Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print April 2, 2008.
Biol Reprod 2008, 10.1095/biolreprod.108.067702
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
79/1/154    most recent
biolreprod.108.067702v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Abbott, D. H
Right arrow Articles by Tarantal, A. F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Abbott, D. H
Right arrow Articles by Tarantal, A. F
Agricola
Right arrow Articles by Abbott, D. H
Right arrow Articles by Tarantal, A. F
BIOLOGY OF REPRODUCTION 79, 154–163 (2008)
DOI: 10.1095/biolreprod.108.067702
© 2008 by the Society for the Study of Reproduction, Inc.

Environment; 

Endocrine Antecedents of Polycystic Ovary Syndrome in Fetal and Infant Prenatally Androgenized Female Rhesus Monkeys1

David H Abbott 2 3 4 5, Deborah K Barnett 3 6, Jon E Levine 7, Vasantha Padmanabhan 8, Daniel A Dumesic 3 4 9, Steve Jacoris 3, and Alice F Tarantal 10

Departments of Neurobiology and Physiology,7 Northwestern University, Evanston, Illinois 60208 Departments of Pediatrics, Obstetrics and Gynecology, Molecular and Integrative Physiology, and Reproductive Sciences Program,8 University of Michigan, Ann Arbor, Michigan 48109 Reproductive Medicine and Infertility Associates,9 Woodbury, Minnesota 55125 Department of Biology,6 University of Alaska-Southeast, Sitka, Alaska 99835 Departments of Pediatrics and Cell Biology and Human Anatomy and the California National Primate Research Center,10 University of California, Davis, California 95616 Wisconsin National Primate Research Center,3 Department of Obstetrics and Gynecology,4 and Endocrinology-Reproductive Physiology Program,5 University of Wisconsin, Madison, Wisconsin 53715

ABSTRACT

Experimentally induced fetal androgen excess induces polycystic ovary syndrome-like traits in adult female rhesus monkeys (Macaca mulatta). Developmental changes leading to this endocrinopathy are not known. We therefore studied 15 time-mated, gravid female rhesus monkeys with known female fetuses. Nine dams received daily s.c. injections of 15 mg of testosterone propionate (TP), and six received injections of oil vehicle (control) from 40 through 80 days of gestation (term, 165 days; range, ±10 days). All fetuses were delivered by cesarean section using established methods at term. Ultrasound-guided fetal blood sample collection and peripheral venous sample collection of dams and subsequent infants enabled determination of circulating levels of steroid hormones, LH and FSH. The TP injections elevated serum testosterone and androstenedione levels in the dams and prenatally androgenized (PA) fetuses. After cessation of TP injections, testosterone levels returned to values within the reference range for animals in these age groups, whereas serum androstenedione levels in PA infants were elevated. The TP injections did not increase estrogen levels in the dams or the PA fetuses or infants, yet conjugated estrogen levels were elevated in the TP-injected dams. Serum levels of LH and FSH were elevated in late-gestation PA fetuses, and LH levels were elevated in PA infants. These studies suggest that experimentally induced fetal androgen excess increases gonadotropin secretion in PA female fetuses and infants and elevates endogenous androgen levels in PA infants. Thus, in this nonhuman primate model, differential programming of the fetal hypothalamo-pituitary unit with concomitant hyperandrogenism provides evidence to suggest developmental origins of LH and androgen excess in adulthood.

androgen excess, early development, environment, fetal programming, LH hypersecretion, LH negative feedback, luteinizing hormone, testosterone

INTRODUCTION

Experimental induction of androgen excess in female rhesus monkeys (Macaca mulatta) during early or late gestation has been shown to reprogram reproductive and metabolic physiology, resulting in polycystic ovary syndrome (PCOS) in adulthood [1]. Such adult, prenatally androgenized (PA) female rhesus monkeys have been documented to exhibit intermittent or absent menstrual cycles [1, 2], ovarian hyperandrogenism [1, 3], and polycystic ovarian morphology [4], all traits that confer the diagnosis of PCOS by either National Institutes of Health [5] or Rotterdam [6] criteria or currently proposed amendments [7, 8]. Comparable to the situation in many women with PCOS [7, 9, 10, 11], profound metabolic dysfunction has been shown to accompany the reproductive disorders of PA female rhesus monkeys [1].

Also, similar to many women with PCOS, PA female monkeys exposed to androgen excess during early gestation exhibit LH excess and increased LH responsiveness to exogenous GnRH (for monkeys, see [1, 12]; for women with PCOS, see [13, 14]), suggestive of enhanced hypothalamic GnRH release and/or increased pituitary sensitivity to GnRH. Diminished sex-steroid negative feedback on LH release is found in PA female monkeys exposed in early or late gestation [1, 15, 16] and in women with PCOS [17, 18], and this may explain the increased pulsatile LH secretion observed in both species (for women with PCOS, see [19]; for PA monkeys, Levine and Abbott, unpublished data). Conversely, circulating FSH levels have been reported to be low to normal in both PA female monkeys [12, 20] and in women with PCOS [14, 21], thereby elevating the serum LH:FSH ratio. It has been proposed that LH hypersecretion in PCOS may originate during adolescence, initially caused by hyperandrogenism and then, potentially, contributing to peripubertal hyperandrogenism (as in some adolescents with PCOS [22, 23]). Such neuroendocrine dysfunction, however, may originate in utero from androgen/estrogen-induced changes in the fetal hypothalamo-pituitary axis [4, 24–27] before any subsequent peripubertal dysfunction.

Based on previous studies that have shown the importance of the rhesus monkey model of fetal androgen excess and its application to understanding the mechanisms associated with PCOS [1–4, 28], the present study was designed to examine whether androgen excess in early gestation during a defined window of development hormonally differentiates LH hypersecretion in PA female rhesus monkeys during fetal life and in early infancy and, if so, whether it is accompanied by endogenous hyperandrogenism during this period of development. Prenatally androgenized monkeys exposed during early gestation were chosen for the present study because adult female monkeys exposed to such fetal manipulation express LH dysfunction (increased basal levels, increased LH response to GnRH, and decreased sensitivity to sex-steroid negative feedback [1, 15, 16]) more clearly than female adults exposed during late gestation. The overall intent was to determine whether androgen-induced reprogramming of the nonhuman primate fetal hypothalamo-pituitary-gonadal axis occurs in the early ontogeny of reproductive endocrine traits found in women with PCOS. Such excessive LH stimulation of the immature ovary could, potentially, contribute to the peripubertal onset of reproductive dysfunction found in women with hyperandrogenic disorders, including those with PCOS [27].

MATERIALS AND METHODS

Animals

All animal procedures conformed to the requirements of the Animal Welfare Act, and protocols were approved before implementation by the Institutional Animal Care and Use Committee at the University of California, Davis. Normally cycling, adult female rhesus monkeys (M. mulatta), housed at the California National Primate Research Center (CNPRC), with a history of previous pregnancy were time-mated and identified as pregnant using established methods [29]. Pregnancy in rhesus monkeys typically is divided into trimesters by 55-day increments, with 0–55 days of gestation representing the first trimester, 56–110 days of gestation representing the second trimester, and 111–165 days of gestation representing the third trimester (term, 165 days; range, ±10 days) [30].

Experimental Design

At approximately 30 days of gestation, an approximate 2-ml blood sample was collected from a peripheral vein to identify female embryos using established PCR-based assays to demonstrate the absence of Y-chromosomal DNA in maternal blood [31]. At 40 days of gestation, 15 dams with confirmed female embryos were assigned on a rotational basis to receive consecutive daily s.c. injections of either 15 mg of testosterone propionate (TP) in 100–200 µl of sesame oil (n = 9 TP-injected dams) or sesame oil alone (n = 6 oil-injected dams), with the last day of treatment occurring at 80 days of gestation (early second trimester). Treatment assignments also were balanced so that the two groups of dams were similar with regard to preconception age (TP-injected dams, 11.2 ± 1.5 yr [mean ± SEM]; oil-injected controls, 8.4 ± 2.0 yr) and body weight (TP-injected dams, 7.0 ± 0.4 kg; oil-injected controls, 6.8 ± 0.5 kg). Once assigned to the present study, dams were monitored sonographically under ketamine (10 mg/kg i.m.) every 7–10 days during gestation using standard protocols to assess fetal growth and development using established growth charts for this species [29].

This early gestation exposure of female monkeys to TP-mediated androgen excess has been shown to result in a hyperandrogenic prenatal environment, equivalent to that found in males during early gestation [32], and occurs in a gestational time period when multiple fetal organ systems, including those involved in reproduction and metabolism, are undergoing development [1]. Elevating fetal female testosterone levels into the fetal male range provides a physiologically relevant test of our hypothesis for fetal androgen excess programming of PCOS, because in humans, congenital virilization of female fetuses commonly programs neuroendocrine and metabolic features of PCOS, leading to an increased incidence of PCOS (25–50% in classical congenital adrenal hyperplasia [27]) compared to that found in nonvirilized women (6–7% PCOS [33–35]). Moreover, 40% of human fetal females have unbound (i.e., free) testosterone values during the early to mid-second trimester that are equivalent to those in normal human fetal males [36], indicating that our use of testosterone levels in the present study equivalent to those of fetal males provides a relevant physiological challenge. The TP dose used in the present study also is within the range of 5–15 mg TP/day used previously for PA female rhesus monkeys with PCOS-like traits in adulthood [1, 2]. This TP dose successfully overcomes the primate placenta's robust ability to aromatize and inactivate androgens, which is considerably greater than that of the placenta in nonprimate species, such as rats [37] and sheep [38]. Sonographic studies were performed under ketamine, as noted above, during gestation, and newborns were then delivered by cesarean section at term (160 days of gestation; range, ±2 days) using standard protocols [29]. Cesarean section was employed to provide a relatively consistent gestational length and to avoid complications associated with spontaneous delivery. Prenatally, all PA female fetuses displayed virilized external genitalia when examined sonographically, and this was confirmed grossly at the time of delivery, thus providing an effective biomarker for the effectiveness of androgen excess during early gestation. Newborns were placed in incubators after delivery and were reared in a nursery using established protocols [29]. At 2 mo of age, infants were given an i.v. overdose of pentobarbital (60 mg/kg) for tissue procurement.

Blood Sample Collection

Fetal blood samples (1–3 ml) were collected by ultrasound-guided cardiocentesis using established protocols [39] at defined time points, as described below. Peripheral blood samples also were collected from the dams (3–10 ml) and infants (~3 ml) at defined time points (see below). All blood samples from all age groups were collected between 0700 and 0900 h, as shown in Figure 1. When multiple maternal or fetal blood samples were collected (80, 120, and 140 days of gestation), dams were sedated with an i.m. injection of Telazol (5–8 mg/kg; Wyeth, Madison, WI). Infants were handheld by experienced personnel and not sedated for blood sampling. These well-established blood sampling techniques provided unique insights regarding the simultaneous progression of fetal and maternal reproductive hormone levels within the same individual nonhuman primates during gestation and without surgical intervention or the need for instrumentation.


Figure 01
View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 Diagrammatic representation of the experimental design, illustrating days of maternal s.c. injections (15 mg of TP, n = 9; oil, control n = 6) during 40–80 days of gestation; days of blood sample collection in dams, fetuses, and infants; timing of cesarean section delivery; and fetal and infant GnRH tests.

Fetal GnRH Tests

To assess pituitary gonadotropin responsiveness to exogenous GnRH in fetal PA female monkeys, an i.v. bolus injection of 5 µg of GnRH (~15–20 µg/kg) was administered in 0.1 ml of sterile saline at approximately 0700 h at 120 days of gestation (third trimester) under ultrasound guidance via the fetal circulation (Fig. 1). Fetal blood samples were collected by ultrasound-guided cardiocentesis immediately before (0 min) and at 10, 20, 30, and 40 min after the GnRH injection. The approximately 15–20 µg/kg dose of GnRH was chosen in an attempt to consistently stimulate LH release, as reported in earlier studies of fetal and infant rhesus monkeys ([40]: 80 µg [~240–320 µg/kg]; [41]: 10–50 µg [~30–200 µg/kg]; and [42]: 500 µg [~625 µg/kg]). Inconsistent LH release occurs when lower doses (e.g., 50 ng/kg [43]) of exogenous GnRH are employed (LH release occurred in only three of seven female infant controls and in none of six female infant PA monkeys [43]). As in that previous study [43], we noted the number of females showing an increase of more than 50% in serum bioactive LH (bio-LH) levels following a GnRH injection.

Infant GnRH Tests

A similar assessment of pituitary gonadotropin responsiveness to exogenous GnRH was performed in infant PA female monkeys. The GnRH was injected via a peripheral vessel, and then blood samples were collected in handheld, unanesthetized animals from a femoral vein. For infants, GnRH tests were administered during both morning (~0700 h) and evening (~2100 h) hours on different days to detect any diurnal changes in pituitary gonadotropin responsiveness (Fig. 1). The morning GnRH test was administered on Postnatal Day 30, when an i.v. GnRH bolus (20 µg, ~25 µg/kg) was given in 0.4 ml of sterile saline. Blood samples were obtained postinjection before (0 min) and at 10, 20, 30, and 40 min after the GnRH injection. The evening GnRH test was administered on Postnatal Day 37 using an identical injection and blood sample collection protocol.

Steroid Hormone Assay Procedures

All hormones were assayed in the Assay Services Laboratories of the Wisconsin National Primate Research Center (WNPRC) [12, 44, 45]. All steroid hormones used as reference preparations in standard curves were obtained from Steraloids. Serum steroid hormone concentrations in all samples, except those used for conjugated estrogen determinations, were analyzed with an Agilent 1100 liquid chromatograph/mass spectrometer (LCMS) equipped with an electrospray ionization source and Chemstation software (Ver A 10.02). The LCMS methods were validated using U.S. Food and Drug Administration protocols (Guidance for Industry Bioanalytical Method Validation, May 2001) and adapted from those described previously for humans [46].

Using positive-ion identification for testosterone (m/z 289.2) and androstenedione (m/z 286.8), the LCMS standard curve for testosterone ranged from 0 to 80 ng/ml, and that for androstenedione ranged from 0 to 2 ng/ml. The calibration plots over 3 days for testosterone and androstenedione showed, respectively, slopes of 0.91 and 1.67, intercepts of 0.0008 and 0.003, and regression coefficients of 0.999 and 0.991. Relevant characteristics for LCMS determination of both testosterone and androstenedione values are presented in Table 1. For sample preparation, 100-µl aliquots from individual serum samples or quality-control rhesus monkey serum pools were placed into 13 x 100 mm borosilicate glass culture tubes, and to each of these, 500 µl of 18.2 M{Omega}-cm filtered (distilled and ultraviolet light-purified) water were added, as were 20 µl of both d5-testosterone (m/z 294.2; CDN Isotopes) and d7-androstenedione solutions (m/z 293.8) at 100 pg/µl of each (in ethanol; Sigma). The deuterated androgens were added as internal standards to monitor recovery. Each standard, pool, or individual sample was then extracted with 2 ml of diethyl ether (Fisher), and the separated solvent fraction was evaporated and reconstituted in 20 µl of water:acetonitrile (1:1, v/v; EMD Chemicals), from which 4 µl (20%) were injected onto a Phenomenex Synergi Max-RP 150 x 1.0 mm column with 4-µm particles at 80 A. All solvents used were HPLC grade. Mobile phase A comprised water/acetonitrile (95%/5%) with 0.01% formic acid, whereas mobile phase B comprised acetonitrile/water (95%/5%) with 0.01% formic acid. Before injecting each sample, the LCMS column was equilibrated for 10 min at 150 µl/min with 60% mobile phase B and 40% mobile phase A, and the column was maintained at 35°C. Serial dilutions of rhesus monkey serum (100–400 µl) yielded testosterone and androstenedione values parallel to the LCMS standards, and regression analyses favorably compared LCMS determinations to those made by our previously validated methods (enzyme immunoassay [EIA] for testosterone and RIA for androstenedione) [44] (Table 1). The only major difference in LCMS-determined compared to EIA- or RIA-determined serum androgen values was reflected in the consistent, but reliable, lower serum values generated by LCMS (Table 1).


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 1 Characteristics of the LCMS methods for determination of serum androgen and estrogen levels in control and testosterone propionate treated female rhesus monkeys and their comparison to previously validated in-house RIAs and EIAs [44].

For estrogen determinations by LCMS, we derivatized estradiol and estrone with dansyl chloride to improve ionization efficiency and LCMS sensitivity, as described previously [47]. Standards, pools, and individual samples were prepared similarly to those for androgens except that 20 µl of ethanol solutions of both d5-estradiol dansyl (m/z 511.4, 2 pg/µl) and d4-estrone dansyl (m/z 504.4, 4 pg/µl) were added as internal standards to monitor recovery. We then extracted each standard, pool, or individual sample as described for androgens but reconstituted in 100 µl of ethanol, which was vortexed briefly before adding 500 µl of 18.2 M{Omega}-cm filtered water with 2 ml of dichloromethane and vortexing again for 1 min. The organic layer was removed, evaporated to dryness, and reconstituted with 20 µl of 100 mM sodium bicarbonate (adjusted to pH 10.5 with 1 M sodium hydroxide). The mixture was vortexed for 1 min before addition of 20 µl of dansyl chloride (1 mg/ml in acetone) and incubation at 33°C for 2.5 min. Twenty microliters (50%) of the derivatized estrogen solution were then injected onto the LCMS column.

Using positive-ion identification for estradiol dansyl (m/z 506.4) and estrone dansyl (m/z 504.4), the standard curves for both estrogens were 0–40 pg/ml. The calibration plots over 3 days for estradiol dansyl and estrone dansyl showed, respectively, slopes of 0.63 and 1.16, intercepts of 0.009 and 0.008, and regression coefficients of 0.926 and 0.927. Relevant characteristics for LCMS determination of both estradiol and estrone are presented in Table 1. Equilibration procedures and mobile-phase dynamics similar to those used for LCMS androgen determinations were employed. Serial dilutions of rhesus monkey serum (50–400 µl) yielded estradiol and estrone values parallel to the LCMS standards, and regression analyses favorably compared LCMS determinations to those made using our previously validated methods (RIA) [12, 44] (Table 1). Whereas LCMS determined values were highly correlated with those from our established assays, they were consistently, but reliably, either lower than those produced by our previously established methods (estradiol RIA) or higher than those produced by previous methods (estrone RIA) (Table 1).

Estrogen Conjugate Determinations

Because nonhuman primates exhibit extensive conjugation of estrogens in the liver, placenta, and other organs during the process of bio-inactivation and excretion of these steroids [48, 49], we employed sequential enzyme hydrolysis and acid solvolysis to maternal serum samples to quantify total conjugate concentrations for both estradiol and estrone [50] that better reflect total estrogen in the circulation. Serum estriol concentrations were not determined, because this estrogen is present only at very low levels in rhesus monkey pregnancies [51]. The remaining sample volumes from fetuses and infants were insufficient to assess estrogen conjugate levels in their serum. All fractions of deconjugated estrogens were resuspended in celite chromatography solvents, and the estrogens were separated through celite column chromatography and assayed by RIA, as described previously [44]. Intraassay coefficients of variation for quality control (QC) values for the single RIAs performed were 5.8% for estradiol and 7.3% of estrone.

Gonadotropin Assay Procedures

Serum bio-LH and immunoactive FSH were determined by mouse Leydig cell bioassay and RIA using the reference preparations of recombinant cynomolgus monkey LH (AFP-6936A) and recombinant cynomolgus monkey FSH (AFP-6940A), respectively, obtained from Dr. Parlow at the National Hormone and Peptide Program [12]. Intra- and interassay coefficients of variation for QC values were as follows: bio-LH, 10.8% and 17.7%, respectively; FSH, 4.6% and 10.9%, respectively. Whereas the mouse Leydig cell bioassay cannot distinguish between bio-LH and bioactive chorionic gonadotropin (bio-CG), rhesus placentae secrete little or no bio-CG by 40 days of gestation [51–53]. Thus, little likelihood exists of placental bio-CG contributing to fetal bio-LH levels at 80–120 days of gestation.

Statistical Analysis

All hormonal data were tested for normality using a Lilliefors test (two-sided) and were log transformed to achieve homogeneity of variance and to increase linearity [54]. All hormonal variables were analyzed using separate two-way ANOVAs (with repeated-measures designs as appropriate) for dams, fetuses, and infants (the latter two sets of ANOVAs reflect distinctly different developmental stages and hormone parameter values within the same individuals), with fetal androgen exposure and time as independent variables. When significant (P < 0.05) statistical interactions were identified by ANOVA, appropriate post hoc analyses (with Bonferroni correction) were performed on the variables (Systat, Ver 5.2; Macintosh). The extreme studentized deviate outlier test (one-sided) [55] identified one control female fetus as an outlier (P < 0.01) for bio-LH values when compared to all other study fetuses combined. The data from this control female thus were excluded from analyses for bio-LH and FSH. All log-transformed parameters are expressed as the antilog of the transformed mean (95% confidence limit) except for fetal and infant bio-LH data when individual values are shown instead of the 95% confidence limit. All other data are shown as the mean ± SEM.

RESULTS

Newborn Weight and Appearance of PA Infants

Immediately following cesarean section at term, the newborn weights of PA female infants (0.44 ± 0.16 kg) did not differ from those of female controls (0.46 ± 0.20 kg). The external genitalia of PA female infants were virilized as anticipated, exhibiting an empty scrotum and a preputial opening housing an immature phallus, which is a typical phenotypic marker of androgen excess during early gestation indicating sufficient testosterone treatment provided at an appropriate gestational age.

Experimentally Induced Changes in Circulating Androgen Concentrations

The TP injections induced an approximately 110-fold increase (P < 0.001) in maternal serum testosterone levels, with a considerably smaller increase (~11-fold, P < 0.001) in fetal levels (fetal androgen exposure x time interaction, P < 0.001) (Fig. 2). The increases in maternal and fetal testosterone levels were restricted to the duration of daily TP injections. Circulating testosterone levels were otherwise similar in both treatment groups of dams, fetuses, and infants.


Figure 02
View larger version (10K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 Serum testosterone levels (backtransformed means, with 95% confidence limits represented by the error bars) in gravid female rhesus monkeys (Macaca mulatta) receiving 15 mg of TP (filled circles, dashed line) or oil (open circles, solid line) injections during 40–80 days of gestation (a) and in their fetuses (b) and infants (c). ***P < 0.001 vs. controls on the same day of gestation following fetal androgen exposure x time interaction (P < 0.001).

Serum androstenedione changes following TP injection mostly paralleled those of serum testosterone except that the magnitude of the serum androstenedione increase was less than that of serum testosterone, being comparable in both dams (~4–5-fold, P < 0.001 vs. controls) and fetuses (~3-fold, P < 0.001 vs. controls; fetal androgen exposure x time interaction, P < 0.001) (Fig. 3). In contrast to serum testosterone concentrations, serum androstenedione levels in young PA infants were elevated (fetal androgen exposure effect, P < 0.019) to approximately 50%–100% above those values normally found in infant controls between Postnatal Days 1 and 30. Serum androstenedione levels also exhibited temporal changes across gestation and early infancy. In both female groups, maternal serum androstenedione levels rose progressively (time effect, P < 0.001) as gestation progressed, whereas serum androstenedione levels declined (time effect, P < 0.03) in early infancy (Fig. 3).


Figure 03
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 Serum androstenedione levels (backtransformed means, with 95% confidence limits represented by the error bars) in gravid female rhesus monkeys (Macaca mulatta) receiving 15 mg of TP (filled circles, dashed line) or oil (open circles, solid line) injections during 40–80 days of gestation (a) and in their fetuses (b) and infants (c). ***P < 0.001 vs. controls on the same day of gestation following fetal androgen exposure x time interaction (P < 0.001), #P < 0.03 vs. controls on Postnatal Days 1 and 30 (fetal androgen exposure effect).

Experimentally Induced Changes in Circulating Estrogen and Progesterone Concentrations

Unlike the case with circulating androgen levels, TP injections did not elevate serum estradiol or estrone levels in dams, fetuses, or infants (Figs. 4 and 5). Nevertheless, maternal circulating levels of total conjugated estrogen were elevated (P < 0.03) by TP injections when measured at 80 days of gestation (TP-injected dams, 3.1 [2.1, 4.7] ng/ml; controls, 1.3 [0.8, 2.1] ng/ml). In TP-injected dams, serum estrone levels were lower (fetal androgen exposure effect, P < 0.036) during gestation (Fig. 5). No between-group differences, however, were found regarding temporal changes in fetuses across gestation and in early infancy for serum estradiol and estrone levels. Maternal estrone levels rose steadily (time effect, P < 0.001) during gestation in both female groups (Fig. 5), whereas maternal estradiol levels reached a plateau by midgestation (Fig. 4). During infancy, circulating levels of both estrogens declined precipitously (time effect, P < 0.01) between Postnatal Days 1 and 30.


Figure 04
View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 Serum estradiol levels (backtransformed means, with 95% confidence limits represented by the error bars) in gravid female rhesus monkeys (Macaca mulatta) receiving 15 mg of TP (filled circles, dashed line) or oil (open circles, solid line) injections during 40–80 days of gestation (a) and in their fetuses (b) and infants (c).


Figure 05
View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 Serum estrone levels (backtransformed means, with 95% confidence limits represented by the error bars) in gravid female rhesus monkeys (Macaca mulatta) receiving 15 mg of TP (filled circles, dashed line) or oil (open circles, solid line) injections during 40–80 days of gestation (a) and in their fetuses (b) and infants (c). §P < 0.036 vs. controls (fetal androgen exposure effect).

Maternal serum progesterone levels did not differ between TP-injected and control females across gestation (40–160 days of gestation), averaging 3.8 (3.4, 4.2) and 3.3 (2.3, 4.3) ng/ml, respectively.

Experimentally Induced Changes in Circulating Bio-LH and FSH Levels in Fetuses and Infants

As expected for rhesus monkey dams, most serum bio-LH values in both female groups were equal to or less than the bioassay sensitivity limit of 0.06 ng/ml from 40 to 160 days of gestation (data not shown).

In fetal subjects, serum bio-LH levels in controls remained relatively constant between 80 and 120 days of gestation (difference in bio-LH levels: between 80 and 100 days of gestation, +0.8 ± 0.8 ng/ml; between 100 and 120 days of gestation, –0.3 ± 0.9 ng/ml), whereas bio-LH levels in fetal PA monkeys consistently increased across the same gestational ages (fetal androgen exposure x time interaction, P < 0.001; difference in bio-LH levels in PA monkeys: between 80 and 100 days of gestation, +2.0 ± 0.6 ng/ml; between 100 and 120 days of gestation, +2.7 ± 0.7 ng/ml) (Fig. 6). In 80-day-gestation (second-trimester) PA fetuses, maternal TP injections reduced (P < 0.001) fetal circulating bio-LH levels to approximately 10% of those found in oil-injected controls. Following cessation of TP injections, bio-LH levels in all PA female fetuses increased to values comparable to those of controls after 20 days (i.e., 100 days of gestation) and then increased again after a further 20 days, resulting in most (six of nine) PA monkey values exceeding (P < 0.042) those in controls by 120 days of gestation (Fig. 6). No relationships between PA monkey maternal and fetal testosterone levels at 80 days of gestation and those of fetal bio-LH on 100 and 120 days of gestation were obvious.


Figure 06
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6 Serum bio-LH levels (backtransformed means and individual values) in PA (filled bars and symbols) and control (open bars and symbols) female (a) fetuses on the last day of TP or oil s.c. injections and 20 and 40 days later and (b) newborn and 30-day-old infants. Each symbol refers to the same individual female at every time point. ***P < 0.001, {partial}P < 0.025 vs. controls on the same day of gestation following a fetal androgen exposure x time interaction (P <0.001), {dagger}P < 0.019 vs. controls on Postnatal Days 1 and 30 (fetal androgen exposure effect).

During the fetal GnRH test administered on Day 120 of gestation, serum bio-LH levels were elevated (fetal androgen exposure effect, P < 0.05) in PA females compared to control females (Fig. 7), resulting in increased (P < 0.048) area-under-the-curve bio-LH values in PA females (PA, 2.4 [1.6, 3.4] ng ml–1 min–1; control: 1.2 [0.7, 2.0] ng ml–1 min–1). The absolute increase in the GnRH-induced increase in serum bio-LH levels, however, paralleled that in controls (time effect, P < 0.003) and exceeded baseline values by 30 min (P < 0.03) in both female groups. Four of five controls exhibited GnRH-induced increases in bio-LH levels that exceeded baseline values by more than 50%, whereas only five of nine PA fetuses showed a similar increase in bio-LH. Additionally, serum FSH levels were elevated (fetal androgen exposure effect, P < 0.05) in PA fetuses during the GnRH test, whereas fetal FSH levels in both female groups were unresponsive to the exogenous GnRH stimulus (no female fetus exhibited an increase in FSH levels exceeding 50% of its baseline value) (Fig. 7).


Figure 07
View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 7 Serum gonadotropin levels (backtransformed means, with 95% confidence limits represented by the error bars) following an i.v. injection of exogenous GnRH in prenatally androgenized (filled circles) and control (open circles) female rhesus monkeys (Macaca mulatta): fetal responses to 5 µg of GnRH at 120 days of gestation in terms of (a) bio-LH and (b) FSH and infant responses to 20 µg of GnRH at (c) 30 days postpartum (bio-LH, ~0700 h) and (d) 37 days postpartum (bio-LH, ~2100 h). In a, *P < 0.05 vs. controls (fetal androgen exposure effect), #P < 0.03 vs. 0 min (time effect); in b, *P < 0.05 vs. controls (fetal androgen exposure effect); in c, following fetal androgen exposure x time interaction (P < 0.008), ***P < 0.001 vs. 0 min (control females only), #P < 0.03 vs. 10 min (PA females only); and in d, ***P < 0.001 vs. 0 min (time effect).

In newborn PA infants, serum bio-LH values remained elevated for the first 30 postnatal days (P < 0.019) (Fig. 6). A decline (P < 0.03) in overall bio-LH levels occurred during this time, but no significant (P < 0.074) interaction of fetal androgen exposure with time was observed. Thus, on Postnatal Day 1, seven of nine androgenized female bio-LH values exceeded the control range, whereas only two of nine PA female bio-LH levels exceeded those of controls by Postnatal Day 30. Morning and evening infant GnRH tests elicited no female group differences in bio-LH responsiveness to GnRH (Fig. 7), but all infants showed an increase of more than 50% in bio-LH levels following each GnRH injection. During the morning GnRH test, serum bio-LH levels in PA infants, however, progressively increased until 30 min following GnRH injection. The bio-LH levels in controls, however, failed to do so after 10 min (fetal androgen exposure x time interaction, P < 0.008), but area-under-the-curve bio-LH values were similar in PA (1.0 [0.6, 1.6] ng ml–1 min–1) and control (1.3 [0.7, 2.3] ng ml–1 min–1) infants. Insufficient sample volumes remained to obtain meaningful infant serum FSH values.

DISCUSSION

Elevated circulating LH levels and rapid, pulsatile LH release are hypothalamo-pituitary hallmarks of adult PA female rhesus monkeys, women exposed to androgen excess during early gestation [1, 19, 24], and women with PCOS [19, 22]. It has been proposed that pre- or perinatal androgen excess [2, 4, 23–26], peripubertal hyperandrogenism [22, 23], or excessive and prolonged adult ovarian production of estrogen from androstenedione [56] may contribute to the persistently rapid GnRH pulse frequency in women with PCOS via impaired hypothalamic feedback inhibition. Such LH excess in women with PCOS contributes to ovarian hyperandrogenism in adulthood [22, 57; but see 58]. The present evidence of fetal and infant LH excess in a nonhuman primate model of PCOS is consistent with early life programming of neuroendocrine dysfunction that will manifest as postpubertal LH hypersecretion [1, 2] in a manner resembling that of PCOS in women.

Hypothalamo-pituitary changes seen in fetal PA female rhesus monkeys resemble those normally observed in fetal males [59], suggesting some degree of masculinized hypothalamo-pituitary function in PA females. Midgestational circulating testosterone levels in normal fetal male monkeys mostly exceed those in normal fetal females [60], but LH levels of such fetal males usually are lower than those of respective fetal females [61]. This sexually dimorphic LH differential probably represents testicular testosterone-mediated negative feedback at the hypothalamic level in fetal males [62]. Our present findings in PA females emulate this LH differential: Specifically, fetal PA females, when exposed to testosterone levels typical of those seen in the lower range of normal fetal males, have low circulating bio-LH levels compared with normal female controls at 80 days of gestation (second trimester). In fetal males and PA females alike, such LH suppression may represent testosterone-mediated changes in hypothalamic regulation of LH secretion [62], altered either directly via androgen receptors [63, 64] or indirectly via estrogen receptors, through target tissue aromatization of exogenous testosterone [65, 66].

Such an explanation is further supported by increases in circulating LH levels observed in fetal male, but not in fetal female, monkeys following gonadectomy at 100 days of gestation [59] that are prevented by testosterone replacement at the time of gonadectomy [67]. Corresponding with fetal androgen programming of the hypothalamo-pituitary unit, reduced LH levels in PA female fetuses at 80 days of gestation were followed by a rise in serum bio-LH levels to those of control females by 100 days of gestation (when circulating testosterone levels in PA females decreased to control female values), after which they exceeded control values by 120 days of gestation. The variability of individual serum bio-LH values in both control and PA female fetuses and infants probably reflects the episodic nature of hypothalamic GnRH stimulating episodic release of LH from gonadotropes in the anterior pituitary. The serum LH response of PA females to diminished testosterone exposure closely resembles that exhibited by fetal male rhesus monkeys (at the same gestational age) following gonadectomy at 100 days of gestation [60], although it remains to be determined whether circulating testosterone levels are equivalent between ovary-intact female and gonadectomized male monkeys at these gestational ages.

Persistence of elevated serum LH levels in newborn, ovary-intact PA female infants confirms an earlier finding of LH excess in infant PA female monkeys exposed to a much lower dose of testosterone during early gestation (weekly i.m. injections of 20 mg of testosterone enanthate given to gravid monkeys [43]). Male infant rhesus monkeys also exhibit elevated circulating LH levels compared to those of normal infant females [68, 69]. Because the lower dose of androgen in the previous study [43] also masculinized components of infant female monkey behavior [70] without virilizing external genitalia [43], elevations of fetal female testosterone insufficient to induce genital masculinization nevertheless are sufficient to reprogram sexually dimorphic aspects of hypothalamo-pituitary-gonadal function and behavior. Short-term loss of bio-LH hypersecretion as PA female infants age in both the present and previous [43] studies probably represents the onset of prepubertal quiescence of hypothalamic GnRH release found in immature, gonadally intact or gonadectomized male and female rhesus monkeys [69, 71, 72]. Elevated circulating androstenedione levels in PA female infants probably do not contribute a sufficient additional negative-feedback component to LH suppression [69, 71, 73].

In concert with LH hypersecretion of female fetal PA monkeys, circulating FSH levels also were elevated in the same monkeys at 120 days of gestation. Concomitant LH and FSH excess likely represents enhanced hypothalamic GnRH stimulation of pituitary function, together with an apparent absence of ovarian hormone negative-feedback regulation. Absence of ovarian feedback may represent an immature stage of ovarian development at this gestational age, because such rhesus monkey ovaries have not yet developed gonadotropin responsiveness or the capacity to secrete estradiol [74]. It is interesting to speculate that subsequent LH excess found in adult PA female monkeys, in the absence of excessive levels of FSH [12, 44], may represent differential negative feedback on FSH from ovarian hormones, including inhibins [75].

In addition to gonadotropin excess, hyperandrogenism also was apparent in infant female PA monkeys. It is possible that elevated androstenedione levels in these young infants represent ovarian responses to LH excess, because congenital LH excess can induce prepubertal ovarian hyperandrogenism in humans [76] that may be conditional on sufficient FSH stimulation of the ovary [77, 78]. Alternatively, adult PA female monkeys exhibit adrenal androgen excess [45], and adrenarche occurs around the time of birth in rhesus monkeys coincident with fetal zone regression [79] well before the conventional prepubertal timing found in great apes and humans [79, 80]. Therefore, the elevated androstenedione levels in PA female infants may indicate an adrenal and/or ovarian endocrine antecedent of adult hyperandrogenism, particularly because the adrenal gland normally contributes to elevated circulating androgen levels in infant male rhesus monkeys [73].

Apart from transient increases in circulating androgen and conjugated estrogen levels during TP injections, little impact on maternal reproductive hormonal profiles was observed during the affected pregnancies. Circulating levels of progesterone and estradiol were normal in TP-injected dams and comparable to values across gestation reported previously in rhesus monkeys [51, 81]. Not surprisingly, bio-LH or bio-CG levels were low to undetectable in the circulation of all pregnant females in the current study, both because CGB mRNA expression is undetectable in rhesus placentae and cultured rhesus syncytiotrophoblast cells by 28 days of gestation [82] and because rhesus placentae secrete little or no bio-CG following this stage of gestation [51–53]. The increased levels of conjugated estrogens in the maternal circulation during the time interval of TP injections, however, may indicate alterations in estrogen bio-inactivation and excretion induced by an androgen excess [48, 49]. The biological relevance of diminished elevation in circulating estrone levels in late-gestation, TP-injected dams after TP injections is unclear, because estradiol is the predominant, more bioactive, placental estrogen in rhesus monkeys [51]. It is unlikely that diminished estrone levels alone in TP-injected mothers reflect diminished placental function [83], because the body weights of PA infants on the day of delivery were similar to those of controls. Furthermore, we observed no obvious fetal growth restriction in PA monkey infants, confirming previous reports [43, 84], unlike nonprimate PA females that have low birth weight indicative of fetal growth restriction (e.g., rats [85] and sheep [86]).

Taken together, our experimental findings provide evidence for the developmental progression of bio-LH hypersecretion with endogenous hyperandrogenism in female monkey fetuses or infants exposed to exogenous androgen excess during early gestation, which resembles that found in adult female monkeys, similarly exposed hyperandrogenic women [1, 27], and many women with PCOS [6]. This further suggests that genetically or environmentally determined differentiation of ovarian function during fetal life may contribute to ovarian hyperandrogenism during mid to late gestation [4], because ovarian steroidogenesis (e.g., rhesus monkeys [74] and humans [87]) and steroid receptors (e.g., humans [88]) exist at this time, when the external genitalia are less responsive to androgen action (e.g., rhesus monkeys: [89] and humans [90]). Such ovarian hyperandrogenism may, in turn, reprogram the fetal hypothalamo-pituitary-gonadal axis to promote exaggerated pulsatile LH—and, presumably, GnRH—release with puberty in a manner that resembles that of PCOS and other hyperandrogenic conditions.

ACKNOWLEDGMENTS

We thank J.M. Turk, S.J. Muller, J.R. Lange, and Assay Services of the WNPRC for assistance with endocrine determinations; RJ Singh, Ph.D., of the Endocrine Laboratory, Mayo Foundation and Clinic (Rochester, MN, USA), for assistance with LCMS development and confirmation of LCMS values at the WNPRC; members of the animal care staff at the CNPRC for expert technical assistance; and R.A. Becker and J.L. Peterson at the WNPRC for assistance with preparation of the figures and manuscript, respectively.

FOOTNOTES

1Supported by National Institutes of Health grants P50 HD044405, U01 HD044650, P51 RR000167 (Wisconsin National Primate Research Center [WNPRC] base operating grant), and RR00169 (California National Primate Research Center base operating grant) and partly conducted at a facility (WNPRC) constructed with support from Research Facilities Improvement Program grants RR15459-01 and RR020141-01. Back

Correspondence: 2David H. Abbott, Department of Obstetrics and Gynecology and Wisconsin National Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison, WI 53715. FAX: 608 263 3524; e-mail: abbott{at}primate.wisc.edu

Received: 21 January 2008.

First decision: 5 February 2008.

Accepted: 17 March 2008.

REFERENCES

  1. Abbott DH, Barnett DK, Bruns CM, Dumesic DA. Androgen excess fetal programming of female reproduction: a developmental etiology for polycystic ovary syndrome. Hum Reprod Update 2005; 11:357–374.[Abstract/Free Full Text]
  2. Abbott DH, Dumesic DA, Eisner JR, Colman RJ, Kemnitz JW. Insights into the development of PCOS from studies of prenatally androgenized female rhesus monkeys. Trends Endocrinol Metab 1998; 9:62–67.[CrossRef][Medline]
  3. Eisner JR, Barnett MA, Dumesic DA, Abbott DH. Ovarian hyperandrogenism in adult female rhesus monkeys exposed to prenatal androgen excess. Fertil Steril 2002; 77:167–172.[CrossRef][Medline]
  4. Abbott DH, Dumesic DA, Franks S. Developmental origin of polycystic ovary syndrome—a hypothesis. J Endocrinol 2002; 174:1–5.[Abstract]
  5. Zawadzki JA, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In: Dunaif A, Givens JR, Haseltine FP, Merriam GR (eds.), Polycystic Ovary Syndrome. Boston: Blackwell Scientific 1992:377–384.
  6. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004; 19:41–47.[Abstract/Free Full Text]
  7. Azziz R, Carmina E, Dewailly D, Diamanti-Kandarakis E, Escobar-Morreale HF, Futterweit W, Janssen OE, Legro RS, Norman RJ, Taylor AE, Witchel SF. Position statement: criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen Excess Society guideline. J Clin Endocrinol Metab 2006; 91:4237–4245.[Abstract/Free Full Text]
  8. Franks S. Diagnosis of polycystic ovarian syndrome: in defense of the Rotterdam criteria. J Clin Endocrinol Metab 2006; 91:786–789.[Abstract/Free Full Text]
  9. Ehrmann DA, Liljenquist DR, Kasza K, Azziz R, Legro RS, Ghazzi MN. PCOS/Troglitazone Study Group. Prevalence and predictors of the metabolic syndrome in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2006; 91:48–53.[Abstract/Free Full Text]
  10. Goodarzi MO, Azziz R. Diagnosis, epidemiology, and genetics of the polycystic ovary syndrome. Best Pract Res Clin Endocrinol Metab 2006; 20:193–205.[CrossRef][Medline]
  11. Dunaif A. Insulin resistance in women with polycystic ovary syndrome. Fertil Steril 2006; 86(suppl 1)S13–S14.[Medline]
  12. Dumesic DA, Abbott DH, Eisner JR, Goy RW. Prenatal exposure of female rhesus monkeys to testosterone propionate increases serum luteinizing hormone levels in adulthood. Fertil Steril 1997; 67:155–163.[CrossRef][Medline]
  13. Barnes RB, Rosenfield RL, Burstein S, Ehrmann DA. Pituitary-ovarian responses to nafarelin testing in the polycystic ovary syndrome. N Engl J Med 1989; 320:559–565.[Abstract]
  14. Christman GM, Randolph JF, Kelch RP, Marshall JC. Reduction of gonadotropin-releasing hormone pulse frequency is associated with subsequent selective follicle-stimulating hormone secretion in women with polycystic ovarian disease. J Clin Endocrinol Metab 1991; 72:1278–1285.[Abstract/Free Full Text]
  15. Steiner RA, Clifton DK, Spies HG, Resko JA. Sexual differentiation and feedback control of luteinizing hormone secretion in the rhesus monkey. Biol Reprod 1976; 15:206–212.[Abstract]
  16. Dumesic DA, Schramm RD, Abbott DH. Early origins of polycystic ovary syndrome. Reprod Fertil Dev 2005; 17:349–360.[CrossRef][Medline]
  17. Daniels TL, Berga SL. Resistance of gonadotropin releasing hormone drive to sex steroid-induced suppression in hyperandrogenic anovulation. J Clin Endocrinol Metab 1997; 82:4179–4183.[Abstract/Free Full Text]
  18. Eagleson CA, Gingrich MB, Pastor CL, Arora TK, Burt CM, Evans WS, Marshall JC. Polycystic ovarian syndrome: evidence that flutamide restores sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone. J Clin Endocrinol Metab 2000; 85:4047–4052.[Abstract/Free Full Text]
  19. McCartney CR, Eagleson CA, Marshall JC. Regulation of gonadotropin secretion: implications for polycystic ovary syndrome. Semin Reprod Med 2002; 20:317–326.[CrossRef][Medline]
  20. Abbott DH, Dumesic DA, Eisner JR, Kemnitz JW, Goy RW. The prenatally androgenized female rhesus monkey as a model for polycystic ovarian syndrome. In: Azziz R, Nesterl JE, Dewailly D (eds.), Androgen Excess Disorders in Women. Philadelphia: Lippencott-Raven Publishers 1997:369–382.
  21. Franks S, Mason H, Willis D. Follicular dynamics in the polycystic ovary syndrome. Mol Cell Endocrinol 2000; 163:49–52.[CrossRef][Medline]
  22. Blank SK, McCartney CR, Marshall JC. The origins and sequelae of abnormal neuroendocrine function in polycystic ovary syndrome. Hum Reprod Update 2006; 12:351–361.[Abstract/Free Full Text]
  23. Escobar ME, Ropelato MG, Ballerini MG, Gryngarten MG, Garcia Rudaz MC, Veldhuis JD, Barontini M. Acceleration of luteinizing hormone pulse frequency in adolescent girls with a history of central precocious puberty with versus without hyperandrogenism. Horm Res 2007; 68:278–285.[CrossRef][Medline]
  24. Barnes RB, Rosenfield RL, Ehrmann DA, Cara JF, Cuttler L, Levitsky LL, Rosenthal IM. Ovarian hyperandrogynism as a result of congenital adrenal virilizing disorders: evidence for perinatal masculinization of neuroendocrine function in women. J Clin Endocrinol Metab 1994; 79:1328–1333.[Abstract]
  25. Xita N, Tsatsoulis A. Review: fetal programming of polycystic ovary syndrome by androgen excess: evidence from experimental, clinical, and genetic association studies. J Clin Endocrinol Metab 2006; 91:1660–1666.[Abstract/Free Full Text]
  26. Sarma HN, Manikkam M, Herkimer C, Dell'Orco J, Welch KB, Foster DL, Padmanabhan V. Fetal programming: excess prenatal testosterone reduces postnatal luteinizing hormone, but not follicle-stimulating hormone responsiveness, to estradiol negative feedback in the female. Endocrinology 2005; 146:4281–4291.[CrossRef][Medline]
  27. Rosenfield RL. Clinical review: identifying children at risk for polycystic ovary syndrome. J Clin Endocrinol Metab 2007; 92:787–796.[Abstract/Free Full Text]
  28. Zhou R, Bruns CM, Bird IM, Kemnitz JW, Goodfriend TL, Dumesic DA, Abbott DH. Pioglitazone improves insulin action and normalizes menstrual cycles in a majority of prenatally androgenized female rhesus monkeys. Reprod Toxicol 2007; 23:438–448.[CrossRef][Medline]
  29. Tarantal AF. Ultrasound imaging in rhesus (Macaca mulatta) and long-tailed (Macaca fascicularis) macaques: reproductive and research applications. In: Wolfe-Coote S (ed.), The Laboratory Primate. San Diego: Elsevier Academic Press 2005:317–352.
  30. Tarantal AF, Gargosky SE. Characterization of the insulin-like growth factor (IGF) axis in the serum of maternal and fetal macaques (Macaca mulatta and Macaca fascicularis). Growth Regul 1995; 5:190–198.[Medline]
  31. Jimenez DF, Tarantal AF. Fetal gender determination in early first trimester pregnancies of rhesus monkeys (Macaca mulatta) by fluorescent PCR analysis of maternal serum. J Med Primatol 2005; 32:315–319.[CrossRef]
  32. Resko JA, Buhl AE, Phoenix CH. Treatment of pregnant rhesus macaques with testosterone propionate: observations on its fate in the fetus. Biol Reprod 1987; 37:1185–1191.[Abstract]
  33. Diamanti-Kandarakis E, Kouli CR, Bergiele AT, Filandra FA, Tsianateli TC, Spina GG, Zapanti ED, Bartzis MI. A survey of the polycystic ovary syndrome in the Greek island of Lesbos: hormonal and metabolic profile. J Clin Endocrinol Metab 1999; 84:4006–4011.[Abstract/Free Full Text]
  34. Asunción M, Calvo RM, San Millán JL, Sancho J, Avila S, Escobar-Morreale HF. A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain. J Clin Endocrinol Metab 2000; 85:2434–2438.[Abstract/Free Full Text]
  35. Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004; 89:2745–2749.[Abstract/Free Full Text]
  36. Beck-Peccoz P, Padmanabhan V, Baggiani AM, Cortelazzi D, Buscaglia M, Medri G, Marconi AM, Pardi G, Beitins IZ. Maturation of hypothalamic-pituitary-gonadal function in normal human fetuses: circulating levels of gonadotropins, their common alpha-subunit and free testosterone, and discrepancy between immunological and biological activities of circulating follicle-stimulating hormone. J Clin Endocrinol Metab 1991; 73:525–532.[Abstract/Free Full Text]
  37. Nakanishi T. The problem of species comparison of developmental toxicity: can we extrapolate human developmental toxicity induced by environmental chemicals from the data on rodents. Yakugaku Zasshi 2007; 127:491–500.[CrossRef][Medline]
  38. France JT, Mason JI, Magness RR, Murry BA, Rosenfeld CR. Ovine placental aromatase: studies of activity levels, kinetic characteristics, and effects of aromatase inhibitors. J Steroid Biochem 1987; 28:155–160.[Medline]
  39. Tarantal AF. Interventional ultrasound in pregnant macaques: embryonic/fetal applications. J Med Primatol 1990; 19:47–58.[Medline]
  40. Norman RL, Spies HG. Effect of luteinizing hormone-releasing hormone on the pituitary-gonadal axis in fetal and infant rhesus monkeys. Endocrinology 1979; 10:655–659.[CrossRef]
  41. Huhtaniemi IT, Korenbrot CC, Seron-Ferre M, Foster DB, Parer JT, Jaffe RB. Stimulation of testosterone production in vivo and in vitro in the male rhesus monkey fetus in late gestation. Endocrinology 1977; 100:839–844.[Abstract/Free Full Text]
  42. Monroe SE, Yamamoto M, Jaffe RB. Changes in gonadotrope responsivity to gonadotropin releasing hormone during development of the rhesus monkey. Biol Reprod 1983; 29:422–431.[Abstract]
  43. Herman RA, Jones B, Mann DR, Wallen K. Timing of prenatal androgen exposure: anatomical and endocrine effects on juvenile male and female rhesus monkeys. Horm Behav 2000; 38:52–66.[CrossRef][Medline]
  44. Dumesic DA, Schramm RD, Peterson E, Paprocki AM, Zhou R, Abbott DH. Impaired developmental competence of oocytes in adult prenatally androgenized female rhesus monkeys undergoing gonadotropin stimulation for in vitro fertilization. J Clin Endocrinol Metab 2002; 87:1111–1119.[Abstract/Free Full Text]
  45. Zhou R, Bird IM, Dumesic DA, Abbott DH. Adrenal hyperandrogenism is induced by fetal androgen excess in a rhesus monkey model of polycystic ovary syndrome. J Clin Endocrinol Metab 2005; 90:6630–6637.[Abstract/Free Full Text]
  46. Wang C, Catlin DH, Demers LM, Starcevic B, Swerdloff RS. Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab 2004; 89:534–543.[Abstract/Free Full Text]
  47. Nelson RE, Grebe SK, O'Kane DJ, Singh RJ. Liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma. Clin Chem 2004; 50:373–384.[Abstract/Free Full Text]
  48. Barbier O, Belanger A. The cynomolgus monkey (Macaca fascicularis) is the best animal model for the study of steroid glucuronidation. J Steroid Biochem Mol Biol 2003; 85:235–245.[CrossRef][Medline]
  49. Leung KC, Lee YT, Fung KP, Yu WN, Yu CC. beta-Glucuronidase and estrogens in hydatidiform mole. Clin Chim Act 1976; 72:295–300.[CrossRef]
  50. Ziegler TE, Scheffler G, Wittwer DJ, Schultz-Darken N, Snowdon CT, Abbott DH. Metabolism of reproductive steroids during the ovarian cycle in two species of callitrichids, Saguinus oedipus and Callithrix jacchus, and estimation of the ovulatory period from fecal steroids. Biol Reprod 1996; 54:91–99.[Abstract]
  51. Atkinson LE, Hotchkiss J, Fritz GR, Surve AH, Neill JD, Knobil E. Circulating levels of steroids and chorionic gonadotropin during pregnancy in the rhesus monkey, with special attention to the rescue of the corpus luteum in early pregnancy. Biol Reprod 1975; 12:335–345.[Abstract]
  52. Hodgen GD. Primate chorionic gonadotropins: their comparative biological, immunologic and chemical properties. In: Nathanielsz PW (ed.), Fetal Endocrinology. New York: Academic Press 1981:95–110.
  53. Hobson BM, Wide L. The similarity of chorionic gonadotrophin and its subunits in term placentae from man, apes, old and New World monkeys and a prosimian. Folia Primatol (Basel) 1981; 35:51–64.[CrossRef][Medline]
  54. Sokal RR, Rohlf FJ. Biometry: The Principles and Practice of Statistics in Biological Research, 3rd ed. New York: W. H. Freeman and Co. 1995.
  55. Iglewicz B, Hoaglin DC. How to detect and handle outliers. ASQC basic references in quality control Vol 16, Milwaukee, WI: American Society for Quality Control 1993.
  56. Baird DT, Corker CS, Davidson DW, Hunter WM, Michie EA, Van Look PF. Pituitary-ovarian relationships in polycystic ovary syndrome. J Clin Endocrinol Metab 1977; 45:798–801.[Abstract/Free Full Text]
  57. Rosenfield RL, Barnes RB, Ehrmann DA. Studies of the nature of 17-hydroxyprogesterone hyperresponsiveness to gonadotropin-releasing hormone agonist challenge in functional ovarian hyperandrogenism. J Clin Endocrinol Metab 1994; 79:1686–1692.[Abstract]
  58. Gilling-Smith C, Story H, Rogers V, Franks S. Evidence for a primary abnormality of thecal cell steroidogenesis in the polycystic ovary syndrome. Clin Endocrinol (Oxf ) 1997; 47:93–99.[CrossRef][Medline]
  59. Ellinwood WE, Baughman WL, Resko JA. The effects of gonadectomy and testosterone treatment on luteinizing hormone secretion in fetal rhesus monkeys. Endocrinology 1982; 110:183–189.[Abstract/Free Full Text]
  60. Resko JA, Ellinwood WE. Sexual differentiation of the brain of primates. In: Serio M, Motta M, Zanisis M, Martini L (eds.), Sexual Differentiation: Basic and Clinical Aspects. New York: Raven Press 1984:169–181.
  61. Ellinwood WE, Resko JA. Sex differences in biologically active and immunoreactive gonadotropins in the fetal circulation of rhesus monkeys. Endocrinology 1980; 107:902–907.[Abstract/Free Full Text]
  62. Plant TM, Dubey AK. Evidence from the rhesus monkey (Macaca mulatta) for the view that negative feedback control of luteinizing hormone secretion by the testis is mediated by a deceleration of hypothalamic gonadotropin-releasing hormone pulse frequency. Endocrinology 1984; 115:2145–2153.[Abstract/Free Full Text]
  63. Abbott DH, Batty KA, Dubey AK, Herbert J, Shiers HM. The passage of 5alpha-dihydrotestosterone from serum into cerebrospinal fluid and LH negative feedback in castrated rhesus monkeys. J Endocrinol 1985; 104:325–330.[Abstract/Free Full Text]
  64. Scott CJ, Clarke IJ, Rao A, Tilbrook AJ. Sex differences in the distribution and abundance of androgen receptor mRNA-containing cells in the preoptic area and hypothalamus of the ram and ewe. J Neuroendocrinol 2004; 16:956–963.[CrossRef][Medline]
  65. Resko JA, Roselli CE. Prenatal hormones organize sex differences of the neuroendocrine reproductive system: observations on guinea pigs and nonhuman primates. Cell Mol Neurobiol 1997; 17:627–648.[CrossRef][Medline]
  66. Resko JA, Pereyra-Martinez AC, Stadelman HL, Roselli CE. Cellular observations and hormonal correlates of feedback control of luteinizing hormone secretion by testosterone in long-term castrated male rhesus monkeys. Biol Reprod 2000; 63:872–878.[Abstract/Free Full Text]
  67. Resko JA, Ellinwood WE. Negative feedback regulation of gonadotropin secretion by androgens in fetal rhesus macaques. Biol Reprod 1985; 33:346–352.[Abstract]
  68. Mann DR, Davis-DaSilva M, Wallen K, Coan P, Evans DE, Collins DC. Blockade of neonatal activation of the pituitary-testicular axis with continuous administration of a gonadotropin-releasing hormone agonist in male rhesus monkeys. J Clin Endocrinol Metab 1984; 59:207–211.[Abstract/Free Full Text]
  69. Plant TM. The effects of neonatal orchidectomy on the developmental pattern of gonadotropin secretion in the male rhesus monkey (Macaca mulatta). Endocrinology 1980; 106:1451–1454.[Abstract/Free Full Text]
  70. Tomaszycki ML, Davis JE, Gouzoules H, Wallen K. Sex differences in infant rhesus macaque separation-rejection vocalizations and effects of prenatal androgens. Horm Behav 2001; 39:267–276.[CrossRef][Medline]
  71. Plant TM. A striking sex difference in the gonadotropin response to gonadectomy during infantile development in the rhesus monkey (Macaca mulatta). Endocrinology 1986; 119:539–545.[Abstract/Free Full Text]
  72. Terasawa E, Bridson WE, Nass TE, Noonan JJ, Dierschke DJ. Developmental changes in the luteinizing hormone secretory pattern in peripubertal female rhesus monkeys: comparisons between gonadally intact and ovariectomized animals. Endocrinology 1984; 115:2233–2240.[Abstract/Free Full Text]
  73. Plant TM, Zorub DS. A study of the role of the adrenal glands in the initiation of the hiatus in gonadotropin secretion during prepubertal development in the male rhesus monkey (Macaca mulatta). Endocrinology 1984; 114:560–565.[Abstract/Free Full Text]
  74. Ellinwood WE, McClellan MC, Brenner RM, Resko JA. Estradiol synthesis by fetal monkey ovaries correlates with antral follicle formation. Biol Reprod 1983; 28:505–516.[Abstract]
  75. Abbott DH, Eisner JR, Groome NP, Dumesic DA, McNeilly AS. Decreased serum levels of inhibin A and inhibin B in anovulatory prenatally androgenized female rhesus monkeys. J Endocrinol 1997; 152(suppl)P258.
  76. Rosenfeld RG, Reitz RE, King AB, Hintz RL. 1980 Familial precocious puberty associated with isolated elevation of luteinizing hormone. N Engl J Med 303;859–862.
  77. Barnes RB, Namnoum AB, Rosenfield RL, Layman LC. The role of LH and FSH in ovarian androgen secretion and ovarian follicular development: clinical studies in a patient with isolated FSH deficiency and multicystic ovaries. Hum Reprod 2002; 17:88–91.[Abstract/Free Full Text]
  78. Latronico AC, Lins TS, Brito VN, Arnhold IJ, Mendonca BB. The effect of distinct activating mutations of the luteinizing hormone receptor gene on the pituitary-gonadal axis in both sexes. Clin Endocrinol (Oxf) 2000; 53:609–613.[CrossRef][Medline]
  79. Conley AJ, Pattison JC, Bird IM. Variations in adrenal androgen production among (nonhuman) primates. Semin Reprod Med 2004; 22:311–326.[CrossRef][Medline]
  80. Cutler GB Jr, Glenn M, Bush M, Hodgen GD, Graham CE, Loriaux DL. Adrenarche: a survey of rodents, domestic animals, and primates. Endocrinology 1978; 103:2112–2118.[Abstract/Free Full Text]
  81. Stanczyk FZ, Hess DL, Namkung PC, Senner JW, Petra PH, Novy MJ. Alterations in sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques. Biol Reprod 1986; 35:126–132.[Abstract]
  82. Golos TG, Handrow RR, Durning M, Fisher JM, Rilling JK. Regulation of chorionic gonadotropin alpha and chorionic somatomammotropin messenger ribonucleic acid expression by 8-bromo-adenosine 3',5'-monophosphate and dexamethasone in cultured rhesus monkey syncytiotrophoblasts. Endocrinology 1992; 131:89–100.[Abstract/Free Full Text]
  83. Albrecht ED, Pepe GJ. Central integrative role of estrogen in modulating the communication between the placenta and fetus that results in primate fecal-placental development. Placenta 1999; 20:129–139.[CrossRef][Medline]
  84. Abbott DH, Bruns CM, Barnett DK, Dumesic DA. Fetal programming of polycystic ovary syndrome. In: Kovacs WG, Norman RL (eds.), Polycystic Ovary Syndrome, 2nd ed. Cambridge, UK: Cambridge University Press 2006:262–287.
  85. Slob AK, den Hamer R, Woutersen PJ, van der Werff ten Bosch JJ. Prenatal testosterone propionate and postnatal ovarian activity in the rat. Acta Endocrinol (Copenh) 1983; 103:420–427.[Abstract/Free Full Text]
  86. Manikkam M, Crespi EJ, Doop DD, Herkimer C, Lee JS, Yu S, Brown MB, Foster DL, Padmanabhan V. Fetal programming: prenatal testosterone excess leads to fetal growth retardation and postnatal catch-up growth in sheep. Endocrinology 2004; 145:790–798.[Abstract/Free Full Text]
  87. Cole B, Hensinger K, Maciel GA, Chang RJ, Erickson GF. Human fetal ovary development involves the spatiotemporal expression of P450c17 protein. J Clin Endocrinol Metab 2006; 91:3654–3661.[Abstract/Free Full Text]
  88. Wilson CM, McPhaul MJ. A and B forms of the androgen receptor are expressed in a variety of human tissues. Mol Cell Endocrinol 1996; 120:51–57.[CrossRef][Medline]
  89. Goy RW, Bercovitch FB, McBrair MC. Behavioral masculinization is independent of genital masculinization in prenatally androgenized female rhesus macaques. Horm Behav 1988; 22:552–571.[CrossRef][Medline]
  90. Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS. Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 1995; 16:271–321.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
E. A. M. Kuijper, J. M. Vink, C. B. Lambalk, and D. I. Boomsma
Prevalence of Polycystic Ovary Syndrome in Women from Opposite-Sex Twin Pairs
J. Clin. Endocrinol. Metab., June 1, 2009; 94(6): 1987 - 1990.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
79/1/154    most recent
biolreprod.108.067702v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Abbott, D. H
Right arrow Articles by Tarantal, A. F
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Abbott, D. H
Right arrow Articles by Tarantal, A. F
Agricola
Right arrow Articles by Abbott, D. H
Right arrow Articles by Tarantal, A. F

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS