C LI NI CA L RE SE AR CH A RT IC LE

A Longitudinal Study of Estrogen-Responsive

Tissues and Hormone Concentrations in Infants
Fed Soy Formula

Margaret A. Adgent,1,2 David M. Umbach,3 Babette S. Zemel,4,5 Andrea Kelly,5,6

Joan I. Schall,4 Eileen G. Ford,4 Kerry James,7 Kassa Darge,5,8 Julianne C. Botelho,9
Hubert W. Vesper,9 Donald Walt Chandler,10 Jon M. Nakamoto,11 Walter J. Rogan,1
and Virginia A. Stallings4,5

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1
Epidemiology Branch, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, North Carolina 27709; 2Department of Pediatrics, Vanderbilt University Medical
Center, Nashville, Tennessee 37232; 3Biostatistics and Computational Biology Branch, National Institute of
Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709;
4
Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, The Children’s Hospital
of Philadelphia, Philadelphia, Pennsylvania 19104; 5University of Pennsylvania Perelman School of Medicine,
Philadelphia, Pennsylvania 19104; 6Department of Pediatrics, Division of Endocrinology, The Children’s
Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; 7Social & Scientific Systems, Inc., Durham, North
Carolina 27703; 8Department of Radiology, The Children’s Hospital of Philadelphia, Philadelphia,
Pennsylvania 19104; 9Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta,
Georgia 30341; 10Endocrine Sciences, LabCorp, Calabasas Hills, California 91301; and 11Quest Diagnostics
Nichols Institute, San Juan Capistrano, California 92675

Purpose: Chemicals with hormonelike activity, such as estrogenic isoflavones, may perturb human
development. Infants exclusively fed soy-based formula are highly exposed to isoflavones, but their
physiologic responses remain uncharacterized. Estrogen-responsive postnatal development was
compared in infants exclusively fed soy formula, cow-milk formula, and breast milk.

Methods: We enrolled 410 infants born in Philadelphia-area hospitals between 2010 and 2014; 283
were exclusively fed soy formula (n = 102), cow-milk formula (n = 111), or breast milk (n = 70)
throughout the study (birth to 28 or 36 weeks for boys and girls, respectively). We repeatedly
measured maturation index (MI) in vaginal and urethral epithelial cells using standard cytological
methods, uterine volume and breast-bud diameter using ultrasound, and serum estradiol and
follicle-stimulating hormone levels. We estimated MI, organ-growth, and hormone trajectories by
diet using mixed-effects regression splines.

Results: Maternal demographics did not differ between cow-milk–fed and soy-fed infants but did
differ between formula-fed and breastfed infants. Vaginal-cell MI trended higher (P = 0.01) and
uterine volume decreased more slowly (P = 0.01) in soy-fed girls compared with cow-milk–fed girls;
however, their trajectories of breast-bud diameter and hormone concentrations did not differ. We
observed no significant differences between boys fed cow-milk vs soy formula; estradiol was not
detectable. Breastfed infants differed from soy-formula–fed infants in vaginal-cell MI, uterine
volume, and girls’ estradiol and boys’ breast-bud diameter trajectories.

Conclusions: Relative to girls fed cow-milk formula, those fed soy formula demonstrated tissue- and
organ-level developmental trajectories consistent with response to exogenous estrogen exposure.
Studies are needed to further evaluate the effects of soy on child development. (J Clin Endocrinol
Metab 103: 1899–1909, 2018)

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: E2, 17-b estradiol; EDC, endocrine-disrupting compound; ER, estrogen
Printed in USA receptor; FSH, follicle-stimulating hormone; IFED, Infant Feeding and Early Development;
Received 11 October 2017. Accepted 26 February 2018. MI, maturation index; SAG, sagittal; TRV, transverse.
First Published Online 1 March 2018

doi: 10.1210/jc.2017-02249 J Clin Endocrinol Metab, May 2018, 103(5):1899–1909 https://academic.oup.com/jcem 1899
1900 Adgent et al Soy Formula and Infant Reproductive Development
ealthy development of estrogen-responsive tissues in
H early life is critical for normal reproductive function
and dependent, in part, on steroid-hormone signaling (1).
Early-life exposure to endocrine-disrupting compounds
(EDCs) may perturb reproductive development in ways
that affect later disease risk. The association of prenatal
exposure to diethylstilbestrol (a potent synthetic estro-
gen) with the subsequent development of cancer, repro-
ductive tract malformations, and infertility supports this
concept (2). Epidemiologic evidence for associations of
other purported EDCs with adverse reproductive de-
velopment is limited.
Presently, the greatest source of human exposure to
estrogenic EDCs, apart from pharmaceuticals such as
oral contraceptives, comes from dietary phytoestrogens
(3). Genistein, a phytoestrogen found in soy protein, can
bind to and activate estrogen receptors (ERs), particularly
ER-b, albeit with much lower affinity than endogenous
estradiol (4). Soy formula, in particular, provides a large,
well-characterized amount of genistein (5). Combining all
such dietary exposures into “estrogen equivalents,” in-
fants fed soy formula have approximately 1000-fold
greater total estrogen exposure (6, 7) and urinary genistein
concentrations 500 times higher than other infants (5).
These higher genistein concentrations overlap genistein
concentrations that induce, for example, changes in re-
productive tract histopathology, estrous cyclicity, and
fertility in rodent models (serum levels of genistein of ;500
to 1800 ng/mL ) (8). Several epidemiologic studies suggest
an association between soy formula use and adult re-
productive health (9–12), but little is known about re-
productive system changes that may occur during infancy,
near the time of soy formula exposure (13–15).
Estrogen-responsive development begins in utero and
continues into the postnatal period. It is closely tied to
exposure to maternal estrogen during pregnancy, with-
drawal from maternal estrogen after birth, and, subse-
quently, in female infants, transient and variable estrogen
production for approximately 2 years, often termed
“minipuberty” (16, 17). Fetuses and infants of both sexes
have estrogen-responsive tissue. Maternal estrogen con-
centration increases steeply through gestation, and male
and female newborns exhibit increasing estrogenization
of breast and external genitalia with advancing gesta-
tional age (18). Then, in the early postnatal period,
concurrent with the loss of maternally sourced estrogen,
infants exhibit declining estrogenization, evidenced by
decreasing size of the breast (15) and uterus (19), and loss
of superficial cells in the urogenital epithelium (15, 20).
We hypothesized that early postnatal exposure to soy
formula prolongs the physiologic estrogenization of the
newborn. This study’s primary aim was to characterize
J Clin Endocrinol Metab, May 2018, 103(5):1899–1909
the postnatal development of estrogen-responsive tissues
(i.e., uterus, breast bud, and urogenital epithelium), serum
estradiol concentrations, and follicle-stimulating hormone
(FSH) concentrations according to infant-feeding prac-
tices, with an emphasis on comparison between infants fed
soy formula and those fed cow-milk formula. Evidence of a
contemporaneous physiologic response in soy formula–
fed infants, who would be predicted to respond on the
basis of the laboratory evidence, would support the con-
cept that endocrine disrupters detected in the laboratory
are active in humans. Such evidence would also contribute
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to our understanding of early-life disruption of steroid-
hormone signaling in humans, not only by soy but also by
estrogenic EDCs more generally.
Materials and Methods
Study design
The Infant Feeding and Early Development (IFED) Study
was an observational study that assessed estrogen-responsive
tissues and organs longitudinally during the postnatal period.
Between August 2010 and November 2013, IFED staff en-
rolled mothers and their neonates from eight Philadelphia,
Pennsylvania, regional hospitals (21) (Supplemental Methods).
Eligible mothers had decided before contact by study staff to feed
their infants soy formula, cow-milk formula, or breast milk
exclusively from birth, spoke English, were 18 years of age or
older, and had no endocrine disorder (including polycystic ovary
syndrome), diabetes (including gestational diabetes), or thyroid
conditions, and no use of steroid medication (except inhaled or
topical glucocorticoids), immunosuppressants, or hormones to
maintain pregnancy during the pregnancy. Eligible infants were
healthy singletons of 37 to 42 weeks’ gestation who weighed
between 2500 and 4500 g at birth. Male infants with hypo-
spadias or nonpalpable testes, and infants with ambiguous
genitalia or serious illness, including major congenital
malformations, chromosomal anomalies, sustained respiratory
distress, or other condition known to affect growth and de-
velopment, were excluded. No siblings were enrolled. We
compensated families for their time and inconvenience. In ad-
dition, incentives such as formula and supplemental diapers
were given (Supplemental Methods).
Abstraction from 26,383 medical charts at delivery iden-
tified 3946 potentially eligible mother-infant dyads who were
approached at birth. Of these, 524 mothers granted consent to
undergo screening for the study and 340 enrolled. An addi-
tional 94 mothers were recruited from prenatal clinics before
giving birth, 70 of whom met birth eligibility criteria and
enrolled following delivery. Mothers provided informed
written consent. Institutional review boards at the Children’s
Hospital of Philadelphia, Virtua Hospitals, Abington Memorial
Hospital, the National Institute of Environmental Health Sci-
ences, and Copernicus Group approved the protocol.
Continued participation in the IFED Study required that the
infant continue to meet the health criteria specified at enroll-
ment and also that the mother adhere to one of the study’s three
simple feeding regimens (i.e., breast milk, cow-milk formula, or
soy formula), allowing for only limited excursions. At the week-
2 visit, we assessed feeding history since birth and placed infants
doi: 10.1210/jc.2017-02249
into feeding groups, using the following criteria: breastfed infants
received 90% to 100% of calories from breast milk and #1% of
calories from soy formula; infants fed cow-milk formula received
90% to 100% of calories from cow-milk formula and #1% of
calories from soy formula; and soy formula–fed infants received
90% to 100% of calories from soy formula. If infants did not
meet these criteria at the week-2 visit, they were withdrawn from
the study. At each subsequent visit, we administered the feeding-
history questionnaire to assess such adherence since the previous
visit. The threshold for feeding-regimen adherence lowered from
90% to 100% of calories to 85% at 13 weeks of age and then
80% at 25 weeks of age to accommodate mothers who were
supplementing, though the strict 1% upper limit on calories from
soy formula for breastfed and cow-milk formula–fed infants was
retained. Nonadherence resulted in withdrawal. To validate
feeding adherence assessed by questionnaire, we measured iso-
flavones in urine samples from a randomly selected subset of
infants fed cow-milk formula (n = 19) and those fed soy formula
(n = 20) at age 12 weeks (Supplemental Methods).
Birth visits were completed within 72 hours of delivery. For
girls, subsequent visits were scheduled at 2 and 4 weeks
(64 days); 8 and 12 weeks (610 days); 16, 20, 24, 28, and
32 weeks (614 days); and 36 weeks (630 days). Before June
2012, subjects were also seen at week 6. Boys’ follow-up visits
were scheduled similarly and were designed to conclude at
28 weeks (630 days) based on pilot data that suggested more
prolonged response in girls than boys (15) (Supplemental Table 1).
At each visit, we verified adherence to feeding regimen by
structured questionnaire, performed anthropometry, and col-
lected urogenital epithelial cells; blood collection began at week 2.
Also at week 2, mothers completed questionnaires and provided
information on reasons for feeding choices. We obtained ultra-
sound images of breast buds and uterus at birth and weeks 4, 16,
24, and 32. Our protocol allowed ultrasound measures to be
performed at the visit immediately before or after the designated
visit to accommodate the scheduling needs of our study subjects.
Before June 2012, we also obtained ultrasound images at week 8.
Optimal participation allowed for many of these measures
(e.g., anthropometry, urogenital cells, blood) to be collected at
every visit, but at minimum, mothers and infants were required
to contribute a core set of three (boys) or four (girls) blood
samples. For boys, required blood-sample collections were set at
2 or 4 weeks; 8, 12, or 16 weeks; and 24 or 28 weeks. For girls,
required blood-sample collections were set at 2 or 4 weeks; 8 or
12 weeks; 20 or 24 weeks; and 32 or 36 weeks. Follow-up visits
ended in March 2014.
Ultrasound imaging
All sonographers were trained and certified according to the
IFED Study protocol. For uterus and breast buds, we obtained
three or four images each in the transverse (TRV) and sagittal
(SAG) views (21). Each dimension—SAG, TRV, and anterior-
posterior (AP)—was measured three or four times from separate
images according to protocol. The geometric mean of the
multiple measurements was used as a dimension-specific di-
ameter. We calculated uterine volume, approximated as a
cylinder, from the diameters as follows [Eq. (1)]:
2pðSAG=2ÞðTRV=2ÞðAP=2Þ:
Breast-bud diameter was calculated as the geometric mean of
the TRV and SAG diameters from both left and right breasts.
Additional details are described in the Supplemental Methods.
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Urogenital cytology
Urogenital epithelial cells were collected via swab of the
vaginal introitus (girls) or urethral meatus (boys) at every study
visit and were processed according to previously described
methods (Tricore Reference Laboratories, Albuquerque, NM)
(20). In brief, samples were stained using standard procedures
(GYN SurePath and SurePath PrepStain; BD, Franklin Lakes,
NJ). Applying established criteria (22), blinded cytotechnol-
ogists scored 100 cells per sample and classified each cell as
either basal/parabasal, intermediate, or superficial. In 6% of
boys and 1% of girls, samples included fewer than 100 cells, so
50 cells were counted and doubled. Superficial cells are in-
dicative of estrogenization (23–25), a phenomenon that is
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widely established in adults and infants (26–28) and has been
measured in studies of dietary soy exposure in postmenopausal
women (29, 30). We quantified estrogenization using a Matu-
ration Index (MI) equal to half the percentage of intermediate
cells plus the percentage of superficial cells (15, 20, 31).
Serum hormones
Serum 17-b estradiol (E2) was analyzed using an isotope-
dilution liquid chromatography–tandem mass spectrometry
method (US Centers for Disease Control and Prevention,
Atlanta, GA) (32). E2 was dissociated from binding proteins,
extracted from serum using liquid-liquid extractions, and
analyzed by liquid chromatography–tandem mass spectrometry
using negative electrospray ionization. The E2 method interassay
coefficient of variation was , 4.6% over 20 analytical runs and
the limit of detection was 2.99 pg/mL. To monitor accuracy, each
run included the reference material BCR576, at 31.1 pg/mL,
from the Institute for Reference Materials and Measure-
ment; the mean bias was 20.7% [95% confidence interval
(CI), 22.0% to 0.5%]. FSH in girls’ serum was analyzed using
an electrochemiluminescence immunoassay (Quest Diagnostics
Nichols Institute, San Juan Capistrano, CA) optimized for
pediatric use (limit of quantitation: 0.05 mIU/mL; limit of detection:
0.007 mIU/mL; intrassay coefficient of variation, 9.75%; and
interassay coefficient of variation, 11%, at an approximate FSH
concentration of 0.5 mIU/mL).
Statistical analysis
Analyses were limited to infants who contributed a minimum
of three (boys) to four (girls) blood samples (Supplemental
Methods). We tested feeding-group differences in maternal and
infant characteristics with x2 tests or t tests. We examined
feeding-group differences in age trajectories using mixed-effects
regression splines under normality assumptions. Our strategy
was similar for all outcomes, though some details differed.
Trajectories were represented as natural cubic splines with four
knots, common to all feeding groups and equally spaced in the
square root of age (days) to accommodate closer visit spacing in
early weeks. The mixed-effects model included subject-specific
random effects for each regression coefficient, allowing each
study subject to have a separate trajectory that deviated from the
average. Uterine volume, breast-bud diameter, and hormone
concentrations were log2 transformed for analyses; MI was
untransformed. We report comparisons between all feeding
groups, but emphasize cow-milk formula as the most appro-
priate comparison diet for soy formula, because women who
breastfeed tend to differ from women who formula feed in
multiple and often unmeasurable ways, and because breast
1902 Adgent et al Soy Formula and Infant Reproductive Development J Clin Endocrinol Metab, May 2018, 103(5):1899–1909

milk may contain estrogenic pollutants or hormones (33), albeit
at low concentrations, that were not measured in this study.
Under the assumption that any differences in MI or organ
size present at birth are unrelated to postnatal feeding regimen,
we compared postnatal trajectories between the feeding groups
for those outcomes, using contrasts among spline coefficients,
excluding the intercept. To display these postnatal trajectory
comparisons (denoted “relative trajectories”), we shifted the
fitted feeding-group–specific trajectories (denoted “absolute
trajectories”) vertically to have a common intercept. In sensi-
tivity analyses, we adjusted all models for neonatal weight and
gestational age. SAS, version 9.3 (SAS Institute, Cary, NC) was
used for statistical analysis.
and 70 breastfed (64%). Infants who did not complete the
study did not differ demographically or by feeding group
from those who completed the study (Supplemental
Table 2). Common reasons for exiting the study included
feeding-method change (38%) and loss of contact or
missed appointments (39%); 69% of exits occurred be-
fore age 8 weeks. Of those who completed the study
(Table 1; Supplemental Table 2), 70% of mothers were
black and 57% had a high school education or less.
Mothers of breastfed infants had higher educational at-
tainment and were more likely to be white. At the birth

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Results
Of 410 mother-infant dyads enrolled, 283 (69%) com-
pleted the study, comprising 111 infants fed cow-milk
formula (73% of enrolled), 102 fed soy formula (68%),
visit, breastfed boys were heavier than boys fed cow-milk
formula or soy formula, whereas girls fed soy formula
were heavier than those fed cow-milk formula. Weight
gain between birth and the 12-week visits did not differ
between feeding groups for boys (P = 0.22) or girls (P =
0.58). Common reasons for choosing soy formula were

Table 1. Maternal and Infant Characteristics by Feeding Group and Infant Sex
Girls Boys

Soy Milk Cow Milk Breast Milk Total Soy Milk Cow Milk Breast Milk Total
No. of dyads 48 56 32 136 54 55 38 147
Maternal race
Black 38 (79) 45 (80) 15 (47) 98 (72) 42 (78) 45 (82) 14 (37) 101 (69)
White 7 (15) 8 (14) 14 (44) 29 (21) 11 (20) 7 (13) 16 (42) 34 (23)
Other/multiracial/unknown 3 (6) 3 (5) 3 (9) 9 (7) 1 (2) 3 (5) 8 (21) 12 (8)
x2 P valuea 1.00 ,0.01 0.38 ,0.0001
Maternal education
Less than high school 14 (29) 8 (14) 0 22 (16) 11 (20) 19 (35) 0 (0) 30 (20)
High school or GED 18 (37) 34 (61) 8 (25) 60 (44) 26 (48) 16 (29) 8 (21) 50 (34)
Some college or Associates 13 (27) 12 (21) 10 (31) 35 (26) 14 (26) 15 (27) 10 (26) 39 (27)
degree
College or postgraduate 3 (6) 2 (4) 14 (44) 19 (14) 3 (6) 5 (9) 20 (53) 28 (19)
x2 P valuea 0.14 ,0.0001 0.19 ,0.0001
Maternal age (y)
Mean (SD) 26.1 (5.0) 25.2 (5.5) 26.9 (5.2) 25.9 (5.3) 25.9 (5.5) 25.7 (6.6) 28.0 (5.4) 26.4 (5.9)
Median 26 24 27.5 26 25 25 28 26
Minimum, maximum 18, 40 18, 40 18, 37 18, 40 18, 43 18, 42 19, 38 18, 43
P valueb 0.38 0.27 0.86 0.05
Neonatal weight,c kg
Mean (SD) 3.24 (0.4) 3.06 (0.4) 3.13 (0.4) 3.14 (0.4) 3.21 (0.4) 3.24 (0.4) 3.42 (0.4) 3.27 (0.4)
Median 3.20 3.03 3.12 3.09 3.27 3.21 3.41 3.28
Minimum, maximum 2.40, 4.09 2.40, 3.87 2.51, 3.99 2.40, 4.09 2.47, 4.12 2.36, 4.28 2.51, 4.20 2.36, 4.28
P valueb 0.02 0.88 0.77 0.01
Gestational age, wk
37 3 (6) 6 (11) 3 (9) 12 (9) 5 (9) 5 (9) 1 (3) 11 (7)
38 6 (13) 16 (29) 7 (22) 29 (21) 9 (17) 9 (16) 4 (10) 22 (15)
39 23 (48) 19 (34) 9 (28) 51 (37) 27 (50) 17 (31) 11 (29) 55 (37)
40 13 (27) 8 (14) 10 (31) 31 (23) 10 (18) 19 (35) 9 (24) 38 (26)
41 3 (6) 7 (12) 3 (9) 13 (10) 3 (6) 5 (9) 13 (34) 21 (14)
x2 P valuea 0.09 0.71 0.25 ,0.01
Data given as no. (%) unless otherwise indicated.
Abbreviations: GED, general equivalency diploma; SD, standard deviation.
a 2
x P values are from exact tests based on likelihood ratio statistics. The P value straddling Cow Milk and Soy Milk columns compares those two feeding
groups; the P value under Breast Milk compares the breast-fed group with the cow- and soy-formula–fed groups combined.
b
P values from t tests of contrasts in a one-way analysis of variance. The P value straddling Cow Milk and Soy Milk columns compares those two feeding
groups; the P value under Breast Milk compares the breast-fed group with the cow- and soy-formula–fed groups combined.
c
Neonatal weight is based on birth-visit weight measurement, conducted within first 72 hours after birth.
doi: 10.1210/jc.2017-02249
successful use with a previous child, perceived health-
fulness, and anticipated lactose intolerance in the infant
based on family history (Table 2).
Our urinary isoflavone validation substudy provided
evidence of high feeding-regimen adherence among those
who completed the study. Median (minimum, maxi-
mum) urinary genistein concentrations, measured in the
feeding-group validation subset, were 9,460 ng/mL (52,
102,000 ng/mL) and 7 ng/mL (1, 102 ng/mL) for the
20 soy formula–fed and 19 cow-milk formula–fed infants,
respectively. Using a 200 ng/mL cut point as evidence of
a soy diet (5), our feeding assessment questionnaire had
specificity 0.95 and sensitivity 1.0.
The relative MI trajectory of soy formula–fed girls
differed significantly from those of girls fed cow-milk
formula (P = 0.01) and those who were breastfed (P =
0.01; Fig. 1a; Supplemental Fig. 1a). Trajectories for each
of the girls’ feeding groups demonstrated an initial decline
in MI; however, with increasing age, the MI trajectory of
girls fed soy formula diverged upward relative to that
of the other feeding groups. Breastfed and cow-milk
formula–fed boys also had MI trajectories with an ini-
tial decline. This decline was absent in soy formula–fed
boys (Fig. 1b; Supplemental Fig. 1b). Ultimately, these
feeding-group MI trajectory differences in boys were not
statistically significant (cow-milk formula vs soy formula
P = 0.08; breast milk vs soy formula P = 0.10; breast milk
vs cow-milk formula P = 0.67).
The uteri of soy formula–fed girls were smaller at the
near-birth measurement (11.2 cm3) than those of cow-
milk formula–fed (14.0 cm3) or breastfed girls (13.5 cm3;
Supplemental Fig. 2a). Trajectories of relative uterine
volume showed that involution was slower among soy
formula–fed girls than among cow-milk formula–fed
(P = 0.01) and breastfed girls (P , 0.01; Fig. 2a). Though
Table 2. Maternal Report of Reasons for Soy Formula Use
Reasona No.b
I fed my other child(ren) soy formula. 102
I think soy is healthier than other types of formula. 88
I suspect my baby had milk intolerance or my family 88
has trouble digesting cow’s milk.
My family or friends recommended it. 88
I chose soy formula for religious reasons. 102
Health care provider recommended soy. 102
I prefer a dairy-free diet. 102
My child had colic or other digestive problem. 102
I suspected my baby had a food allergy or 102
intolerance (other than milk).
Other 102
Family follows a vegan diet. 102
a
Mothers evaluated each reason independently as a major reason, minor reason, or not a reason for choosing soy formula. More than one major or minor
reason could be selected.
b
Owing to questionnaire version changes, some responses were only captured for 88 of 102 soy formula–feeding mothers.
https://academic.oup.com/jcem 1903
trajectories for cow-milk formula–fed and breastfed girls
were similar, breastfed girls had lower uterine volumes
later in infancy (P = 0.07).
Girls demonstrated a transient peak in breast-bud di-
ameter at 4 weeks of age, then stable diameters, with no
evident feeding-group differences (Fig. 2b; Supplemental
Fig. 2b). Boys demonstrated a transient peak at 4 weeks of
age, then falling diameters (Fig. 2c; Supplemental Fig. 2c).
After the transient peak, there was a larger decrease in
breast-bud diameter in breastfed boys than boys fed soy
formula (P = 0.02) or cow-milk formula (P = 0.05).
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Approximately 33% of girls had detectible concen-
trations of E2 at week 2, increasing to 85% at week 16
and decreasing to 50% by week 36 (Supplemental Fig. 3).
The proportion of samples having detectible E2 did not
differ by feeding group (P = 0.69). Median (25th, 75th
percentile) E2 concentrations were 6.0 pg/mL (2.1,
10.7 pg/mL) and 6.4 pg/mL (3.9, 10.0 pg/mL) at 12 and
16 weeks, respectively. E2 trajectories did not differ
between soy formula–fed and cow-milk formula–fed girls
(P = 0.44) or between cow-milk formula–fed and
breastfed girls (P = 0.17) but did differ between breastfed
and soy formula–fed girls (P = 0.02) (Fig. 3). We saw no
differences in girls’ FSH concentration trajectories among
feeding groups (Supplemental Fig. 4). Among boys, we
detected E2 in only 10 of 1150 samples (0.9%) from
10 subjects, all at week 2 or 4.
Results for MI, organ size, and E2 and FSH levels were
insensitive to neonatal weight and gestational age adjust-
ment, with minor exceptions for borderline associations:
Differences between MI trajectories of soy-fed boys and both
cow-milk formula–fed and breastfed boys more closely
approached significance after gestational age adjustment
(P = 0.05 for both comparisons); differences between
uterine volume trajectories for cow-milk formula–fed and
Major Reason, (%) Minor Reason (%) Not a Reason (%)
54 6 40
54 15 31
52 14 34
27 7 66
17 4 79
15 10 75
12 14 74
7 5 88
7 13 80
4 1 95
1 3 96
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Figure 1. Relative trajectory of MI vs age by feeding group. (a) Girls: soy formula vs cow-milk formula, P = 0.01; soy formula vs breast milk, P =
0.01; breast milk vs cow-milk formula, P = 0.23. (b) Boys: soy formula vs cow-milk formula, P = 0.08; soy formula vs breast milk, P = 0.10; breast
milk vs cow-milk formula, P = 0.67. Feeding-group–specific trajectories fitted to data were shifted vertically to a common intercept at zero
(dashed line) to better display postnatal changes (i.e., fitted MI at a given age minus fitted MI at birth). Thus, the vertical axis represents change
in MI since birth. Shaded bands represent 95% pointwise confidence limits for each feeding-group–specific trajectory. Absolute trajectories (i.e.,
those without vertical shift to common intercept) are shown in Supplemental Fig. 2.

breastfed girls reached statistical significance after ad-
justment for neonatal weight (P = 0.04) and gestational
age (0.03).
Discussion
Compared with infants fed cow-milk formula, infants fed
soy formula from birth demonstrated evidence of es-
trogen response in the urogenital epithelium and uterus
but did not demonstrate differences in breast or serum
E2 concentrations, or, in girls, FSH concentrations. Cells
from the vaginal introitus had an increasingly higher MI in
soy formula–fed girls than in cow-milk formula–fed girls
through 36 weeks. Cells from the distal urethra had a higher
MI in soy formula–fed boys than cow-milk formula–fed
boys through approximately 6 weeks, after which the
difference diminished and then disappeared by approx-
imately age 28 weeks. In addition, uterine volumes in-
voluted more slowly in soy formula–fed girls. These effects
are accepted as evidence of estrogen exposure (34, 35).
E2 is known to be detectable at highly variable con-
centrations among infant girls (16, 17). At 12 weeks of
doi: 10.1210/jc.2017-02249
Figure 2. Relative trajectories of organ size via ultrasound vs age by
feeding group. (a) Uterine volume in girls (soy formula vs cow-milk
formula, P = 0.01; soy formula vs breast milk, P , 0.01; breast milk
vs cow-milk formula, P = 0.07). (b) Breast-bud diameter in girls (soy
formula vs cow-milk formula, P = 0.72; soy formula vs breast milk,
P = 0.73; breast milk vs cow-milk formula, P = 0.92). (c) Breast-bud
diameter in boys (soy formula vs cow-milk formula, P = 0.37; soy
formula vs breast milk, P = 0.02; breast milk vs cow-milk formula,
P = 0.05). Feeding-group–specific trajectories fitted to data were
shifted vertically to a common intercept at a log2-transformed value
of zero (dashed line) to better display postnatal changes (i.e., fitted
log2-transformed organ size at a given age minus fitted log2-
transformed organ size at birth). Thus, the vertical axis represents
the ratio of current size to size at birth. Shaded bands represent
95% pointwise confidence limits for each feeding-group–specific
trajectory. Absolute trajectories (i.e., those without vertical shift to
common intercept) are shown in Supplemental Fig. 3.
https://academic.oup.com/jcem 1905
age, median E2 concentrations have been estimated at
approximately 8 to 9 pg/mL (30 to 31 pmol/L) (36, 37);
FSH concentrations have been estimated to be 3.8 mIU/mL
(95% CI, 1.2 to 18.8 mIU/mL) at this age (36). However,
many previous studies of E2 in infancy relied on methods
such as radioimmunoassay, which may lack appropriate
sensitivity to detect E2, particularly at low concentrations
(32). We used a state-of-the-art mass spectrometry method
to measure E2; our E2 results at 12 weeks were slightly
lower than those in some previous reports and this po-
tentially is attributable to improved accuracy. Notably, the
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most dynamic period with respect to organ and tissue
trajectories were in the immediate postpartum period
during withdrawal from maternal estrogen; later responses
to endogenous estrogen during proposed minipuberty were
not noted, although the different responses to soy formula
between girls’ and boys’ MI may be due, in part, to the girls’
concurrent E2 production.
Our study was not a randomized trial, and our results
may be influenced by differences in families or mothers
choosing the different feeding regimens, differences be-
tween infants, or other potentially confounding factors.
Postnatal urogenital cytological changes and uterine size
in infants have not been much studied, and there are no
established confounding factors related to these mea-
sures. The two formula groups did not differ signifi-
cantly in maternal race, age, or education. The soy-fed
girls were heavier at the birth visit, but adjustment for
this did not change our findings. In general, few mothers
in the United States choose soy formula as their first
formula. In our study, mothers’ stated reasons for
choosing to start with soy formula included successful
previous use, perceived healthfulness, and family his-
tory, or suspicion of future onset of, lactose intolerance
in their infants. Confounding by indication could pro-
duce our findings if these reasons are associated with an
estrogen effect on urogenital cytology and uterus in-
dependently of actual soy formula use. Because we did
not study infants whose mothers preferred soy formula
but who did not use it, we cannot rule out such con-
founding, but we suggest it to be unlikely. An additional
source of confounding may be exposure to other estro-
genic compounds from the home environment or else-
where, particularly if substantial exposure was more
likely to occur among soy-fed infants. Conversely, if these
exposures were more likely to occur in nonsoy-feeding
groups, we may be underestimating the effects of soy.
However, the current study was not designed to char-
acterize such exposures.
Using an ultrasound protocol similar to ours (19),
Gilchrist et al. (13) found no difference in uterine or
breast volume among 4-month-old girls (soy formula
fed, n = 20; cow-milk formula fed, n = 23; or breastfed,
1906 Adgent et al Soy Formula and Infant Reproductive Development J Clin Endocrinol Metab, May 2018, 103(5):1899–1909

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Figure 3. Fitted E2 trajectory vs age by feeding group in girls. Colors code feeding groups: black, breast milk; red, cow-milk formula; blue, soy
formula. Values below the limit of detection (2.99 pg/mL) are modeled at the limit of detection divided by the square root of 2. Shaded bands
represent 95% pointwise confidence limits for each feeding-group–specific trajectory. Each subject contributed up to 11 measurements through
time. Soy formula vs cow-milk formula, P = 0.44; soy formula vs breast milk, P = 0.02; breast milk vs cow-milk formula, P = 0.17.

n = 20). Some of these children were evaluated again at
age 5 years (38) and showed no feeding-group differ-
ences. The inconsistency between our findings and theirs
may be due to the smaller sample size of their cohort, the
later introduction of soy formula in their cohort, or to the
absence of repeated measures in their study design; they
could not characterize differences in organ growth tra-
jectories nor allow adjustment for feeding-group differ-
ences present at birth. An Israeli study found greater
prevalence of palpable breast tissue in 2-year-old children
fed soy formula as infants (14), and premature thelarche
was noted in another study among children fed soy
formula (among other exposures) (39). Although we did
not observe any difference in breast-tissue growth in
infant girls, we did observe that those fed soy formula
tended to have larger breast diameter. The transient peak
in breast diameter we describe in all feeding groups has
been previously reported (40), although our ultrasound-
based measurements yielded slightly larger diameters
than those determined by palpation in previous studies
(37). We know of no other study besides our small pilot
(15) that used urogenital cytology as an outcome in re-
lation to soy-formula exposure. Results from that pilot are
consistent with those described here. In addition, we have
reported altered DNA methylation in vaginal cells of some
girls fed soy formula in this current study and provided
experimental evidence that such changes are associated
with decreased gene expression (41).
Several studies have examined soy-formula use in in-
fancy in relation to conditions that occur later in life (9–12,
42). The only longitudinal study focused on soy formula
and adult health included young adults (ages 20 to 34 years)
who were fed soy formula or cow-milk formula as infants
(12). Women fed soy formula as infants (n = 128) reported
longer menstrual cycles and more menstrual pain than
women fed cow-milk formula (n = 268); no significant
differences were seen in men. Notably, adverse menstrual
cycle characteristics were also associated with reported
soy formula use among black women (10). Reported
soy-formula use has been associated with endometriosis
(9), with the size of uterine fibroids (11), and with early
and late age at menarche (43, 44).
Administration of pharmaceutical estrogen to pre-
mature infants produces results qualitatively similar to
our findings with soy formula in term infants. In a
randomized trial (35), girls born prematurely who were
given estradiol and progesterone had vulvar cell samples
exhibiting more estrogenization, larger uteri, and wider
breast-bud diameter than those given placebo. These
estrogen-mediated changes in urogenital cytology and
uterus size are consistent with our findings and support our
conclusions that soy formula exerts estrogenic effects at
these sites. In contrast to this trial and our expectations,
however, we did not observe effects on breast tissue.
Two North American advisory groups have consid-
ered the safety of soy infant formula. The Committee on
doi: 10.1210/jc.2017-02249
Nutrition, American Academy of Pediatrics, recommends
soy formula only for infants with galactosemia or he-
reditary lactase deficiency and mentions that soy formula
might be useful for families wishing to avoid formula
containing animal products (45). A US National Toxi-
cology Program expert panel concluded that there was
“minimal concern” for adverse developmental effects in
infants who consume soy formula, based on data avail-
able up to 2009 (8). However, it also stated:
The existing epidemiological literature on soy in-
fant formula exposure is insufficient to reach a
conclusion on whether soy infant formula does or
does not cause adverse effects on development in
humans. There is ‘clear evidence’ for adverse effects
of genistein on reproductive development and
function in female rats and mice manifested as
accelerated puberty (i.e., decreased age at vagi-
nal opening), abnormal estrous cyclicity, cellular
changes to the female reproductive tract, and de-
creased fecundity (i.e., decreased fertility, implants,
and litter size). Also, infants fed soy infant formula
can have blood levels of total genistein that exceed
those measured in neonatal or weanling rodents
following treatment with genistein at dose levels
that induced adverse effects in the animals (8).
Public concern about bisphenol A, a chemical used in
plastics that has documented estrogen activity, centers on
exposures to infants and young children (46). Concern
about the risks of early-life exposure to xenoestrogens
dates to the experience with diethylstilbestrol, a potent es-
trogen given in the 1950s to prevent miscarriage (2). It
resulted in the occurrence of vaginal adenocarcinoma in
some young women born of such pregnancies, establishing
the possibility of delayed toxicity from perinatal exposure to
an estrogen.
Genistein is a weaker estrogen than diethylstilbes-
trol and endogenous estradiol, but is stronger than
chemicals such as bisphenol A (4); here, we offer evi-
dence that soy formula, a known source of genistein,
provokes an estrogenlike response in infants. In neo-
natal mice, genistein exposure can produce substan-
tial reproductive tract toxicity (47, 48). Whether our
findings presage long-term effects in infants similar to
those of genistein exposure in rodents is yet to be
established. Epigenetic alterations in the vaginal cells of
soy formula–fed girls (41) provide a possible mecha-
nism for such delayed effects.
In summary, we show that infants who consume
soy formula present with changes to tissue consistent
with those seen with exogenous estrogen. Genistein is
the major isoflavone in soy formula and is capable of
ER binding. Experimentally, genistein has increased
https://academic.oup.com/jcem 1907
murine uterine wet weight and produces downstream
transcriptional activation of estrogen responsive
genes (34). Animal evidence predicts that genistein can
induce developmental and reproductive toxicity at
doses relevant to soy formula–fed infants (49). Our
findings are consistent with those in the animal liter-
ature in that we detected several developmental pertur-
bations of estrogen-responsive tissue. Our methods
targeting subclinical metrics of tissue growth and response
may be useful tools for identifying early responses to
suspected estrogenic agents.
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Acknowledgments
We thank all patients and families for their participation; the care
providers and staff at our main study site, Children’s Hospital of
Philadelphia (CHOP), as well as our birth/recruitment hospital
sites: the University of Pennsylvania Hospital, Pennsylvania
Hospital and Virtua Voorhees Hospital, Virtua Memorial
Hospital, Abington Memorial Hospital, Cooper Memorial
Hospital, and Holy Redeemer Hospital. We thank Els Nijs;
Adeka McIntosh; Laura Poznick, RDMS; Trudy Morgan,
RDMS; Marcy Hutchinson, RDMS, RVT; Danielle Drigo,
Infant Feeding and Early Development (IFED) team of research
assistants; and the Department of Radiology at CHOP for their
work with study design, quality assurance, data procedures, and
collection. We thank our collaborators at the Department of
Radiology at Virtua Voorhees; Elizabeth Fong-deLeon, De-
partment of Pediatrics at Virtua Voorhees Hospital; and Steven
Shapiro, Department of Pediatrics at Abington Memorial
Hospital. We thank Dr. Michael Rybak and Patrick Simon of
the Centers for Disease Control and Prevention for their work
on urinary isoflavone analysis; Donna Baird and Retha Newbold,
MS, at National Institute of Environmental Health Sciences; and
Stacy Patchel for her careful data management and statistical
programming.
Formula for study participants was a gift from Nestlé and
Mead Johnson Nutrition. The IFED advisory board included
Sheri Berenbaum, Russ Hauser, Michael DiPietro, Linda Adair,
Laurie Moyer-Mileur, RD, and our late colleagues Judson Van
Wyk, Laurence Finberg, and Melvin Grumbach. The findings
and conclusions in this manuscript are those of the authors and
do not necessarily represent the official views or positions of the
US Centers for Disease Control and Prevention/Agency for
Toxic Substances and Disease Registry. Use of trade names and
commercial sources is for identification only and does not
constitute endorsement by the US Department of Health and
Human Services and the US Centers for Disease Control and
Prevention.
Financial Support: This research was supported in part by
the Intramural Research Program of the National Institutes of
Health (NIH), National Institute of Environmental Health
Sciences (Project No. Z01-ES044006 to W.J.R.). Data collec-
tion at the Children’s Hospital of Philadelphia (CHOP) was
supported through Subcontract PHR-SUPS2-S-09-00196 under
Contract HHSN291200555546C between the National Institute
1908 Adgent et al Soy Formula and Infant Reproductive Development
of Environmental Health Sciences and Social & Scientific
Systems Inc. This project was supported by the Nutrition Center
at the CHOP and by the National Center for Research Re-
sources, Grant UL1TR000003 (National Center for Advancing
Translational Sciences).
Correspondence and Reprint Requests: Margaret A. Adgent,
PhD, 1313 21st Avenue S, Suite 313, Nashville, Tennessee 37232.
E-mail: margaret.a.adgent@vanderbilt.edu.
Disclosure Summary: The authors have nothing to
disclose.
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