Toxicologic Pathology, 36: 164S-170S, 2008

Copyright © 2008 by Society of Toxicologic Pathology

ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623308326152

Recommendations for Routine Sampling, Trimming, and

Paraffin-Embedding of Female Reproductive Organs,
Mammary Gland, and Placenta in the Cynomolgus Monkey
ERIC VAN ESCH,1 EVELINE P.C.T. DE RIJK,2 EBERHARD BUSE,3 MARTINA ZÖLLER,4 AND J. MARK CLINE5
1
Principal Pathologist, Department of Toxicology and Drug Disposition, Schering-Plough,
Oss, the Netherlands
2
Department of Toxicology and Drug Disposition,
Schering-Plough, Oss, the Netherlands
3
Covance Laboratories GmbH, Münster, Germany
4
Covance Laboratories GmbH, Münster, Germany
5
Wake Forest University Primate Center,
Winston-Salem, North Carolina, USA

ABSTRACT

In toxicity studies, the nonhuman primate is often the species of choice to evaluate the toxicologic potential of chemicals and drugs. Especially in
the case of effects on female reproductive organs and mammary glands, other animal species are less predictive for man. To enable reliable histopatho-
logic interpretation allowing a solid safety assessment, it is a prerequisite to obtain material of consistently high quality. Standardization of autopsy
techniques, tissue sampling, and fixation and staining procedures will help significantly to obtain the quality that is needed. For this purpose, a detailed
description of the procedures from necropsy to microscopic slide preparation of the female reproductive organs of the cynomolgus monkey is
given. Procedures to sample and process the placenta are included. These recommendations can be used to achieve consistent, high-quality tissue
preparations, allowing pathologists to conduct sensitive, accurate, and meaningful evaluations of the study material.

Keywords: dissection; female reproductive organs; placenta; tissue sampling; histotechnique; nonhuman primates; cynomolgus monkey.

INTRODUCTION the tissues are adequately dissected and trimmed. Nevertheless,

to reduce intra- and interstudy variability, and to fulfill quality
In general, regulatory guidelines, intending to aid the approval
assurance requirements (good laboratory practice, or GLP), it is
and registration of pharmaceutical and other chemical products,
important to standardize sampling, trimming, and embedding
specify only the organs and tissues that must be evaluated grossly,
procedures as much as possible. This paper is intended to be a
weighed, and microscopically examined. Such guidelines do not
guide for the toxicologic pathologist involved in studies with
specify the sample sizes and numbers, nor do they give indica-
nonhuman primates. The procedures are focused on the female
tions of the parts of the appropriate organs and tissues that have
reproductive organ system and mammary glands and ensure the
to be examined by the pathologist. As a consequence, possible
preparation of well-oriented and standardized microscopic slides
regional intratissue differences in susceptibility of the cellular
that facilitate thorough routine histopathologic examination of
(sub)components within certain organs may be overlooked.
these complex tissues by the pathologist. Because nonhuman pri-
Sample size and number are more critical when processing small
mates are, to an increasing extent, used in reproductive toxicity
organs/tissues, since when not correctly oriented, essential struc-
studies or in other studies with pregnant animals, it can be useful
tures might be lost, for example, when dealing with small labora-
to make slides of the placenta. For this reason, sampling, trim-
tory animals. In larger laboratory animals such as dogs and
ming, and embedding procedures for the placenta have also been
monkeys, the reproductive organs and mammary glands are
included. For illustration, the cynomolgus monkey is chosen as
greater in volume, and therefore sample size is not limiting when
an example of a typical nonhuman primate.
Competing Interests: This article was sponsored by Covance Inc. and Schering-
Plough. Martina Zölle and Eberhard Buse are employed by Covance Inc. Eveline FIXATIVE
P. C. T. de Rijk and Eric Van Esch are employed by Schering-Plough. No other
For fixation, we generally recommend 4% formaldehyde
competing interests were declared.
Address correspondence to: Eric Van Esch, Schering-Plough, Department of made fresh from paraformaldehyde if immunostaining is
Toxicology and Drug Disposition, P.O. Box 20, 5340 BH Oss, the Netherlands; intended (Williams 1977), otherwise commercial 10% neutral
e-mail: eric.van.esch@spcorp.com buffered formalin is acceptable.

164S
Vol. 36, No. 7S, 2008 ROUTINE SAMPLING, TRIMMING, AND EMBEDDING 165S

FIGURE 1.—Schematic overview of the dissection of the reproductive tract (including mammary gland) from a female cynomolgus monkey. (A)
Sampling of the mammary gland and opening of the abdominal cavity. (1) Take nipple and surrounding tissue; (2) open the abdominal cavity
along the “linea alba”; (3) transect the large intestine at the colorectal junction and extract the gastrointestinal tract; (4) transect the caudal abdom-
inal wall and uncover the bony pelvis. (B) Opening of the pelvis and sampling of the ovaries and oviduct. (5) Remove the ovaries; (6) Remove
the oviducts; (7) open the pelvic cavity by cutting through the pubic symphysis; (8) remove the genital tract with the adherent urinary bladder
and rectum from the pelvic cavity. (C) Separation of the rectum and urinary bladder from the uterus. (8) see (B); (9) separate the rectum from the
uterus; (10) remove the urinary bladder from the uterus. (D) Separation of the vagina from the cervix. (11) Cut through the cranial part of the
vagina by cross-section, and remove it from the cervix. (E) Opening of uterus and vagina. (12) Open the uterus by a ventral longitudinal section
to examine the mucosa; (13) open the vagina by a ventral longitudinal section to examine the mucosa. a, anus; aw, abdominal wall; b, urinary
bladder; c, cervix; co, colon; f, oviduct (fallopian tube); o, ovary; rl, round ligament; r, rectum; s, symphysis; u, uterus; vu, vulva; v, vagina.

MACROSCOPIC PROCEDURES • Cut a square piece of skin around both nipples

Mammary Glands
Prior to opening the thoracic cavity, the mammary glands
should be sampled. If possible, the animal should be clipped to
make mammary gland manipulation easier. Both mammary glands,
including the overlying skin, should be collected. This can be done
in the following way:
(approximately 4x4cm or at least a size that
includes the visibly developed mammary glands)
(Figure 1A). The samples also have to include
the subcutaneous fat tissue, but not the underlying
muscle, which is recognized by its dark red
color.
• Report visible abnormalities, and inspect the under-
side of the skin flaps.
166S VAN ESCH ET AL. TOXICOLOGIC PATHOLOGY

• Place the two skin flaps containing the mammary

glands onto a piece of fiberglass screen, or card stock,
to prevent curling up of the tissue, and fix them.

Abdominal Reproductive Organs

When the abdominal cavity is opened and the organs
inspected, the gastrointestinal tract and associated organs can be
removed. To be able to inspect and dissect the whole genital tract,
first locate the obturator foramina on one side of the pelvis. Place
the tip of a bone-cutting shear into one foramen and cut the pubis
(caudal) and ischium (cranial). Repeat this procedure on the
opposite side, and remove the pubic bone to expose the pelvic
canal. Alternatively, the pubic symphysis can be separated in
younger animals by cutting with a sharp scalpel and pushing both
pubic bones apart. The whole genital tract now can be inspected
in situ (Figure 2). (Alternatively, the ovaries and oviducts can be
sampled before the pelvic bone is removed.)
After recording the findings, first the ligaments are dis-
sected from the uterus visible and cervix and then the whole
tract, including ovaries, oviducts, cervix, bladder, and vagina,
can be further dissected from its surrounding tissues. To pre-
vent loss of small or fragile tissues, some tissues are placed in
a prelabeled histology (or embedding) cassette that is put into
the fixative jar.

Ligaments
Although for most toxicologic pathologists, ligaments are
not a standard tissue to sample, they can be taken for further
histopathologic investigation (see also the paper on the monkey
model of prolapse by Shahryarinejad and Vardy, this mono-
graph).
FIGURE 2.—The female genital tract of a mature cynomolgus monkey
in situ. O, ovary; U, uterus; Od, oviduct; RL, round ligament; BL,
broad ligament; B, urinary bladder; C, cervix; PB, pubic bone (pubic
symphysis removed); AW, ventral abdominal wall; R, rectum.

• Identify the round ligaments of the uterus, which extend

from the widest part of the uterus (Figure 2).
• Cut both left and right round ligaments, and put them
in fixative in a prelabeled histology cassette.
• Then identify the cardinal-uterosacral ligament com-
plex, which forms folds on either side of the colon
when the uterus is pulled forward (Figure 2). Cut the
ligaments free, and fix them in a prelabeled histology
cassette.
Because the ligaments are rather thin structures, the preferred
technique for fixation is the Swiss-roll technique (Moolenbeek
and Ruitenberg 1981). This means that (samples of) the ligaments
are rolled on a toothpick or cotton swab (or comparable tool) and
placed, coiled, into the fixative. The Swiss-roll technique is rec-
ommended to be sure that a considerable amount of tissue can be
obtained. After removal of the ligaments, the ovaries and oviducts
can be removed.
• Dissect the ovaries free and separate them from the
attached oviduct.
• Inspect the ovaries for visible cyclic changes and/or
lesions, and document them. Routine photography of
the ovaries may be useful, since asymmetrical struc-
tures such as follicles or corpora lutea may be missed
in sectioning.
• Weigh the ovaries together. For practical reasons, some
laboratories prefer to weigh the ovaries after fixation.
• Fix both ovaries, preferably in a prelabeled histology
cassette.
Oviducts
The oviducts are coiled structures attached to the uterus
with the ovarian ligaments, ending in the reddish fimbriae ven-
trolateral to the ovaries (Figure 2).

• Cut the oviducts from the uterus.

Ovaries
• Document additional changes and/or lesions.
The ovaries are easily recognizable as pale/yellowish, • Place both oviducts in a prelabeled histology cassette,
more or less oval, smooth or nodular structures (Figure 2). and place the cassettes in the fixative.
Vol. 36, No. 7S, 2008 ROUTINE SAMPLING, TRIMMING, AND EMBEDDING 167S

The remaining reproductive organs can now be taken from

the abdominal cavity (uterus, cervix, and vagina, with urinary
bladder still adherent) by pulling while cutting them free from
the surrounding tissues using a sharp scalpel. Keep the bladder
intact with the vaginal wall. The different components can now
be sampled as described below and depicted in Figure 1C.

Uterus and Cervix

The uterus is a pear-shaped organ that continues caudally
into the cervix (Figure 2).

• Separate the caudal part of the vagina from the cervix,

taking care not to damage the exocervix (Figure 1D).
• Remove any superfluous attached fat tissue.
• Separate the uterus from the cervix, and weigh it.
• Carefully, partially incise the uterus longitudinally to
achieve proper fixation of the endometrium, as indi-
cated by the dotted line in Figure 1E.
• Check the inside for any lesions or excessive blood, FIGURE 3.—Placenta at the maternal side with the different structures
and record findings. to be sampled. Slicing enables macroscopic observations within the
• Place the incised uterus and cervix in fixative. placental tissue.

Vagina TRIMMING AND EMBEDDING

Remove the urinary bladder from the vagina. The most
important parts of the vagina to be inspected are the mucosal
folds and ridges.
• Carefully incise longitudinally at the ventral side and
open the vaginal wall (Figure 1E).
• Inspect the mucosa and record abnormal findings if
present.
• Place the opened vagina in fixative.
Before the tissues are processed, they are trimmed and put into
histology cassettes. After processing (dehydrated and saturated
with liquid paraffin), the trimmed tissues are manually put into
paraffin blocks. Both trimming and embedding are essential
steps, because proper tissue orientation enables the pathologist to
examine the most interesting parts of the tissue and ensures stan-
dardization. Standardization is a necessity when comparing tis-
sues from control versus treated animals and drawing the right
conclusions on treatment-related findings.

Vulva and Clitoris Mammary Glands

Examine the vulvar opening and clitoris; if androgenic
effects are of concern, photographic documentation, or clitoral
weight measurement, may be useful.
Placenta
Like other tissues, the placenta should be carefully
inspected (completeness, shape, size, number of discs, lesions)
before removing it from the uterus.
• Start by checking the umbilical cord (length, number
of vessels).
• To be able to check for lesions within the placenta
itself, one can incise it every 2 cm on the fetal surface
while leaving the membranes intact, maintaining the
slices in sequence (Figure 3).
• The placenta can be fixed in toto, or the slices can be
cut and fixed separately.
Cut through both skin flaps in such a way that the nipple is
positioned in the middle of the strip of skin (thus paramedian).
Trim the flaps to fit into a paraffin block, and embed them on
one of the cut surfaces. Slides prepared in this way will contain
skin and adnexa, subcutaneous and mammary gland tissue. The
nipple, with its lactiferous ducts, is included in each slide
(Figure 4A).
Ligaments
Cut transverse through the fixed Swiss-roll and embed the
cut roll of tissue flat in paraffin.
Ovaries
Look for signs of follicular development, corpora lutea, or
lesions to be included in the microscopic slide(s). Cut the
ovaries paramedian to the long axis, and sample the largest
168S VAN ESCH ET AL.
FIGURE 4.—Microscopic slides with sections of the different female repro-
ductive organs and mammary gland showing the orientation when ideally
sampled and processed. (A) A section through the skin, nipple, and under-
lying subcutaneous and mammary gland tissues. (B) Paramedian sections
through both ovaries. Developing follicles and a corpus luteum are visible.
(C) A cross-section through one oviduct. (D) Transverse section through
the fundus of the uterus. (E) Longitudinal, median section through the
endocervix, the coiled cervical channel, exocervix, and the caudal part of
the vaginal wall (from left to right). (F) Transverse section through the
vaginal wall.
TOXICOLOGIC PATHOLOGY
piece. After processing, orient the pieces in paraffin block(s) in
such a way that they stand on the cut surfaces. Slides from
ovaries ideally contain cortex, medulla, and the hilus with the
rete ovary and ovarian vessels (Figure 4B).
Oviduct
Because the oviducts are coiled structures, it is best to
embed both oviducts in their entirety in a paraffin block. Slides
prepared in this way will always contain tangential or cross-
sections of the tube (Figure 4C).
Uterus and Cervix
After fixation, cut a sample from the uterus by cutting the
fundus transversely at the level where the ovarian ligaments
and oviducts are attached (this is more or less the broadest part)
and a few millimeters caudal (Figure 4D). The second uterine
sample is taken longitudinally from the body and includes the
transition toward the cervix (Figure 4E). When taking the sam-
ple from the cervix, it is important to include a small rim of
vaginal wall, ensuring that the transformation zone is included
in the sample (see the paper titled “The Vagina and Cervix of
Macaques” by Charles E. Wood, this monograph). When sam-
ples are too large, they have to be cut in to two or more pieces
and embedded in an equal number of blocks.
Vagina
By making two transverse sections through the middle por-
tion of the vagina, a strip of vaginal wall is cut, and the sample
is placed in a paraffin block and embedded on one of the cut
surfaces (Figure 4F).
Placenta
Ideally, blocks from placental tissue should be taken from dif-
ferent parts (Figure 3): (1) central and peripheral part from both
maternal and fetal aspects; (2) from all parts with macroscopic
lesions; (3) from the umbilical cord; and (4) from the amniotic and
chorioallantoic membranes (for illustrative microscopic images,
see the paper titled “The Macaque Placenta” by de Rijk and
Van Esch, this monograph). For the umbilical cord, a cross-section
is made so the vessels are clearly visible. As for the ligaments, the
Swiss-roll technique can be used to obtain a significant amount of
membrane within the transverse-cut section.
Slides prepared from the paraffin-embedded tissues routinely
are stained using hematoxylin and eosin, although all kinds of
other staining techniques can be applied to visualize specific struc-
tures or substances within these tissues (e.g., reticulum fibers,
glycogen, mucopolysaccharides). Examples of routinely sampled
and stained paraffin sections from the reproductive organs and
mammary glands of a mature cynomolgus monkey are illustrated
Vol. 36, No. 7S, 2008 ROUTINE SAMPLING, TRIMMING, AND EMBEDDING 169S

in Figure 4. Also, immunohistochemical staining methods for the

detection of the estrogen receptors, progesterone receptors, and the
KI67-protein (a marker for cell proliferation) can be very helpful.
(Nowadays, such immunohistochemical techniques can well be
performed on formalin-fixed tissues). Examples of such tech-
niques are given in the different papers within this monograph.

OTHER RELEVANT TISSUES

Pituitary Gland
After removal of the brain, the pituitary should be carefully
dissected from the sella turcica by cutting the overlying dura,
fixed in toto, weighed, and trimmed coronally to include the
neurohypophysis and adenohypophysis (a low-power micro-
scopic image of a pituitary gland prepared in this way is given
in the paper by Weinbauer et al., this monograph).

Adrenal Glands
The adrenal cortex is an important source of androgens and
may be profoundly affected by hormonally active agents. Both
adrenals should be weighed, fixed, and trimmed for histologic
evaluation. Adrenals should be cut in half to allow examination
of both medulla and cortex.

IN-LIFE TECHNIQUES
Endometrial Biopsies
Endometrial biopsies must be taken laproscopically under deep
anesthesia, because the cervical canal of the macaque is too coiled
to be used for transvaginal biopsy techniques. A wedge-shaped
biopsy is carefully cut from the uterine body. One should be cer-
tain that the biopsy includes a significant amount (preferably the FIGURE 5.—Examples of endometrial biopsies from mature cynomolgus
monkeys. (A) An adequate biopsy containing all layers needed for a reli-
full thickness) of the endometrium, including glands of the zona
able evaluation (myometrium, basalis, functionalis, and luminal epithe-
functionalis. The biopsies are placed in 4% paraformaldehyde in
lium). (B) Inadequate biopsy, containing mainly myometrium and only
phosphate-buffered saline (PBS) (pH 7.4) (Lobie 1997) for fixa- few glands of the basalis. Functionalis and luminal epithelium are miss-
tion in a prelabeled histology cassette. ing completely. Original magnification 4X.
After processing, the biopsy is embedded in a paraffin
block. Orientation of the tissue in the block is of utmost impor-
or 95% ethanol. It is important that these vaginal swabs are
tance, since slides prepared from the block need to contain a
collected from approximately the same site in each animal.
significant amount of endometrium. Ideally, the full thickness
Smears are best stained using the Papanicolaou (Pap) stain
of the endometrium, including luminal epithelium, is present in
(Lillie and Fullmer 1976), although a modified Wright’s stain
the slide (Figure 5A).
(Lillie and Fullmer 1976) may suffice.
Incorrectly taken biopsies often consist mainly of
myometrium or include the zona basalis of the endometrium SUMMARY
only (Figure 5B). The presence of a significant amount of zona
functionalis in the final slide is a prerequisite for a sound In this paper we describe the standardized procedures, from
microscopical judgment. Biopsies always have to be handled necropsy techniques to microscopic slide preparation of cynomol-
with care to avoid squeezing artifacts, which can hamper gus monkey female reproductive organs, mammary glands, and
microscopic evaluation. In Figure 6, two examples are given of placenta. The procedures described herein help to obtain repre-
the microscopic appearance of such an artifact. sentative, high-quality microscopic specimens necessary for reli-
able histopathologic evaluation by the pathologist. High-quality
specimens also facilitate the comparison of changes and/or find-
ings among animals within a study (or between different studies).
Vaginal/Cervical Cytology Collection (Smears)
Nevertheless, the qualifications, training, and experience of the
The vaginal vault is to be swabbed, and the cotton swab then pathologist represent the most essential factors in the quality of the
rolled onto two prelabeled glass slides. After drying, the slides histopathology evaluation, interpretation (Crissmann et al. 2004),
need to be fixed, which can be done using Spray-cyte fixative and risk assessment.
170S VAN ESCH ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 6.—Two cases of telescoping artifacts caused by biopsy handling. Glands are squeezed into the glandular lumen. This phenomenon may
mimic hyperplastic or neoplastic lesions (Mazur and Kurman 1995). Original magnification 63X.

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