all of these patients went on to develop breakdown with
fistula. We now feel that if a child is intermittent catheter dependent, and requires phallic
construction, that catheter access to the bladder be accomplished through some form of
abdominal catheterizable stoma. Likewise, if the children are not voiding, we cannot
construct a neourethra which is defunctionalized. These blind urethras, because of
colonization, form abscess, break down, and have in all patients proven to be a
considerable problem.
While much of this report has been devoted to describing problems and lessons
learned, we do feel that phallic construction is an alternative to gender reassignment. Not
all of our patients were offered gender reassignment as an alternative, prior to being
referred to our center. Obviously, 2 of our patients underwent gender reassignment and
requested of their parents that they be re-reassigned. All of the aphallic patients were
offered gender reassignment as were the circumcision accidents, the micropenis patients,
and the cloacal exstrophy patients. None of the post trauma patients were offered gender
reassignment. The decision, however, to not undergo gender reassignment was, with the
exception of the 2 patients that were re-reassigned, the decision of the parents. However,
as previously stated, no patient in our series was operated on before the age of 5 years.
We found that 5 year old patients seem to be remarkably aware of their situation, and as
much as a 5 to 10 year old can give "informed consent", they all did. In fact, many of the
children were remarkably enthusiastic about undergoing surgery, and many on being
worked up with enthusiasm stated that "this was the day that they were going to get their
penis."
278 G. H.JORDAN
A.
B.
c.
Figure 1. Collage illustrating various modifications of the radial foreann flap. A. The foreann flap as described
by Chang (Chang et al, 1984). Notice the shaft paddle is centered over the radial artery with the paddle, which
will become the urethra, becoming quite far distant on the ulnar aspect of the foreann. B. Cricket bat
modification as proposed by Farrow and Boyd (Farrow et al, 1990). This modification centers the urethra over
the radial artery. C. Modification described by Biemer (1988). This modification centers the urethral strip over
the radial artery; shaft paddles are then lateral. As originally described by Siemer, this flap also incorporated a
portion of the radius.
TOTAL PHALLIC CONSTRIJCTION 279
Figure 2. A. Collage illustrating the ulnar forearm flap for phallic construction as utilized at our institution.
Notice the urethral strip is centered over the ulnar artery. Notice the distal continuation to form an integral
glans as described by Puckett. Basically, the flap design is per Biemer; however, centered over the ulnar artery.
B. Illustrates the tubularization of the urethra. The shaft paddles are then closed over the urethra. The integral
glans is then "flipped".
REFERENCES
Biemer E. Penile construction by the radial arm flap. C/in Plnst Surg 15:425, 1988.
Bogoraz NA. Plastic restoration of the penis. Sov Khir. 1936.
Chang TS, Hwant WY. Forearm flap in a one-stage reconstruction of the penis. Plnst Reconst Surg 74:251,
1984.
Farrow GA, Boyd JB. Semple JL. Total reconstruction of a penis employing the "cricket bat flap" single stage
forearm free graft. AUA Today3:1, 1990.
Lovie MJ. Duncan GM, Gleason OW. The ulnar artery forearm flap. Br J Plast Surg 37:486, 1984.
280 G. ".JORDAN
Puckett CL. Montie lE. Construction of the male genitalia in the transexual using a tubed groin flap for the
penis and a hydraulic inflative devie. Plast Reconst Surg 61 :523. 1978.
Sadove RC. Sengezer M. McRoberts JW. et al. One stage total penile reconstruction with a free sensate
osteocutaneous fibular flap. J Plast Reconstr Surg 92: 1314m 1993.
Questions for Dr. Jordan
Question - Do you get insurance companies to pay for this kind of reconstructive
surgery?
Answer - Yes, in fact, even in the transsexual population, there are insurance companies
that consider that a reimbursable disease. Clearly in the issues of trauma, Intersex
congenital anomaly and those kind of things, the insurance companies do review those
claims. When you think about the resources that are expended, this certainly is not a
socioeconomic form, it is a long day in the operating room for not a lot reimbursement;
but at least it is considered a disease process that should be reviewed and reimbursed by
the insurance companies. They just do not really understand what goes in to doing this
surgery.
Question - It is a very interesting population; but I was just wondering - you must have
some data as to whether or not these people, after you go through these reconstructions,
felt it was worthwhile - whether or not it worked?
Answer - Actually, we do have that data and the problem with a lot of the children is that
they are malleable that it is hard to really get from the child whether he thinks it is
worthwhile. I can tell you that we have had some problems with children who had
difficulty adapting to the size of the phallus early on because it was inappropriately large
for their age. Keep in mind that the phallus does grow somatically but it does not have a
hypertrophic growth spurt so there are a little big at first to hopefully the child then will
appropriately grow into what would be an adolescent young adult size phallus. We have
had at least one child that I can think of who had problems adapting to that. Even
children who were early in the series, and clearly from a cosmetic standpoint, have
terrible results compared to what we are getting now. It is funny how these kids turn up.
A kid from Ireland that we operated on suddenly turns up in L.A. and I had not seen him
for years and years. Although he has a poor cosmetic result, he still was very happy that
he had gone through surgery. Therefore, the ones that we can keep hold of long enough
to talk to them when they are adults, they seem to be happy that they underwent the
operation.
Question - This is just a follow-up on Mike's questions. You did not show any slides of
the forearms from which you had taken these grafts. Obviously you were short of time,
but has that been a major problem psychologically?
Answer - Actually not and you would wonder why. I did show you the defect. Early on
what we were doing was using split-thickness skin and the scarring was very unsightly.
TOT AL PHALLIC CONSTRUCTION 281
We now use full-thickness skin and these kids do well. Particularly after the time that we
allow them to take off long sleeves and to go ahead and let it tan as their forearm
ordinarily would. It is a big scar. It is obviously to anyone that looks, but they seem to
deal with it well, with very imaginative stories. (Example: I was surfing in Hawaii and a
great white ... )
Question - I encountered one patient (now an adult) who had had a radial forearm flap
and surprised me by saying that he was functioning well sexually without a prosthesis or
anything rigid within the flap. I wonder how often that happens and how often you do
you recommend a prosthesis.
Answer - It does not happen terribly often, but there are patients who can be sexually
active that way. I do not have good numbers to tell you, but in what is a series of about
120 patients, we have only done 35 prosthesis and we have offered it to all that are of an
appropriate age to have a prosthesis. I am sure that many of these patients are asexual,
but I still think that there are some who can have acceptable sexual activity; but I do not
have a good number for you.
Question - You have touched on the differential growth and size problem in childhood, a
little bit. Are you of the opinion, therefore that the definitive penile reconstruction should
be deferred until puberty or post-puberty? A temporizing phalloplasty which could leave
the field clear and not interfere with the prime sites for doing it until after puberty - or are
you of the opinion that you would go for the prime site produced penis which is too big
with a high complication rate in childhood?
Answer - Right now our opinion is that this can be done pre-pubertally, but I think you
raise a very cogent issue. I guess that the biggest thing is that accessory or temporary
phalloplasty's are so miserable in just about every aspect. For example, John Pryor has a
very innovative technique of phalloplasty but the urethral complication rate is 100%. It is
insensible with the exception of the urethral tube itself, so I cannot imagine that a child
who is going to go through that phase of gender focus, that what he sees will be
satisfactory and of course that is what has driven us to try and do more earlier. But again,
I do not have any data to support our thoughts and we do not have the ability to keep firm
enough hold of these patients to get that good follow-up. I get follow-up on our overseas
patients when I happen to be in that country and th{m they kind of show up and I get to
see them, so there are years in between that I have no follow-up. I think you raise a very
good question. I do not have any better answer than I have just given you.
Question - These patients come to you after they have been psychologically worked-up
locally by the individuals referring them to and you get them when they are primed and
ready to have the phallic reconstruction?
In terms of the donor site, have you experimented with tissue expansion to reduce the
scarring effect? By placing a tissue expander on the other side of the arm to rotate the
flap down might one reduce the scar?
282 G. H.JORDAN
Answer - Let me answer the second questions first. Yes we have reduced the size of the
scar using a tissue expansion and you can do it again. It takes probably 5 or 6 sessions to
expand what is left, enough to get what is a reasonable linear scar, but it can be done.
Tissue expansion obviously is not without its own complications and some of this skin is
less then optimally vascularized and so we have had erosions of the tissue expander of
the tissue that we were trying to expand, but yes, if they are motivated we think that that
is reasonable to do.
As far as psychological counseling, we get them anywhere in the spectrum. We really

get some of these kids within 4 to 6 months of trauma and have had absolutely no
counseling and their parents have not had any counseling. In fact, there have been some
terrible family situations involved with our even seeing the patient to start with. All of
the patients that we see are counseled. We have good pediatric psychologists available.
The re-reassignments we treat for all intense purposes as transsexuals, so they truly have
to go through the Harry Benjamin criteria. Now they are kids, so they are not going to
work in gender, but they have to dress in gender and they have to make the change before
we ever consider re-reassignment. Interestingly, the only re-reassignments that we have
in our series are those two that I just showed you. They were children who were gender
reassigned.
PENILE RECONSTRUCTION WITH A FREE SENSATE
OSTEOCUTANEOUS FIBULA FLAP IN THE
SURGICAL MANAGEMENT OF THE INTERSEX
PATIENT
J. William McRoberts, M.D. and Richard C. Sadove, M.D.

Divisions of Urology and Plastic Surgery, University of Kentucky School of Medicine,
Lexington, KY
INTRODUCTION
The birth of an infant with ambiguous genitalia presents an urgent dilemma, perhaps
more complex today than previously. Prior to the past 20 years, the prevailing belief was
that infants were essentially psychosexually neutral at birth and the child could be
"nurtured" in either the male or female direction if the decision was made early and the
genitalia were appropriate (Diamond and Sigmundson, 1997). Accordingly, the
appearance of the genitalia became a stronger influence than the chromosomal karyotype
in the decision as how to proceed with gender assignment and genitoplasty. The child
was raised male if the phallic size portended adequate urinary and sexual function. If the
phallus was deemed inadequate, the operative belief was that it was far simpler to
construct a functional vagina and normal appearing vulva than to attempt to fashion a
functional and cosmetically acceptable penis from inadequate tissue.
Today, while the classification of ambiguous genitalia have become more refined the
decision of gender remains complex but on sounder ground increasing our ability to
make an accurate diagnosis (Glassberg, 1998;1999. As we've learned from the recent
literature and at this conference, the recent "John-Joan" report resurrected the nature
versus nurture debate, not only in regard to gender assignment in a normal male infant
who lost his penis secondary to electrocaudery circumcision injury but also in gender
assignment in the newborn with ambiguous genitalia. While some self-help
organizations, such as the Intersex Society of North America, have suggested treatment
delay to allow the individual to decide his or her own sex when older, there are no long-
term longitudinal studies to support the benefits of delayed therapy. On the other hand,
instances of patients wanting to subsequently change their own infant gender assignments
are virtually unheard of if assigned a male gender (Glassberg, 1999). Similarly, patients
with congenital adrenal hyperplasia, if assigned a female gender early in life, also do well
provided they faithfully take their suppressive adrenal medications. Failure to classify the
newborn infant as male or female presents insurmountable problems in future child
rearing (Izquierdo and Glassberg, 1999).
Recently a number of longitudinal studies have shed new light on three specific types
of male pseudohermaphroditism: ie. 5 a reductase deficiency; 17 ketosteroid reductase

Edited by Zderic et at., Kluwer Academic/Plenum Publishers, 2002
284 J. W. MCROBERTS, ET AL.
deficiency; and incomplete androgen sensItIvIty (Izquierdo and Glassberg, 1999).

Patients with either of these three forms of male pseudohermaphorditism who are only
mildly or moderately virulized will do well when raised as a female provided the
testicular tissue is removed early. On the other hand, psychosexual problems are bound
to occur if the patient is not proper! y diagnosed as an infant, and the patient is raised as a
female without removing functioning testicles. The following case report illustrates these
points as well as the use of the fibula flap in penile reconstruction in a patient with male
pseudohermaphroditism.
Case Report
SH was born at home in a rural setting in 1957. The infant's genitals were thought to
be somewhat ambiguous secondary to a large clitoris but because of a normal female
introitus and without benefit of karyotype studies, the patient was raised female. SH's
childhood was characterized by "tomboyish" behavior, preference for "boy games and
toys", and a strong dislike at wearing dresses. Her wishes to be a boy were strong
enough that her mother took her to a pediatrician who diagnosed the patient as being
male on the basis of a 46XY karyotype; thereafter the patient was "allowed to be a boy."
The patient consulted one of the authors (JWM) at age 25 in 1985 with a diagnosis of
pseudovaginal perineal hypospadias, later updated to 5 a reductase deficiency. The
diagnosis was based on a 46XY karyotype, microphallus (2.0 em relaxed; 3.0 em
stretched), perineal hypospadias, bilaterally descended testes, a low normal serum
testosterone and normal FSH, scant body hair with female distribution. The patient never
shaved. Patient subsequently underwent a successful Tiersch-Duplay proximal
urethroplasty combined with a free buccal tube graft distally and was now able to stand
up to void for the first time. Encouraged by these tum of events, the patient subsequently
married and remains so with the same person. The patient was able to have "so-so sex"
including orgasm, despite a small, 3cm phallus on stretch. However, the patient was
extremely dissatisfied with the overall penile length and appearance. Accordingly in
1995 the patient underwent penile reconstruction with a free sensate osteocutaneous
fibula flap utilizing buccal graft for the neourethra at a second stage 4 months later. The
small native phallus had been preserved at the base of the new penis which measured
10cm. in stretched length. The patient encountered no post-operation complications, no
strictures or fistulas, and he is able to stand up to void and states he has much more
satisfactory sexual relationships with his wife who concurs in this assessment.
Operative Procedure
The original operative procedure (fig. 1) as applied to the first five patients has been
described previously (Sadove et aI, 1993). Because of high rates of urethro-cutaneous
fistulas and strictures, procedure has since been modified: the construction of the
neourethra and glans corona are deferred until a second stage; buccal mucosa, which is
four times thicker than normal skin, is used to construct the neourethra rather than a full-
TISSUE ENGINEERING APPROACHES 285
thickness skin graft from the hairless groin that was employed previously. The modified
procedure is described in this section.
A cutaneous island is marked on the lower leg skin over the fibula. This island is
raised at the subfacial level, carefully protecting the posterolateral intermembranous
septum and the nerve. The fibula is transected both 6cm proximal to the lateral malleolus
and distal to the knee. Access to the pedicle is facilitated by resection of proximal fibula
bone. The peroneal artery is ligated 6cm proximal to the ankle. It is carefully dissected to
the bifurcation of the tibioperoneal trunk and its division from the posterior tibial artery
where it is transected. This length is necessary to allow the peroneal artery to reach the
superficial femoral artery after transfer. Vascular pedicle is lengthened by subperiosteal
resection of the proximal fibula bone in the flap. Removal of bone to lengthen the
vascular pedicle leaves approximately lOcm of bone available for the neophallus.
Figure 1. The skin island of the flap is wrapped around the fibular bone, the peroneal artery and vein. the
lateral sural cutaneous nerve, and the reconstructed urethra in the course of the phalioplasty as originally
described.
The skin island of the flap is wrapped around the fibula bone. The neophallus is then
transferred to the perineal region and inset. It is revascularized in an end to side manner
to the superficial femoral artery and the saphenous vein. Tension on the vascular
anastomosis is avoided by subperiosteal resection of proximal bone in the flap if
necessary. The two dorsal penile nerves are coapted to the lateral sural cutaneous nerve
from the flap. The perisosteum of the fibula is sutured to the tunica albuginea at the site
of amputation of the corpora. Drill holes are placed in the fibula for passage of sutures
that anchor the bone to the tunica. A split-thickness skin graft is placed on the leg donor
site. In about 4 months, after all wounds are well healed, the glans corona and
neourethra, utilizing buccal mucosa (Duckett, 1992), are constructed.
Results
Our series of total penile reconstruction with the free sensate osteocutaneous fibula
flap now numbers seven consecutive cases: 3 adult patients with traumatic amputation of
the penis; 3 biological female transsexuals; and this most recently reported case of a
patient with 5 a reductase deficiency associated with a microphallus.
All seven grafts are viable with normal sensation. Complications were limited to
urethral strictures and urethral-cutaneous fistulas (table 1). Strictures occurred in 43%
(3/7) of patients and fistulas 57% (4/7). Most of these complications were amenable to
secondary operative correction leaving only one patient with persistent strictures
requiring self catheterizations to maintain patency and one patient with a persistent fistula
requiring the patient to sit down to void. Graft sensitivity was categorized into protective
and erogenous. One hundred percent (717) had protective penile graft sensibility, while
only one patient (14%) had erogenous sensibility. Eighty-six percent (6/7) of patients
experienced orgasms not as a result of stimulation of the patient's neophallus but rather
stimulation of the patient's native erogenous tissues, i.e., the penile stump in cases of
traumatic amputation, clitoris in the instances of biological female transsexuals, and the
retained small penis in the patient with 5 a reductase deficiency.
TABLE 1: RESULTS
BIOLOGICAL TRANSSEXUALS TOTALS
MALES
VIABLE GRAFT 4/4 3/3 7/7 (\00%)
STRICTURES 114 2/3 3/7 (43%)
FISTULAS 3/4 I /3 4/7(57%)
SENSIBILITY
PROTECTIVE 4/4 3/3 7/7 (\00%)

EROGENOUS I /4 0/3 1/7(14%)
ORGASMS 4/4 2/3 6/7 (86%)
In conclusion, the main advantage of the fibula flap phalloplasty is its intrinsic
rigidity, its superior donor site location, and its long vascular pedicle. The fibula flap
provides better bone volume than does the radial forearm flap which commonly results in
a floppy phallus in the absence of bone. Penile prosthesis in other flaps have enjoyed
limited success. Forearm donor site complications can be avoided. The donor site on the
lower extremity can be readily covered with a sock. The vascular pedicle of the fibula
flap is of sufficient length to allow end-to-side anastomosis of the flap to the femoral
artery. Interpositional vein grafts are unnecessary and dissection of the inferior upper
epigastric artery system to serve as a donor may be avoided. The appearance of the
neophallus is excellent. Patient satisfaction and the advantages of the modified technique
convince us that the fibula osteocutaneous flap is an excellent choice for total penile
reconstruction that may be appropriately considered for highly selected intersex patients
if the phallic size portends inadequate urinary and sexual function as well as those who
have sustained traumatic amputation of the penis.
REFERENCES
Diamond M, Sigmundson HA. Sex reassignment at birth. Arch Pediatr Adolesc Med 151 :298-307, 1997.
Duckett JW. Successful hypospadias repair. Contemporary Urology 4:42-55, 1992.
Glassberg Kl. The intersex infant: Early gender assignment and surgical reconstruction. J Pediatr Gynecol
11:151-154,1998.
Glassberg Kl. Editorial: Gender assignment and the pediatric urologist. J Uro1161: 1308-1309, 1999.
Izquierdo G, Glassberg KJ. Gender assignment and gender identity in patients with ambiguous genitalia.
Urology 42:232-242, 1999.
Sadove Re, Sengezer M, McRoberts JW. One-stage penile reconstruction with a free sensate osteocutancous
fibula flap. Plast ReconstrSurg92:1314-1323, 1993.
Questions for Dr. McRoberts
Question - What is most interesting is this question of sensation because I have always
worked on the idea that something that was small but worked as in micropenis was rather
better than something that was big but did not work. When you say they are sensate, you
are quite correct in emphasizing it is protective sensation is something that they can feel
and they won't get it caught in their zipper or something like that, but it is correct, is it
not, that their sexual function, in fact, depends on what you have preserved from the past,
rather than what you have created?
Answer - Absolutely and that is very important not to throw anything away because that
is going to be their erogenous tissue, not the phalloplasty, and not the new penis at all.
And they are essentially hinged so they become stuffers rather than penetrators because if
you had them to be penetrators, it would be impossible to dress them and they could not
bring up their penis on their lower abdomen. But they are probably not unlike people
who use a vacuum erection device and capture their erection with a rubber band at the
base. Essentially they are hinged and they do not really penetrate. They place their
phallus in the vagina and if it is adequate length they can keep it in there and by butting
up against their symphysis pubis have adequate thrusting. But the orgasm again comes
from the native tissues that are preserved.
Question - Do you know anything about what the partners think of it?
Answer - Yes we have interviewed 5 of the 7 partners and they are quite happy with it.
But we have only done 7 cases now in 15 years. I want to emphasize these are very
special cases and I agree with you, the vast majority of the people with the so-called
small penis can get along very well and you pointed that out to us very nicely. So I think
if the patients are cruising along nicely it is only the (terribly-or-totally) dissatisfied
patients who are candidates and I put them off as best I can. And the vast majority of
them I talk out of the procedure because it is a very expensive, long, drawn-out,
committed, painful long period without ambulation, so they really have to be terribly
committed to undergoing the procedure. So you definitely do not want to over-sell it.
Question - What about the application of this technique to an aphalia patient and when
would you consider doing something like that?
Answer - Well, I think I have learned from the conference here that we can wait a lot
longer than we thought we could and I think we can have a patient help decide what
direction this individual wants to go. Certainly the aphalia patient - you probably want to
go with a male gender assignment. We have done these all in - the youngest patient I am
anticipating is 14. The rest of them have been young adults, so essentially their growth is
already done. So I would counsel that these are probably inappropriate procedures for
younger individuals and they ought to be reserved for people who are at least well into
their adolescence.
TISSUE ENGINEERING APPROACHES FOR
GENITAL RECONSTRUCTION
Anthony Atala
Laboratory for Tissue Engineering and Cellular Therapeutics, Department of Urology,
Children's Hospital and Harvard Medical School, Boston, MA,
INTRODUCTION
Genital reconstructive surgery is required for a wide variety intersex anomalies. One
of the major limitations of phallic reconstructive surgery is the availability of sufficient
autologous tissue. Non-genital autologous tissue sources have been used for decades.
Phallic reconstruction was initially attempted in the late 1930s using rib cartilage as a
stiffener in patients with traumatic penile loss (Fmmpkin et ai, 1994; Goodwin et ai,
1952) This method, involving multiple staged surgeries, was soon discouraged due the
unsatisfactory functional and cosmetic results. Silicone rigid prostheses were
popularized in the 1970s and have been used widely (Bretan et aI, 1989; Small et aI,
1975). However, biocompatibility issues have been a problem in selected patients (Nukui
et ai, 1997; Thonrnalla et ai, 1987). Tissue transfer techniques using flaps from various
non-genital sources, such as the groin, dorsalis pedis and forearm, have been used for
genital reconstruction (Jordan et al, 1999). However, operative complications, such as
infection, graft failure and donor site morbidity are not negligible. Phallic reconstruction
using autologous tissue, derived from the patient's own cells, may be preferable in
selected cases.
Reconstruction of Corporal Tissues
Reconstruction of Corporal Smooth Muscle
One of the major components of the phallus is corporal smooth muscle. The creation
of autologous functional and structural corporal tissue de novo would be beneficial.
Initial experiments were performed in order to determine the feasibility of creating
corporal tissue in vivo using cultured human corporal smooth muscle cells seeded onto
biodegradable polymers (Kershen et ai, 1998). Primary normal human corpus cavernosal
smooth muscle cells were isolated from normal young adult patients after informed
consent during routine penile surgery. Muscle cells were maintained in culture, seeded
onto biodegradable polymer scaffolds, and implanted subcutaneously in athymic mice.
Implants were retrieved at 7, 14 and 24 days after surgery for analyses. Corporal smooth
muscle tissue was identified grossly and histologically. Intact smooth muscle cell

290 A. ATALA
multilayers were observed growing along the surface of the polymers throughout all time
points. Early vascular ingrowth at the periphery of the implants was evident by 7 days.
By 24 days, there was evidence of polymer degradation. Smooth muscle phenotype was
confirmed immunocytochemically and by western blot analyses with antibodies to alpha-
smooth muscle actin. This study provided evidence that cultured human corporal smooth
muscle cells may be used in conjunction with biodegradable polymers to create corpus
cavernosum tissue de novo.
Endothelial cell expansion and characterization. In order to engineer functional
corpus cavernosum, both smooth muscle and sinusoidal endothelial cells are essential.
However, penile sinusoidal endothelial cells had not been extensively cultured in the
past, and had not been fully characterized. A method of isolation and expansion of
sinusoidal endothelial cells from corpora cavernosa was devised, and cell function and
gene expression were characterized.
Corpus cavernosum tissue were digested with collagenase type 2, and the cells were
grown in culture. The endothelial cells were isolated from primary culture by magnetic
beads coated with anti bovine E-selectin antibodies. To verify the phenotype, endothelial
cells were immuno-stained with antibodies that recognize endothelial cells-specific
markers. To test if the express functional receptors for vascular endothelial growth factor
(VEGF), cross-linking of 1251-VEGF165 to the cells was performed, and 125 1_
VEGFl65lVEGF-receptor complexes were analyzed by SDS-PAGE and
autoradiography. The mitogenic response of the cells to increasing concentrations of
VEGF and basic fibroblast growth factor (bFGF) was tested, as well as their ability to
form capillaries in three dimensional collagen gels.
Figure I. Human corporal cavernosal endothelial cells form capillary-like structures in vitro (lOOX).
Immuno-isolated endothelial cells from corpus cavernosum had endothelial cells-like

morphology and formed a cobblestone-like structure when they reached confluence. The
cells were positively stained with anti E-selectin, Factor VIII and Flk-l antibodies,
indicating their endothelial origin. Cross-linking of 1251-VEGF165 to VEGF receptors
on the cell surface indicated the expression of two VEGF receptors, Flk-l and neuropilin-
1. Increasing concentrations of VEGF and bFGF resulted in 40 and 70% increase in
DNA synthesis of corporal cavemosal endothelial cells respectively. A similar response
to VEGF and bFGF was observed with endothelial cells derived from adrenal capillaries.
However, endothelial cells did not respond mitogenic ally to neither of the growth factors.
When grown on collagen, corporal cavemosal endothelial cells formed capillary
structures which created a complex three-dimensional capillary network (Figure 1).
These results demonstrate that immuno-isolation is a reliable method to obtain
endothelial cells from corpus cavemosum. Cultured endothelial cells express specific
markers, have an endothelial morphology and express functional VEGF receptors. The
cells proliferate in response to endothelial cell growth factors and they have the capability
to form a three dimensional capillary network. Thus, corpus cavemosum-derived
endothelial cells may be obtained from a biopsy, grown in culture, and expanded, while
retaining their phenotypic characteristics.
Engineering of corporal smooth muscle and endothelium in vivo. In a subsequent
study, the possibility was investigated of developing human corporal tissue in vivo by
combining smooth muscle and endothelial cells (Park et ai, 1999). Primary normal
human corpus cavemosal smooth muscle cells and ECV 304 human endothelial cells
were seeded on biodegradable polymers at concentrations of 20 x 10 6 cells/cm3 and lOx
10 6 cells/cm3 , respectively. A total of 80 polymer scaffolds (60 seeded with cells and 20
without cells) were implanted in the subcutaneous space of 20 athymic mice. Each
animal had 4 implantation sites consisting of 3 polymer scaffolds seeded with muscle and
endothelial cells, and a control (polymer alone). Mice were sacrificed at 1, 3, 5 and 7
days, and at 14, 21, 28 and 42 days after implantation. The retrieved structures were
analyzed grossly and histologically. Immunocytochemical analyses were performed on
cultured cells and the retrieved specimens using several specific antibodies. Polyclonal
Anti-vWF was used to identify infiltrating host vessels. Broadly reacting monoclonal
anti-pancytokeratins AE1/AE3 were used to identify ECV 304 human endothelial cells.
Corporal smooth muscle fibers were labeled with monoclonal anti-alpha smooth muscle
actin. The tissue composition of the retrieved specimens were analyzed by computerized
morphometry. The percentage and the ratio of muscle and endothelial tissue were
calculated using computerized morphometry imaging software.
Human corpus cavemosal smooth muscle cells in culture showed homogenous
populations of spindle shaped cells under phase contrast microscopy. ECV 304 human
endothelial cells were observed as cobblestone monolayers initially and progressively
aggregated and formed extensive capillary-like networks by 27 days of culture.
Immunocytochemical analyses of the cells in vitro were able to identify the ECV 304
human endothelial cells with anti-pancytokeratins and the smooth muscle cells with alpha
smooth muscle actin. Polyclonal anti-vWF antibodies did not stain the ECV 304 cells.
At retrieval all polymer scaffolds seeded with cells had formed distinct tissue
structures and maintained their pre-implantation size. The control scaffolds without cells
had decreased in size with increasing time. Histologically, all of the retrieved polymers
seeded with corporal smooth muscle and endothelial cells showed the survival of the
implanted cells. The presence of penetrating native vasculature was observed 5 days
after implantation. The formation of multilayered strips of smooth muscle adjacent to
endothelium was evident by 7 days after implantation. Increased smooth muscle
292 A.ATALA
organization and accumulation of endothelium lining the luminal structures were evident
14 days after implantation. A well organized construct, consisting of muscle and
endothelial cells, was noted at 28 and 42 days after implantation. A marked degradation
of the polymer fibers was observed by 28 days. There was no evidence of tissue
formation in the controls (polymers without cells).
Immunocytochemical analyses using anti-vWF (identifying native vasculature) and
anti-pancytokeratins (identifying ECV 304 endothelial cells) distinguished the origin of
the vascular structures in each of the constructs. Anti-vWF antibodies stained the native
vessels positively, but failed to stain the implanted endothelial cells and reconstituted
vascular structures. In contrast, anti-pancytokeratin antibodies identified the implanted
endothelial cells and the reconstituted vessels, but did not stain the native vascular
structures. Anti- alpha actin antibodies confirmed the smooth muscle phenotype.
Smooth muscle fibers were progressively organized with time.
Computer assisted quantitative morphometric analysis of the retrieved specimens
showed that the tissue was composed of 31.2% + 1.6% muscle and 16.4% + 1.5%
endothelium. These results were consistent throughout the study. The muscle to
endothelial tissue ratio was 1.98 + 0.16 : 1. This ratio was approximately equivalent to
the ratio of muscle and endothelial cell seeding prior to implantation (2 : 1).
Figure 2. Scanning electron microscopy of human cavemosal smooth muscle and endothelial cells seeded
on acellular matrices.
These experiments showed that human corporal smooth muscle cells and endothelial
cells seeded on biodegradable polymer scaffolds are able to fonn vascularized cavemosal
muscle when implanted in vivo. Endothelial cells are able to act in concert with the
native vasculature. The results of these studies suggested that the creation of well
vascularized autologous corporal-like tissue, consisting of smooth muscle and endothelial
cells, may be possible.
Tissue engineering of structural corporal tissue. The aim of phallic reconstruction is to

achieve structurally and functionally normal genitalia. It had been shown that human
cavernosal smooth muscle and endothelial cells seeded on polymers would form tissue
composed of corporal cells when implanted in vivo. However, corporal tissue
structurally identical to the native corpus cavernosum was not achieved, due to the type
of polymers used. Therefore, a naturally derived acellular corporal tissue matrix that
possesses the same architecture as native corpora was developed (Figure 2). The
feasibility of developing corporal tissue, consisting of human cavernosal smooth muscle
and endothelial cells in vivo, using an acellular corporal tissue matrix as a cell delivery
vehicle was explored (Falke et aI, 1999).
Acellular collagen matrices were derived from processed donor rabbit corpora using
cell lysis techniques. Human corpus cavernosal muscle and endothelial cells were
derived from donor penile tissue, the cells were expanded in vitro and seeded on the
acellular matrices (figure 1). A total of 80 matrices, 20 without and 60 with cells, were
implanted subcutaneously in 20 athymic mice. 36 matrices with cells were maintained in
culture for up to 4 weeks. Hydroxy-proline quantification, western blot analysis, RT-
PCR and scanning electron microscopy of the matrices, with and without cells, were
performed at various time points. Animals were sacrificed at 3 days, 1, 2, 3, 4, 6 and 8
weeks after implantation. Immunocytochemical and histological studies were performed
to confirm the muscle and endothelial phenotype.
Western blot analysis detected alpha actin, myosin and tropomyosin proteins from
human corporal smooth muscle cells. Expression of muscarinic acetylcholine receptor
(mAChR) subtype m4 mRNA was demonstrated by RT-PCR from corporal muscle cells
prior to and 8 weeks after seeding. The implanted matrices showed neovascularity into
the sinusoidal spaces by 1 week after implantation. Increasing organization of smooth
muscle and endothelial cells lining the sinusoidal walls was observed at 2 weeks and
continued with time. The matrices were covered with the appropriate cell architecture 4
weeks after implantation. The matrices showed a stable collagen concentration over 8
weeks, as determined by hydroxy-proline quantific.ation. Immunocyto-chemical studies
using alpha-actin and Factor VIII antibodies confirmed the presence of corporal smooth
muscle and endothelial cells, both in vitro and in vivo, at all time points. There was no
evidence of cellular organization in the control matrices.
This study demonstrated that human cavernosal smooth muscle and endothelial cells
seeded on acellular corporal tissue matrices are able to form vascularized corporal
structures in vivo. The use of these tissue matrices as cell delivery scaffolds allowed for
the development of adequate structural constructs. The formation of corporal tissue,
similar to that of the native erectile tissue, may provide an additional armamentarium in
the management of complex penile reconstructive challenges.
To apply tissue engineering techniques to reconstruct corporal tissue clinically, further
studies must be performed. These include the further development of cell delivery
vehicles identical to that of native corpus cavernosal architecture, and functional and
biomechanical studies of the nea-corpora. Although smooth muscle and endothelial cells
are the major components of erectile tissue, other structures, such as connective tissue
and nerves are needed in order to achieve normal anatomical and functional corpora.
Engineered clitoral tissue. The availability of clitoral smooth muscle tissue for use in
patients with intersex anomalies may be of clinical utility. Primary cultures of human
294 A.ATALA
clitoral smooth muscle cells were derived from operative biopsies obtained during
genitoplasty. Cells were maintained in culture for a period of 75 days after the first
passage, during which time they multiplied into multilayered "tissue-like" structures
which were readily lifted off the culture plate as a stretchable layer. The layers of clitoral
muscle were divided and implanted subcutaneously in athyrnic mice. Animals were
sacrificed at 7, 14, 21, 42, and 84 days after surgery. Implants were examined
histologically with hematoxylin and eosin and masson's trichrome staining, as well as
immunohistochemically with antibodies to the intermediate filaments alpha-smooth
muscle actin and desmin.
Morphological analysis of the "neo-tissue" in culture via phase contrast microscopy
revealed characteristic spindle shaped smooth muscle ceIIs growing on top of each other
in the typical "hill and valley appearance." Tissue specimens analyzed prior to
implantation demonstrated the presence of an extraceIIular matrix as well as positive
staining for the intermediate filaments alpha-smooth muscle actin and desmin. Seven
days after implantation, intact multilayered smooth muscle strips were identified with
evidence of early vascular in-growth at the periphery of the tissue. By 21 days post
implantation, there was evidence of more extensive neovascularization and the smooth
muscle maintained its multilayered architecture. The specimens remained
immunohistochemicaIIy positive for alpha-smooth muscle actin and desmin post
implantation.
This study showed that surgicaIIy obtained clitoral smooth muscle may be cultured in
vitro, expanded, and developed into multilayered sheets of muscle tissue which can be
retransplanted into the in vivo environment. Tissue transplants are neovascularized, and
can survive in vivo, maintaining smooth muscle phenotype and architecture.
The Tissue Engineered Penile Prostheses
Although silicone is an accepted biomaterial for penile prostheses, biocompaubility is a

concern (Nukui et aI, 1997; Thomalla et aI, 1987) The use of a natural prosthesis composed of
autologous ceIIs may be advantageous. A feasibility study for creating natural penile
prostheses made of cartilage was performed initially. (Yoo et aI, 1998).
Cartilage, harvested from the articular surface of calf shoulders, were isolated, grown
and expanded in culture. The cells were seeded onto pre-formed cylindrical polyglycolic
acid polymer rods (1 cm in diameter and 3 cm in length). The cell-polymer scaffolds
were implanted in the subcutaneous space of 20 athymic mice. Each animal had 2
implantation sites consisting of a polymer scaffold seeded with chondrocytes and a
control (polymer alone). The rods were retrieved at 1, 2, 4 and 6 months post
implantation. Biomechanical properties, including compression, tension and bending
were measured on the retrieved structures. Histological analyses were performed to
confirm the ceIIular composition. At retrieval, all of the polymer scaffolds seeded with
cells formed milky-white rod shaped solid cartilaginous structures, maintaining their pre-
implantation size and shape (figure 3). The control scaffolds without cells failed to form
cartilage. There was no evidence of erosion, inflammation or infection in any of the
implanted cartilage rods.
Figure 3. Cylindrical polymer scaffolds seeded with chondrocytes and implanted in vivo formed milky-white
rod shaped solid cartilaginous structures.
The compression, tension and bending studies showed that the cartilage structures
were readily elastic and could withstand high degrees of pressure. Biomechanical
analyses showed that the engineered cartilage rods possessed the mechanical properties
required to maintain penile rigidity. The compression studies showed that the cartilage
rods were able to withstand high degrees of pressure. A ramp compression speed of 200
um/sec, applied to each cartilage rod up to 2000 urn in distance, resulted in 3.8 kg of
resistance. The tension relaxation studies demonstrated that the retrieved cartilage rods
were able to withstand stress and were able to return to their initial state while
maintaining their biomechanical properties. A ramp tension speed of 200 um/second
applied to each cartilage rod created a tensile strength of 2.2 kg, which physically
lengthened the rods an average of 0.48 cm. Relaxation of tension at the same speed
resulted in retraction of the cartilage rods to their initial state. The bending studies
performed at two different speeds showed that the engineered cartilage rods were
durable, malleable, and were able to retain their mechanical properties. Cyclic
compression, performed at rates of 500 um/sec and 20,000 um/sec, demonstrated that the
cartilage rods could withstand up to 3.5 kg of pressure at a predetermined distance of
5000 urn. The relaxation phase of the cyclic compression studies showed that the
engineered rods were able to maintain their tensile strength. None of the rods were
ruptured during the biomechanical stress relaxation studies.
Histological examination with hematoxylin and eosin showed the presence of mature
and well formed cartilage in all the chondrocyte-polymer implants. The polymer fibers
were progressively replaced by cartilage with time progression. Undegraded polymer
fibers were observed at 1 and 2 months after implantation. However, remnants of
polymer scaffolds were not present in the cartilage rods at 6 months. Aldehyde fuschin-
alcian blue and toluidine blue staining demonstrated the presence of highly sulfated
296 A.ATALA
mucopolysaccharides which are differentiated products of chondrocytes. There was no

evidence of cartilage formation in the controls.
In a subsequent study using an autologous system, the feasibility of applying the
engineered cartilage rods in-situ was investigated. (Yoo et aI, 1999). Autologous
chondrocytes harvested from rabbit ear were grown and expanded in culture. The cells
were seeded onto biodegradable poly-L-lactic acid coated polyglycolic acid polymer rods
at a concentration of 50 x 10 6 chondrocytes/cm3 . Eighteen chondrocyte-polymer
scaffolds were implanted into the corporal spaces of 10 rabbits. As controls, two corpora,
one each in 2 rabbits, were not implanted. The animals were sacrificed at 1, 2, 3 and 6
months after implantation. Histological analyses were performed with hematoxylin and
eosin, aldehyde fuschin-alcian blue, and toluidine blue staining. All animals tolerated the
implants for the duration of the study without any complications. Gross examination at
retrieval showed the presence of well formed milky white cartilage structures within the
corpora at 1 month. All polymers were fully degraded by 2 months. There was no
evidence of erosion or infection in any of the implant sites. Histological analyses with
alcian blue and toluidine blue staining demonstrated the presence of mature and well
formed chondrocytes in the retrieved implants. Subsequent studies were performed
assessing the functionality of the cartilage penile rods in vivo long term. To date, the
animals have done well, and can copulate and impregnate their female partners without
problems. Further functional studies need to be completed before applying this
technology to the clinical setting.
Urethra
Various strategies have been proposed over the years for the regeneration of urethral
tissue. Woven meshes of PGA (Dexon®) were used to reconstruct urethras in dogs.
Three to four centimeters of the ventral half of the urethral circumference and its adjacent
corpus spongiosum was excised, and the polymer mesh was sutured to the defective area.
After 2 weeks, the animals were able to void through the neourethra. At two months, the
urothelium was completely regenerated. The polymer meshes were completely absorbed
after three months. No complications occurred. However, the excised corpus spongiosum
did not regenerate (Bazeed et ai, 1983).
PGA has been also used as a cell transplantation vehicle to engineer tubular
urothelium in vivo. Cultured urothelial cells were seeded onto tubular PGA scaffolds and
implanted into athymic mice. At 20 and 30 days, polymer degradation was evident and
tubular urothelium formed in which cells were stained for a urothelium-associated
cytokeratin (Atala et ai, 1992).
PGA mesh tubes coated with polyhydroxybutyric acid (PHB) were used to reconstruct
urethras in dogs. PHB is a biodegradable thermoplastic polymer produced microbially.
PHB degrades by both hydrolysis and enzyme reaction. The hydrolized product, 3-
hydroxybutyric acid, is a natural metabolite that is contained in human blood. Eight to
twelve months later, complete regeneration of urothelium and adjacent connective tissue
occurred. All of the polymers disappeared after one year. There were no anastomotic
strictures or inflammatory reactions.
SIS was utilized as an onlay patch graft for urethroplasty in rabbits (Kropp et aI,
1998). A comprison among the collagen sponges, SIS, and PGA fiber based matrices is
shown in figure 4. SIS was compared to full thickness preputial skin grafts and shams
(simple urethrotomy and closure). Animals were sacrificed between 8 and 12 weeks.
Histologic evaluation demonstrated that SIS promoted urethral regeneration.
Regenerated urethras contained 3-4 layers of stratified columnar urothelium that was
indistinguishable from the normal rabbit urothelium. There was also evidence of circular
smooth muscle regeneration underneath the urothelium. This regenerated muscle was
contained within an abundant amount of collagen and fibrous connective tissue. Grossly,
there was no evidence of diverticular formation. In contrast, all grafts in the preputial
skin group had evidence of diverticulum formation.
Figure 4. Scanning electron micrographs of biomaterials: (top) collagen sponge, (center) acellular matrix
prepared from pig bladder submucosa, (bottom) polyglycolic acid fiber-based matrix. (the size bars = 100 J-lm).
A homologous free graft of acellular urethral matrix was used in a rabbit model (Sievert
et aI, 2000). A 0.8 to 1.1 cm. segment of the urethra was resected and replaced with an
acellular matrix graft of 1.0 to 1.5 cm. Histological examination showed complete
298 A.ATALA
epithelialization and progressive vessel infiltration. At 3 months, smooth muscle bundles

were first observed infiltrating the matrix at the anastomosis; after 6 months, the smooth
muscle bundles had grown into one-third of the matrix. By 8 months, the host and
implant could not be differentiated by urethrography. All tissue components were seen in
the grafted matrix after 3 months, with further improvement over time; however, the
smooth muscle in the matrix was less than in normal rabbit urethra and was not well
oriented.
Figure 5. Urethrogram 6 months post-operatively of a patient who had a portion of his urethra replaced using
tissue engineering techniques.
Recently, another acellular collagen matrix has proven to be a suitable graft for
repairing urethral defects both experimentally and clinically. The acellular collagen
matrix was obtained from porcine bladder for the animal studies. The neourethras
demonstrated a normal urothelial luminal lining and organized muscle bundles, without
any signs of strictures or complications. The animals were able to void through the
neourethra (Chen et aI, 1999). These results were confirmed clinically in a series of
patients with a history of failed hypospadias reconstruction wherein the urethral defects
were repaired with human bladder acellular collagen matrices (Atala et el, 1999). The
neourethras were created by anastomosing the matrix in an onlay fashion to the urethral
plate. The size of the created neourethra ranged from five to fifteen centimeters. After a
three year follow-up, three of the four patients had a successful outcome in regards to
their cosmetic appearance and function (figure 5). One patient who had a 15 cm
neourethra created developed a subglanular fistula. The acellular collagen based matrix
eliminated the necessity of performing additional surgical procedures for graft harvesting,
and operative time, as well as the potential morbidity due to the harvest procedure, were
decreased. Similar results were obtained in pediatric and adult patients with urethral
stricture disease (Kassaby et aI, 2000). Over 60 pediatric and adult patients with urethral
T1SSliE ENGINEERING APPROACHES 299
disease have been succesfully treated using the collagen based matrix. One of its
advantages over non-genital tissue grafts used for urethroplasty, e.g., bladder mucosa and
buccal mucosa, is that the material is "off the shelf'. This eliminates the necessity of
additional surgical procedures for graft harvesting, which may decrease operative time, as
well as the potential morbidity due to the harvest procedure.
Testicular Hormonal Replacement
Leydig cells are the major source of testosterone production in males. Intersex patients
with testicular dysfunction may require androgen replacement for somatic development.
Conventional treatment for testicular dysfunction consists of periodic 1M injections of
chemically modified testosterone, or more recently, of skin patch applications. However,
long term non-pulsatile testosterone therapy is not optimal and can cause multiple
problems, including erythropoiesis and bone density changes.
A system was designed wherein leydig cells were microencapsulated for controlled
testosterone replacement. Microencapsulated leydig cells offer several advantages, such
as serving as a semipermeable barrier between the transplanted cells and the host's
immune system, as well as allowing for the long term physiological release of
testosterone.
Purified leydig cells were isolated, characterized, suspended in an alginate solution
and extruded through an air jet nozzle into a 1.5% CaClz solution were they gelled; and
were further coated with 0.1% poly-L-Iysine (figure 5). The encapsulated cells were
pulsed with HCG every 24 hr. The medium was sampled at different time points after
HCG stimulation and analyzed for testosterone production. Cell viability was confirmed
daily. The encapsulated leydig cells were injected in castrated animals and serum
testosterone was measured serially. The castrated animals reCeIVIng the
microencapsulated cells were able to maintain testosterone levels long term (Machluf et
aI, 1998). These studies suggest that microencapsulated leydig cells may be able to
replace or supplement testosterone in patients with intersex anomalies. A similar system
is cunently being applied for estrogen.
Gene Therapy and Tissue Engineering Applications for Intersex

Anomalies
Genetically Engineered Cells
Cells can be engineered to secrete growth factors for various applications, such as for
promoting angiogenesis for tissue regeneration. Angiogenesis, the process of new blood
vessel formation, is regulated by different growth factors (Klagsbrun et aI, 1991;
Hanahan et aI, 1996; Risau et aI, 1997). These growth factors stimulate endothelial cells
which are already present in the patients body to migrate to the implanted area of need,
300 A. ATALA
where they proliferate and differentiate into blood vessels (Polverini et aI, 1996). One of
the major molecules which promote and regulate angiogenesis is vascular endothelial
growth factor (VEGF). (Klagsbrun et aI, 1991; Polverini et aI, 1996) VEGF is the only
pro angiogenic factor known to act specifically on endothelial cells. Extent amounts of
VEGF in patients may induce rapid formation of new blood vessels and capillaries for
tissue regeneration. Several methods have been used experimentally to deliver VEGF in
vivo. The growth factor protein can be directly injected into tissues (Bauters et aI, 1994),
however, the rapid clearance of VEGF proteins from the vascular system limits its effect
to only minutes. The VEGF gene could be delivered to tissues using various techniques;
however, the transfection efficiency is low, the onset of action is delayed for up to 48 to
72 hours after the VEGF cDNA is incorporated, and the effect is transient, lasting only
several days (Bauters et aI, 1994; Taub et aI, 1998; Takeshita et aI, 1996; Isner et aI,
1999).
An approach which has been pursued to increase and stimulate rapid vascularization in
vivo was to engineer a cell line to secrete high levels of vascular endothelial growth
factor proteins by gene transfecting the cells with the VEGF cDNA. The VEGF secreting
cells where encapsulated in polymeric microspheres. The microspheres allowed nutrients
to reach the cells, while the VEGF proteins secreted from the cells diffused into the
surrounding tissues. The micro spheres protect the coated cells from the host immune
environment. This novel system of neovascularization was tested in vitro and in vivo in
an animal model.
CHO cells were chosen for the expression of recombinant VEGF. We have previously
cloned the human cDNA encoding VEGF I65 (Soker et aI, 1996) and sub cloned it into
pRclCMV expression vector (Invitrogen). In the resulting pCMV -VEGF plasmid, the
expression of VEGF is driven by the cytomegalovirus promoter. VEGF expression
vector was used to transfect CHO cells and neomycin resistant clones were selected.
Conditioned media was collected from individual clones and proteins were absorbed on
heparin Sepharose. Clones that secreted high levels ofVEGF (CHOIVEGF) as measured
by western blot analysis, were selected for subsequent experiments.
CHOIVEGF cells were encapsulated within micro spheres composed of Ca-alginate,
and were coated with the positively-charged polyelectrolyte PLL, and recoated with
alginate. (Machluf et ai, 2000). The microcapsules containing the CHOIVEGF cells had
a spherical shape with an average diameter of 0.6 mm +1- 0.05 mm. Western blot
analyses for human VEGF, performed on the cultured medium of the encapsulated cells,
depicted high levels of VEGF at all retrieval time points.
In vivo injected Microencapsulated CHOIVEGF cells and the surrounding tissues
were harvested from each animal and processed with OCT. Immunostaining with anti
human VEGF showed high levels of VEGF at the inner core of the microcapsules at days
3, 7 and 14 post implantation. Tissues surrounding the microcapsules also stained
positively for VEGF. Empty microspheres without CHOIVEGF cells stained negatively
forVEGF.
Macroscopic examination of the CHOIVEGF microencapsulated cell implant sites
showed a progressive increase in vascularization. At days 7 and 14, extensive
vascularization was evident. Control groups which received the empty microcapsules
showed only minimal vascularization.
Microscopic examination using immunostaining with CD-31 showed positive staining

for endothelial cells in the skin harvested from the study groups at all time points. An
increase in clusters and sinusoidal structures of newly formed capillaries was seen in the
skin over time. A comparison between H&E staining and CD-31 staining of the skin
showed that the newly formed capillaries were scattered around existing blood vessels.
Positively stained sinusoidal structures were less numerous in tissues harvested from the
control animals.
The microencapsulated engineered cells are a novel system for the delivery of VEGF
proteins. The encapsulation of these cells in alginate-PLL capsules protect the
encapsulated cells from the host immune system, while allowing the constant release of
VEGF as needed. The release of VEGF over a period of two weeks stimulated
endothelial cell migration, cluster formation and newly formed capillaries at the implant
sites. The degree of VEGF secretion and the period of delivery can be regulated by
modulating the number of engineered cells which are encapsulated per microsphere, as
well as the number of micro spheres injected. A similar strategy has also been pursued for
the genetic engineering of anti-angiogenic factor secreting cells (loki et aI, 2001).
Gene Therapy for Tissue Engineered Constructs
Based on the feasibility of tissue engineering techniques in which cells seeded on

biodegradable polymer scaffolds form tissue when implanted in vivo, the possibility was
explored of developing a neo-organ system for in vivo gene therapy (Yoo et aI, 1997).
In a series of studies conducted in our laboratory, human urothelial cells were
harvested, expanded in vitro and seeded on biodegradable polymer scaffolds. The cell-
polymer complex was then transfected with PGL3-luc, pCMV -luc and pCMVB-gal
promoter-reporter gene constructs. The transfected cell-polymer scaffolds were then
implanted in vivo and the engineered tissues were retrieved at different time points after
implantation. Results indicate that successful gene transfer may be achieved using
biodegradable polymer scaffolds as a urothelial cell delivery vehicle. The transfected
cell/polymer scaffold formed organ-like structures with functional expression of the
trans fected genes (Y 00 et aI, 1997).
This technology is applicable throughout the spectrum of intersex anomalies which
may be manageable with tissue engineering. For example, one can envision the use of
effecting in vivo gene delivery through the ex vivo transfection of tissue engineered
cell/polymer scaffolds for the genetic modification of diseased corporal smooth muscle
cells harvested from intersex patients. Patients with intersex anomalies often present with
corporal fibrosis. Studies of human corpus cavemosum smooth muscle cells have
suggested that cellular overproduction of the cytokine transforming growth factor-l
(TGF-1) may lead to the synthesis and accumulation of excess collagen in patients with
arterial insufficiency resulting in corporal fibrosis. Prostaglandin E 1 (PGE 1) was shown
to suppress this effect in vitro. Theoretically, the in vitro genetic modification of corporal
smooth muscle cells harvested from an interesex patient, resulting in either a reduction in
the expression of the TGF-l gene, or the overexpression of genes responsible for PGEI
production, could lead to the resumption of erectile functionality once these cells were
used to repopulate the diseased corporal bodies.
302 A.ATALA
The evolution of the science of tissue engineering and cellular therapy has allowed
new approaches for the reconstruction of genital tissues. Engineering efforts, focused on
creating genital structures, have been pursued for several years. One could envision using
this system to treat intersex patients who requires phallic reconstruction. Small phallic
tissue biopsies could be obtained through minimally invasive techniques under local
anesthesia. The autologous cells could be isolated, grown and expanded in culture. The
cells could be seeded on pre-configured polymer scaffolds, engineered in vitro, and
implanted back into the host. It may also be possible to deliver specific genes regulating
fibrosis and inflammation to the newly formed tissues using already established gene
delivery methods.
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Index
Ablatio penis, 2\0 Bed nucleus of the stria terminal is (BSTc)

phallic reconstruction, 276, 286 se~ual dimorphism (figure), 87
Activists, 202, 206; see also Intersex Society of and transsexualism, 92, 93
North America YIP innervation, 87
Alpha-fetoprotein Behavioral instruments 214-217
estrogen binding effects, 62 gender identity, 215
rat brain, 62 gender role behavior, 215
Alzheimer's disease, sex differnces, 87, 88 sexuality, 216
Ambiguous genitalia Biochemical evaluation, in genital ambiguity, 113
and CAH, \09 Bioethics, defined, 151
evaluation (flowchart), 112
maternal/familial medical history, 112, 113 Cartilage rods, as penile prostheses, 294-295
neonatal evaluation, 112 Child Game Participation Questionnaire, 215
prenatal diagnosis, 227, 229 Chorionic villus sampling (CYS)
Androgen biosynthesis, inadequate, 114, 121, 122 for diagnosis of CAH, 229
Androgen insensitivity syndromes (AIS), 114, 126- Clitoral function, neurovascular bundle, 205
127 Clitoral recession, complications of (figure), 253
sex assignment in, 126, 186 Clitoroplasty
and spatial performance testing, 127, 128 and glanular dissection, 247, 248
Androgen receptor influence of prenatal therapy in CAH, 265-266
human phallic growth, figure, 47 neuroanatomic concepts, 242, 243
murine phallic growth, figure, 47 surgical techniques for, 251-254
penile, downregulation, 48 Cloacal exstrophy
and penile growth, 46 antenatal diagnosis, 136, 229
penile, murine, 49 associated findings, 135, 136, 267
Androgen-receptor gene, structure, 127 embryology of, 267
Anecdotal evidence, 162 figure, 229
importance of, 170 gender identity disorder, 191
Anterior commissure, sex differences, human, 93 gender management, 138, 139, 268, 269
Auditory sexual dimorphisms, testing for, 182, 183 gender reassigned, long term outcomes 269, 273
Authority impact of new surgical techniques, 268, 269
clinical decision making, 157 importance of osteotomy in males, 271
defined, 156 as an intersex condition, 268
invoking as an emergency, 168, 171 male sex of rearing followup study, 138, 140, 145
physcian and changes in, 167 neonatal management, 137
305
306 Index
Cloacal exstrophy (cont.) Sa-reductase deficiency (cont.)

and phallic reconstruction, 2276, 277 gender identity, 121, 124
self reassignment, 140 and gender identity formation, 127
testicular histology, 141 and genital ambiguity, 121
Complete androgen insensitivity (CAIS), 126 New Guinean community, 130
and testicular neoplasms, 128 point mutations, 138
gonadotrophin levcis, 127 prostatic volumes, 122
Confidentiality, 166 semen analysis, 138
Conforming genitalia, imporlance of, 195 sex of rearing dilemma, 124-126
Congenital adrenal hyperplasia (CAH), 113,225 and sex assignment 121
biochemical studies, 114 Fluorescent in situ hybridization (FISH), 1I3
as a disease, 156
U-hydroxy1ase deficiency, 110 Gender assignment
external female phenotype, 113 general guidelines, ISS
and female gender identity, 207-209 iatrogenic risk, 156
fetal adrenal transplant for, 234 parental demmands, 185
internal female phenotype, 113 Gender identification, outcomes in intersex patients,
long term medical complications, 226 122, 127, 209, 210
long term psychosocial outcomes, 207-209 Gender identity, 154, 179
metabolic pathways (figure), 111 age of awareness, 125, 196
prenatal diagnosis 226-228 defined. 117
prenatal therapy, 230-234 discordance with gender role, 155
prenatal therapy and CNS imprinting, 235 etiologies, 180, lSI
prenatal therapy, multicenter outcome study, 233 in androgen insensitivity, 127
salt wasters, 115, 207, 208 and micropenis, 51, 52
and sexual orientation, 117 predictors of outcomcs, 182, 1113
surgery for, 116 scale for men, 217
tomboyish behavior, 117, 207, 208 self deciaration, 190, 191
21-hydroxylase deficiency, 108 Gender identity disorder (GID), 199
Gender Identity Interview, 217
Dexamethasone, in prenatal therapy of CAH, 232, Gender role, 117, 154
235 defined, 117
Differential attrition, 213 Genital surgery
Dysgcnetic gonads, and malignant potential, 1I5 long term goals, 204-206
postponed to adnlthood, controversy, 116, 204,
Erotic sensitivity, 205 283
Ethics psychosocial goals, 206
defined, 150 timing of, 116, 263
intellectual objectives, 150 Genotypic sex
defined, 152
Feminizing genitoplasty, 272; also see Clitoroplasty; limitations, 152
Urogenital sinus Gonadal biopsies, role for, 115
neuroanatomical concepts, 242, 243, 246, 247 Gonadal sex
surgery for (figure), 265 and sexual differentiation, 1, 109
timing of surgery, 263 defined, 1
Fetal clitoris, human, neural localization (figures), 243 Gonadectomy, timing in androgen resistance cases,
Fetal penis, human, neural localization (figure), 241 116, 128
5a-dihydrotestosterone, 108, 121, 122
Sa-reductase hCG stimulation test, 114
male pseudohermaphroditism,l21 protocol (figure), 115
type I and 2 gene structure, 122 Hormonal replacement, via encapsulated Leydig
5a-reductase deficiency, 121-126 cells, 299
biochemical findings, 122 Hypertension, and CAR 110
change in gender role, 123, 124 Hypospadias
Dominican community, 123, 124 neuroanatomical findings (figure), 244
Index 307
Hypospadias (COllt.) Miillerian inhibiting substance (MIS) (COllt.)

surgical anatomy, 245, 246 ELISA determination, 29
vascularity, 245 gene structure and map, 27
Hypothalamus: also see SDN-POA knockout mouse model, 29
and sexual behavior, 63-65, 90-94 laterality of effects, 38, 39
sexual differentiation, 59, 60, 83-85 levels in granulosa cell tumors, 32, 33
levels in intersex conditions, 31, 32, 34
Informed consent, 207-277 levels with ovarian carcinoma, 33
Innovation, ethical aspects of, 168 loss of function mutations, 29
Intersex conditions molecular characteristics, 26
etiologies of (table), 108 non Miillerian actions, 28, 29
as normal or abnormal states, 155 receptor gene map, 28
and unequivocal disease, 156 Murine models intersex conditions
Intersex Society of North America (ISNA), 202, 207 hypo gonadotrophic hypogonadal, 45
recommendations for genital surgery, 206, 207 MIS, 29
Long term outcome studies Nature nurture

study methods, 211 Money, John, 177, 201
study vs control groups, 212
Optimal gender policy, 200
Magnetic resonance imaging (MRI), in evaluation of Orgasmic capacity, 205
genital ambiguity, 115 Outcome assessments
Male external genitalia, differentiation of, 42, 109, measurements, 214-217
241 need for blinded conditions, 217
Male pseudohermaphrodites, geographical clustering,
122, 123 Panhypopituitarism, medical management, 115
Malignant transformation Parental rights, 160
in androgen resistance syndromes, 128 Parental support, 118, 161, 162, 184, 185, 191
in dysgenetic gonads, 115 Pediatric assent, 158, 277
Medial preoptic nucleus, sex differences in rat, 59, and self declaration of gender, 158
60 Penile anatomy, surgical applications, 245, 246
Medical ethics, defined, 150 Penile growth
Mice, hypogonadotrophic hypogonadal, 45 androgen dependent, 42, 109
penile androgen receptors, 47, 49 androgen independent, 43
phallic growth, 46, 47 fetal development, 42
testosterone effects, 48 insulin like growth factor (ILGF-l), 43
Micropenis neonatal, 42
animal model, 52 serum testosterone, figure, 49
biochemical evaluation, 114, 115 Penile prosthesis
controversies in manargement, 41 biomechanical studies, 295
defined, 41 tissue engineering, 294-296
and gender conversion, 51, 52 Persistent Miillerian duct syndrome, 19, 25
and gender identity, 51 PET-scanning, eNS, sex differences, 101
human, etiologies, 45 Phallic reconstruction, 275, 283
long term follow up studies, 51, 52 fibula flap, benefit of, 286
and male sexual role, 51 free osteocutaneous fibula flap, 285
prepubertal androgen therapy, 48 goals of surgery, 276
and sex assignment, 51, 52, 186 long term outcomes, 276, 286
testosterone trial, 48, 115 radial forearm flap (figure), 278
Moratorium, on early surgery, 161, 166 timing of surgery, 276, 283, 281
Miillerian ducts, 2, 109 tissue engineering methods, 294-296
Miillerian inhibiting substance (MIS), 2, 26-28, 109 Phenotypic sex, I, 153
biosynthesis, diagram, 27 defined, 1
clinical measurments, 30 components of, 153
etfects of, 25, 28, 29, 109 Prader grading, degree of virilization (figure), 111
308 Index
Prenatal therapy Sex determining genes (cont,)

adrenal transplantation for CAH, 234 SRY, 3, 4,14
clitoral reduction with, 265 steroidogenic factor I (SFI), 6, 7
dexamethasone for CAH, 231, 232 table of, 8
Psychologic counseling, family, 118 Wilms tumor I (WTl), 6, 7
Sex differences. 57, 58, 65
Quigley Scale, 209, 212 Alzheimer's disease 87
human brain, 65
Reproductive potential, role in sex assignment, 206 human studies, problems with, 65
Re-reassignment, preoperative assessment, 282 neurological disorders (table), 76, 87
Resiliency, 196, 273 rat brain, 59. 60
Sex of rearing, defined, I
Salt wasting CAH, lIO, 113 Sexual differentiation
and masculine behaviors, 207, 208 embryology, 226
and 21-hydroxylase activity, 113 time course (figure), 109
Sampling procedures, potential problems, 212, 213 Sexual dimorphism
SDN-POA and sexual behavior, 59
androgen receptor, 84 functional, 60, 61
apoptosis, 63 hypothalamic anatomy 59, 60, 80, 81, 83, 86
and castration, 61 Sexual function, 205
effects of testosterone, 60 Sexual orientation, 86
estrogen receptor, 84 anterior commissure, 93
fetal estradiol effects, 60, 61 cell number, SCN, 91
and fetal tamoxifen, 61, 62 and changes in INAH-3, 93
fetal testosterone exposure, 60 defined, 78, 79
human cell number (figure), 85 factors in, 77
human fetal brain, 80, 81, 85 genetic determinanats, 79
human sexual behavior, 82 hypothalamic findings, 90-93
human volume changes (figure), 90 and prenatal drug exposure, 79
human volume distrbution, 90 SDN-PO cell number (figure), 90, 91
and male behavior, 63, 64 SDN-POA, 90
peptide hormone expression, 83,91 suprachiastmatic nucleus, 90, 91
and rat sexual behavior, 64, 82 vasopressin neurons in SCN, 91
rat versus human, 83 Sexual partner, and genital appearance, 206
steroid actions on, 63 Sexual satisfaction. 205, 206
stimulation studies, 64 Sexually dimorphic nucleus, of preoptic area (SDN-
Selection bias, 213 POA). 59, 60, 81, 83
Self reassignment Smooth muscle
during adolescence, 117, 133, 134, 140 clitoral, in culture, 293, 294
process of, 282 corporal, 290-292
(17) keto reductase deficiency, and gender identity, corporal, and endothelial cells, 392
125, 133, 134 corporal, and growth on polymers, 290
Sex assignment corporal, and VEGF effects, 290, 291
on basis of external genitalia, 116, 183, 200, 202 corporal, growth in acellular matrix (figure), 290-
and cultural biases. 193-194 292
factors influencing, 1I6 SRY,4
long term goals, 203-204 expression in brain (murine), 4, 13, 14
Money's influence, 177,201 expression in brain (human), 79, 80, 62
in Prader 5 patients with CAH, 208 functional cofactors, 5
psychosocial outcomes, 181, 207, 208 and HMG motif, 4
team, 1I6, 1I8 molecular properties, 3
traditional management, 181, 182 point mutation effects, 4
Sex determining genes SRY expression
DAXI,5 in the brain, 4, 13, 14, 62, 79, 80
SOX9.6 and hermaphroditism, 14
Index 309
Suprachiasmatic nucleus (SCN) True-sex policy, 199

development, hormonal, 91, 92
shape differences, 86 Urethroplasty, and tissue engineering techniques,
volumes in homosexual men, 90 297, 298
Surgical reconstruction, goals of, 204-205 Urogenital sinus
ASTRA approach, 263
Testicular development classification scheme, 254
abnormal, 17 embryology, 226
and cell types, 15, 16 Hendren Crawford technique, 267
in complete AIS, 17 high vaginal repairs, 257
Klinefelter's syndrome, 18, 19 perineal approach, 263
mixed gonadal dysgenesis, 19, 20 perineal transvesical approach, 257, 259
normal, 15, 16 posterior sagittal approach, 259-263
persistent Miillerian duct syndrome, 19 Rink approach, 263, 264
and post natal gonadotrophin surge, 17
prepubertal changes, 16
Vasoactive intestinal polypeptide (VIP) neurons, sex
pubertal changes, 16
differences in human brain, 87
and seminiferous tubules, 15, 16, 17
Vasopressin, plasma levels, sex differences, 101
Testis determining factor (TDF), 2
Virilization
Testosterone
metabolic pathways (figure), III
prenatal surge/rat, 59
and sex assignment, 185, 186
as prohormone in CNS, 62
as prohormone, genital development, 63, 108, 121
Third sex, 91, 166 Withholding of information, 203
Tissue engineering Wolffian ducts, 2
penile implants, 295, 296
and urethral reconstruction, 297, 298 XX males, I, 3, 4, 5
Transsexualism, 93, 94 identification of SRY, 3
hypothalamic findings, 93, 90, 103 XXY Klinefelter syndrome, 18

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PEDIATRIC GENDER

Recent Volumes in this Series

Springer-Science+Business Media, LLC

All rights reserved

Wilson C.l. Chung

Frank P.M. Kruijver

Heino F.L. Meyer-Bahlburg

Howard McC. Snyder, III

Stephen A Zderic, M.D.

SRY AND THE GENETICS OF SEX DETERMINATION ................. .

GONADAL DIFFERENTIATION - NORMAL AND

MULLERIAN INHIBITING SUBSTANCE:

MICROPENIS: AN ANIMAL MODEL AND ITS

HYPOTHALAMIC IMPRINTING BY GONADAL

SEXUAL DIFFERENTIA TION OF THE

THE ENDOCRINOLOGIST'S APPROACH TO THE

5a-REDUCTASE-2 DEFICIENCY AND COMPLETE

THE CHOP EXPERIENCE WITH CLOACAL

A FRAMEWORK FOR THE ETHICALLY JUSTIFIED

GENDER IDENTITY AND SEX ASSIGNMENT:

GENDER ASSIGNMENT AND REASSIGNMENT IN

PRENATAL DIAGNOSIS AND TREATMENT OF

ANA TOMICAL STUDIES OF THE FETAL

FEMINIZING GENITOPLASTY ................................................ 251

MANAGEMENT OF CLOACAL EXSTROPHy............................. 267

TOTAL PHALLIC CONSTRUCTION, OPTION

PENILE RECONSTRUCTION WITH A FREE

TISSUE ENGINEERING APPROACHES FOR

INDEX ................................................................................. 305

Brian K. Jordan and Eric Vilain

Genotypic and Phenotypic Sex

Pediatric Gender Assignment: A Critical Reappraisal

In mammals, particularly in humans, embryos are initially not only sexually

Modern Concepts of Sexual Development

In 1947, Alfred lost performed castrations in fetal rabbits, showing that if

SRY, the Primary Trigger

(McElreavey et aI, 1992a). The variability of the phenotype observed in XX sex-reversed

Non-Y Chromosomal Genes and Sex Determination

Chromosomal rearrangements of chromosome 17 were observed in patients with

SFl and WT-l:

in a mouse knock-out model, the observed phenotype is the absence of gonad

Sex Determination, a Complex Network of Genes

Genes Involved in Sexual Differentiation

In contrast to sex determination, most genetic players of sexual differentiation are

Table 1. Genes Involved in Sex Detennination

Gene Localization Gene family Putative Phenotype of mutations

Conclusion: A Molecular Approach to the Diagnosis of Intersex

Normal renal andlor

XV gonadal dysgenesis Androgen

Abbas N, McElreavey K, Leconiat M, Vilain E, Jaubert F, Berger R, Nihoul-Fekete C, Rappaport R, Fellous M.

Tommerup N, Schempp W, Meinecke P, Pedersen S, Bolund L, Brandt C, Goodpasture C, Guldberg P, Held

Questions for Dr. Vilain

Question - How rapidly can you make a molecular diagnosis?

Answer - It depends. If it is peR based it is a matter of days. If it is like looking for a

Faruk Hadziselirnovic, Dale Huff

This chapter concerning normal and abnormal testicular development represents 28

Normal Testicular Development

Testicular Development During the First Year of Life

Pediatric Gender Assignment: A Critical Reappraisal 15

Development During the Fourth Year of Life

Changes Occurring During Puberty

The Significance of Postnatal Gonadotropin Surge for Testicular

ABNORMAL TESTICULAR DEVELOPMENT