Developmental Programming: Impact of Excess Prenatal ... - CiteSeerX

BIOLOGY OF REPRODUCTION 84, 87–96 (2011) Published online before print 25 August 2010. DOI 10.1095/biolreprod.110.086686

Developmental Programming: Impact of Excess Prenatal Testosterone on Intrauterine Fetal Endocrine Milieu and Growth in Sheep1 Almudena Veiga-Lopez,4,5 Teresa L. Steckler,3,4,5 David H. Abbott,9 Kathleen B. Welch,8 Puliyur S. MohanKumar,10 David J. Phillips,12 Kent Refsal,11 and Vasantha Padmanabhan2,5,6,7 Departments of Pediatrics,5 Obstetrics and Gynecology,6 Reproductive Sciences Program,7 and Center for Statistical Consultation and Research,8 University of Michigan, Ann Arbor, Michigan Department of Obstetrics and Gynecology and Wisconsin National Primate Research Center,9 University of Wisconsin, Madison, Wisconsin Department of Pathobiology and Diagnostic Investigation,10 and Diagnostic Center for Population and Animal Health,11 College of Veterinary Medicine, Michigan State University, East Lansing, Michigan Monash Institute of Medical Research,12 Monash University, Melbourne, Victoria, Australia reproductive and metabolic pathology in prenatal testosteronetreated sheep.

ABSTRACT Prenatal testosterone excess in sheep leads to reproductive and metabolic disruptions that mimic those seen in women with polycystic ovary syndrome. Comparison of prenatal testosterone-treated sheep with prenatal dihydrotestosteronetreated sheep suggests facilitation of defects by androgenic as well as androgen-independent effects of testosterone. We hypothesized that the disruptive impact of prenatal testosterone on adult pathology may partially depend on its conversion to estrogen and consequent changes in maternal and fetal endocrine environments. Pregnant Suffolk sheep were administered either cottonseed oil (control) or testosterone propionate in cottonseed oil (100 mg, i.m. twice weekly), from Day 30 to Day 90 of gestation (term is ;147 d). Maternal (uterine) and fetal (umbilical) arterial samples were collected at Days 64–66, 87–90, and 139–140 (range; referred to as D65, D90, and D140, respectively) of gestation. Concentrations of gonadal and metabolic hormones, as well as differentiation factors, were measured using liquid chromatography/mass spectrometer, radioimmunoassay, or ELISA. Findings indicate that testosterone treatment produced maternal and fetal testosterone levels comparable to adult males and D65 control male fetuses, respectively. Testosterone treatment increased fetal estradiol and estrone levels during the treatment period in both sexes, supportive of placental aromatization of testosterone. These steroidal changes were followed by a reduction in maternal estradiol levels at term, a reduction in activin A availability, and induction of intrauterine growth restriction in D140 female fetuses. Overall, our findings provide the first direct evidence in support of the potential for both androgenic as well as estrogenic contribution in the development of adult

activin, androgens, developmental biology, estrogens, PCOS

The maternal environment in which a fetus develops has a profound effect on its developmental trajectory, impacting not only offspring viability, but also expression of its adult phenotype. Changes in fetal environment, which include poor nutrition, hypoxia, and endocrine imbalance, have been found to alter the normal ontogeny of fetal organ differentiation and growth in several species, culminating in adult dysfunction [1, 2]. Exposure to such adverse conditions during development results in fetal growth retardation, and fetal responses to such insults lead to faulty or altered development of endocrine systems and organs [1, 2]. Steroids play a central integrative role and orchestrate the dialog that occurs between the environment in which a fetus develops and differentiation of organ systems. For instance, studies in sheep found that prenatal exposure to testosterone results in intrauterine growth restriction (IUGR) and low-birth weight female offspring that manifest subsequent catch-up growth, functional hyperandrogenism, hypergonadotropism and neuroendocrine feedback defects, multifollicular ovaries, increased follicular recruitment and persistence, and early reproductive failure, as well as metabolic perturbations, such as insulin resistance [3–6]. The adult reproductive and metabolic phenotype of prenatal testosterone-treated sheep, similar to prenatal testosterone-treated monkeys [3, 7], recapitulates the phenotype seen in women with polycystic ovary syndrome (PCOS) [8–10]. Parallel studies in sheep with prenatal dihydrotestosterone (DHT; nonaromatizable) [11–13] or prenatal exposure to testosterone plus an androgen antagonist (flutamide) [14] have found that some adult reproductive defects may stem from aromatization of testosterone to estrogen. As such, while the adult consequences of exposure to prenatal testosterone excess have been investigated in depth, it remains to be established whether the fetus is exposed to both increased androgens and estrogens following gestational testosterone treatment, and, if so, at what site conversion to estrogen occurs. Furthermore, the reprogramming of fetal organ differentiation and function from exposure to excess testosterone during gestation may be mediated by steroid action at various levels. First, such changes can be induced by altered maternal endocrine milieu and metabolism. Maternal insults have an

1 Supported by National Institutes of Health/U.S. Public Health Service grant P01-HD44232 to V.P. 2Correspondence: Vasantha Padmanabhan, Pediatrics, Obstetrics, and Gynecology, Molecular and Integrative Physiology, and the Reproductive Sciences Program, University of Michigan, 300 N. Ingalls Building, Rm 1138, Ann Arbor, MI 48109-5404. FAX: 734 615 5441; e-mail: [email protected] 3 Current address: Extension and Outreach, University of Illinois, Urbana, IL. 4 These authors contributed equally to this work.

Received: 14 June 2010. First decision: 29 July 2010. Accepted: 20 August 2010. Ó 2011 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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INTRODUCTION

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TABLE 1. Number of control (C) and testosterone (T)-treated dams used at each gestational time point (Day 65, 90, and 140), and the number and gender (F: female; M: male) of fetuses included per treatment group at each gestational time point.a Group Mothers C T Fetuses C C T T

Gender

D65

D90

D140

-

16 (10) 13 (8)

13 (9) 15 (12)

10 (9) 14 (9)

F M F M

8 13 9 9

8 10 8 12

10 6 11 9

(6) (9) (6) (6)

(6) (7) (6) (8)

(6) (4) (7) (7)

Collection of Plasma and Tissues Fetal tissues and maternal and cord samples were collected from control and testosterone-treated dams on Days 64–66, 87–90, and 139–140 (range) of gestation (referred to as D65, D90, and D140, respectively). All dams were sedated with 20–30 ml of pentobarbital i.v. (Nembutol Na solution, 50 mg/ml; Abbott Laboratories, Chicago, IL), and subsequently maintained under general anesthesia (1–2% halothane; Halocarbon Laboratories, River Edge, NJ). The gravid uterus was exposed through a midline incision and the uterine wall incised to obtain blood from the umbilical artery from each fetus. Fetuses were removed for body measures and tissue harvest. D140 fetuses were given an intracardiac barbiturate (2 ml; Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI) prior to tissue harvest. Dams were euthanized with an overdose of Fatal Plus.

a

First number represents sample size for body measures and number in parentheses represents sample size for hormonal measures.

MATERIALS AND METHODS Breeding and Prenatal Treatments All procedures were approved by the University of Michigan Animal Care and Use Committee. Suffolk ewes (2–3 yr old) were purchased locally and group fed a ration consisting of 0.5 kg of shelled corn and 1.0–1.5 kg of alfalfa hay/ewe/day, providing 2.31 Mcal/kg of digestible energy. The day of mating was determined by visual confirmation of paint markings left on the rumps of ewes by the raddled rams. To compensate for increased nutritional demand of the dam during the last 6 wk before the expected lambing date, ewes assigned for Day 140 collection were group fed 0.5 kg shelled corn, 2 kg alfalfa hay/ ewe/day (2.6 Mcal/kg of digestible energy). Both diets meet early- and lategestation nutrient requirements for sheep, as defined by the National Research Council [24]. Aureomycin crumbles (chlortetracycline, 250 mg/ewe/day) were administered to reduce losses due to abortion from diseases, such as Campylobacter and Chlamydia. Breeder animals assigned to generate control and testosterone-treated fetuses were balanced for maternal weight, body score, and animal providers. Details of testosterone treatment have been described previously [21]. Briefly, testosterone treatment involved twice weekly i.m. injections of 100 mg testosterone propionate (Sigma-Aldrich Corp., St. Louis, MO) in cottonseed oil (2 ml volume) from Day 30 to Day 90 of gestation (term, ;147 days). Control ewes received the same volume of vehicle.

The number and gender of fetuses/gestational age from each treatment group are summarized in Table 1. Fetal measures included weight, crown-rump length (from the highest midpoint on the top of the head to the base of the tail), and head circumference. Fetal uteri and gonads (ovaries/testes) were collected and weighed at D90 and D140. The anogenital ratio (the ratio of anourethral to anonavel distances) was determined as described previously [21] to assess the degree of masculanization of the external genitalia.

Hormone Assays Maternal and fetal plasma at all gestational time points were analyzed for circulating concentrations of steroids: dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone, E2, estrone, and cortisol. In addition, concentrations of thyroid hormones (free and total thyroxine [free T4 and total T4, respectively], total 3,5,3 0 triiodothyronine [total T3]), hormones and factors implicated in energy homeostasis (glucose, insulin, leptin, insulin-like growth factor 1 [IGF1]), and differentiation factors (activin A [ActA], immunoreactive inhibin [Ir-Inh], and follistatin [FS]) were measured only in D140 samples, due to insufficient volume of blood being available at other gestational ages. DHEA, A4, testosterone, E2, estrone, and cortisol were assayed in the Assay Services Laboratories of the Wisconsin National Primate Research Center using an Agilent 1100 liquid chromatography (LC)/mass spectrometer (MS) equipped with an electrospray ionization source and Chemstation software version A 10.02. Reference steroid hormones used for generating standard curves were obtained from Steraloids Inc. (Newport, RI). The LC/MS methods were validated using Federal Drug Administration protocols (May 2001) [25]. Serial dilutions of sheep plasma (100–400 ll) yielded DHEA, A4, testosterone, and cortisol values that diluted parallel to LC/MS standards (accuracy, 102%, 99%, 92%, and 103%, respectively). For determination of unconjugated estrogens by LC/MS, E2 and estrone were derivatized with dansyl chloride to improve ionization efficiency and LC/MS sensitivity [25]. Serial dilutions of sheep plasma (50–400 ll) yielded E2 and estrone values that diluted parallel to the LC/MS standards (accuracy, 109% and 96%, respectively). The lower limits of LCMS quantification were: DHEA, 1 ng/ml; A4, 30 pg/ml; testosterone (T), 50 pg/ml; E2 and estrone, 4 pg/ml; and cortisol, 1 ng/ml. The within-day and between-day coefficient of variation (CV) were: DHEA, 5.5% and 19.6%; A4, 10.2% and 17.7%; T, 5.1% and 14.0%; E2, 7.7% and 10.5%; estrone, 8.3% and 7.0%; and cortisol, 6.5% and 10.3%, respectively. Plasma-free and T4 concentrations were measured using validated radioimmunoassays (RIAs) (GammaCoat Free T4 [Two-Step] and GammaCoat M Total T4; DiaSorin, Stillwater, MN). The detection limit, and intra- and inter-assay CVs for free T4 were 3 pmol/L, 12.0%, and 19.0%, and for total T4 were 4 nmol/L, 6.0%, and 9.0%, respectively. Total T3 concentrations were measured with a competitive RIA [26] using a T3 antibody (MP Biomedicals, Orangeburg, NY) and 125I T3 (New England Nuclear, Boston, MA). The detection limit, and inter- and intra-assay CVs for this assay were 0.3 nmol/L, 12.0%, and 11.0%, respectively. Plasma glucose levels were measured by the Glucose Oxidase Method (Pointe Scientific, Inc., Canton, MI). The intra-assay CVs for glucose measures based on three quality control pools measuring 103, 304, and 797 mg/dl were 1.8%, 1.2%, and 1.7%, and the interassay CVs were 1.6%, 1.2%, and 1.8%, respectively. Plasma insulin levels were measured by a RIA (MP Biomedicals). The sensitivity of the insulin assay was 3.0 6 1.0 lU/ L (mean 6 SEM). Mean CVs, based on two quality control pools measuring 30 and 107 lU/L, were 11% and 5% for intra-assay, and 3.5% and 8.1% for interassay, respectively. Plasma leptin was measured using a double-antibody RIA [27]. The sensitivity and the intra-assay CVs based on three quality control pools averaged 0.05 ng/ml, 9.1%, 5.6%, and 4.2%, respectively. Plasma IGFI concentrations were measured using an RIA [21, 28]. Sensitivity, and intra- and


impact on fetal development and adult phenotype [15, 16], and can be gender specific [17, 18]. Second, effects of prenatal testosterone excess may be mediated via altered nutrient transfer, hypoxia, and/or hormone transport at the placental level. Our studies have found that there is an advanced placental differentiation (Astapova and Padmanabhan, unpublished results) (i.e., increased percentage of everted placentomes [19]), suggestive of deficiency in nutrient transfer in testosterone-treated females [20, 21]. The placenta is also the site of conversion of testosterone to estradiol (E2), due to the presence of aromatase [22, 23]. Third, excess testosterone or the aromatized E2 may cross the placenta and directly influence fetal steroidal/metabolic/endocrine milieu, as well as organ growth and differentiation. In order to identify mediators involved in altering fetal developmental trajectory and adult dysfunction, it is essential to gain an understanding of changes in fetal and maternal levels of androgens and estrogens, changes in maternal and fetal endocrine milieu, and the growth trajectory of the fetus following gestational testosterone treatment. The primary goal of this study was therefore to determine if the fetus is actually exposed to increased estrogen in addition to androgen levels following gestational testosterone treatment, as well as to study the impact of gestational testosterone excess on maternal and fetal endocrine milieu and relate it to the growth trajectory of the fetus.

Fetal Measures

PRENATAL STEROID EXCESS DISRUPTS FETAL MILIEU

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FIG. 1. Fetal measures. Mean 6 SEM of fetal weight (FW; kg), crown to rump length (cm), head circumference/FW, and anogenital ratio in female (Females, left) and male (Males, right) fetuses at gestational D65, D90, and D140. Controls (C) are shown as white bars, and testosterone (T) treated as black bars. Asterisks above bars denote significant treatment differences (P , 0.05) within gender and a given developmental time point. #Significant gender differences (P , 0.05) within treatment group and developmental time point; significant differences between gestational time points are depicted by bracketed areas (C, T, both groups). Number of fetuses assessed was as follows: Control females, 8, 8, 10; control males, 13, 10, 6; T females, 9, 8, 11; and T males, 9, 12, 9 (for D65, D90, and D140, respectively).

interassay CVs for the IGF assay were 2.9 ng/ml, 12.0%, and 22.4%, respectively. Plasma ActA was measured using ELISA [29], according to the manufacturer’s instructions (Oxford Bio-Innovations, Oxfordshire, UK), and involved previously described modifications [30]. The detection limit, and mean intra- and interplate CVs were 0.01 ng/ml, 6.9%, and 9.6%, respectively. Ir-Inh was measured by a heterologous RIA [31] using an in-house human recombinant (hr)-inhibin A as standard and hr-inhibin A as tracer. The assay cross-reacts 288% with pro-aC, the prosequence of the inhibin a subunit. Castrate ram plasma with no detectable inhibin A was used as the diluent to eliminate matrix effects. The limit of detection, and the average intra- and interassay CVs, averaged 0.21 ng/ml, 8.1%, and 3.3%, respectively. Total (activin bound plus free) plasma FS concentrations were measured using a discontinuous RIA [32] that uses a dissociating reagent to dissociate the activin-FS complex. The limit of detection, and the average intra- and interassay CVs were 1.8 ng/ml, 9.2%, and 5.3%, respectively.

Statistical Analysis Differences in body and hormonal measures between control and testosterone-treated groups within gestational age and sex were analyzed using analysis of covariance (ANCOVA), after adjusting for the number of fetuses and applying appropriate transformations to account for nonnormality. The ANCOVA was also used to test for differences between gender (males vs. females) within gestational age and treatment group. Two-way ANOVA models adjusting for the number of fetuses were used to assess the effects of models with different two-way interactions for all fetal body measures, steroid measures, and other hormone measures: 1) gender, treatment and the gender by treatment interaction within each gestational age; and 2) gestational age, treatment, and the gestational age-by-treatment interaction within each gender. A three-way ANOVA model was used to analyze the gestational age by treatment by gender interaction and lower-order terms for all steroid measures. The normality assumption for the residuals was checked using residual plots. For all measures, the experimental unit was the mother. Outliers (mean 6 three times the interquartile range [IQR]); where Q1 and Q3 are the first and third quartiles, respectively, and IQR ¼ Q3 Q1) were excluded based on the Tukey criteria [33]. Results are presented as mean 6 SEM. Significance was defined as P , 0.05. All analyses were carried out using SAS for Windows version 9.2 (2002–2008; SAS Institute, Cary, NC).

RESULTS Fetal Characteristics Fetal weight, body measures, and anogenital ratios of control and testosterone groups are shown in Figure 1. Fetal weight increased in control fetuses throughout gestation and up to 10-fold during the last third of the gestation (P , 0.05).


FIG. 2. Mean 6 SEM of fetal reproductive organ weights/fetal weight (FW; kg); ovary/FW and uterus/FW in females, and testis/FW in males at gestational D90 and D140. Controls (C) are shown as white bars, and testosterone (T) treated as black bars. *Significant differences (P , 0.05) between treatment groups within developmental time point; significant differences (P , 0.05) between gestational time points are depicted by bracketed areas (C, T, both groups). Number of fetuses assessed was as follows: Control females, 8, 10; control males, 10, 6; T females, 8, 11; and T males, 12, 9 (for D90 and D140, respectively).

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FIG. 3. Mean 6 SEM concentrations of testosterone (T) (ng/ml), E2 (pg/ml), and estrone (pg/ml) in maternal uterine artery (Maternal, left) and umbilical artery of female (Females, middle), and male fetuses (Males, right) at gestational D65, D90, and D140. Controls (C) are shown as white bars, and testosterone treated as black bars. *Significant treatment differences (P , 0.05) within gender and developmental time point; #significant gender differences (P , 0.05) within treatment group and developmental time point. Significant differences between gestational time points are depicted by bracketed areas (C, T, both groups; T, T only). Number of fetuses assessed was as follows: Control females, 6, 6, 6; control males, 9, 7, 4; T females, 6, 6, 7; and T males, 6, 8, 7 (for D65, D90, and D140, respectively). Number of maternal samples assessed was as follows: C, 10, 9, 9; T, 8, 12, 9 (for D65, D90, and D140, respectively).

females remained constant between the two gestational ages, and was not affected by testosterone treatment. Maternal Steroid Measures Maternal steroid measures are summarized in Figures 3 and 4 (left panels). Maternal testosterone concentrations were below assay sensitivity in control females at all gestational ages. Maternal concentrations of testosterone in testosteronetreated mothers were higher compared with controls at D65 and D90 (P , 0.001), but not D140 of gestation. In both control and testosterone-treated females, maternal concentrations of E2 and estrone increased with advanced gestation (P , 0.05). E2 and estrone concentrations, however, were significantly lower on D140 of gestation in the testosterone-treated mothers compared with controls (P , 0.05). Maternal DHEA concentrations were lower on D140 of gestation compared with D65 and D90 in both control and testosterone-treated mothers (P , 0.05). While maternal A4 concentrations remained constant across gestational ages in control mothers, they were higher in testosterone-treated mothers on D65 of gestation compared with controls (P , 0.05). Maternal cortisol concentrations in both control and testosterone-treated animals followed a similar pattern, with levels at D140 being higher compared with earlier gestational ages (P , 0.05). Gestational testosterone treatment had no effect on maternal cortisol concentrations. Fetal Steroid Measures Fetal steroid concentrations are also summarized in Figures 3 and 4 (middle and right panels for female and male fetuses,


Crown-to-rump size increased, while head circumference-tofetal weight ratio decreased over time in both control males and females (P , 0.01). Testosterone-treated female fetuses followed a similar trajectory with fetal weight increasing only 7-fold during the last trimester (P , 0.05). On gestational D140, testosterone-treated female fetuses had reduced body weight, reduced crown-to-rump length, and increased ratio of head circumference-to-fetal weight compared with controls (P , 0.05). Testosterone-treated females were also smaller compared with their testosterone-treated male counterparts at D140 (P , 0.05). Contrary to our earlier study involving a larger cohort of animals [21], testosterone-treated male fetuses in this study did not differ in weight or body measures relative to control males. As expected, anogenital ratio was diminished in control females compared with control males at all gestational ages (P , 0.001). Anogenital ratio was increased in testosterone-treated female fetuses compared with controls at all three gestational ages (P , 0.001), but was similar to that in control and testosterone-treated males. The gonadal weight-to-fetal weight ratio at D90 and D140 of gestation is summarized in Figure 2. Ovarian weights for a subset of animals have been reported previously [13, 34]. As expected, gonadal (ovaries and testes)-to-fetal weight ratios decreased from D90 to D140 of gestation in control fetuses (P , 0.01). This pattern was also observed in testosterone-treated fetuses (P , 0.01). There were no significant differences in the ratio of ovarian-to-fetal weight between control and testosterone-treated females at D140. In contrast, testosterone-treated male fetuses had lower testes-to-fetal weight ratio compared with controls at D90 (P , 0.05), but not at D140. The ratio of uterine-to-fetal weight in control and testosterone-treated


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FIG. 4. Mean 6 SEM concentrations of DHEA (pg/ml), A4 (pg/ml), and cortisol (pg/ ml) in maternal uterine artery (Maternal, left) and umbilical artery of female (Females, middle), and male (Males, right) fetuses at gestational D65, D90, and D140. Controls (C) are shown as white bars, and testosterone (T) treated as black bars. *Significant treatment differences (P , 0.05) within gender and developmental time point; significant differences between gestational time points are depicted by bracketed areas (C, T: both groups). Number of fetuses assessed was as follows: Control females, 6, 6, 6; control males, 9, 7, 4; T females, 6, 6, 7; and T males, 6, 8, 7 (for D65, D90, and D140, respectively). Number of maternal samples assessed was as follows: C, 10, 9, 9; T, 8, 12, 9 (for D65, D90, and D140, respectively).

evident only in female fetuses (P , 0.01). A three-way (treatment by age by gender) interaction was also found in fetal E2 concentrations. An effect of offspring number was only evident for female fetuses in umbilical measures of estrone at D65. No treatment or gender differences in DHEA concentrations were found at any fetal age. Testosterone treatment did not induce changes in A4 concentrations in female fetuses, but increased A4 concentrations at D140 male fetuses compared with corresponding controls (P , 0.05). Fetal cortisol concentrations increased 7- to 10-fold at D140 compared with earlier gestational ages in both control and testosterone-treated fetuses (P , 0.05). Other Maternal and Fetal Measures (D140 only) The effects of gestational testosterone treatment on other maternal and fetal hormonal measures are summarized in Figure 5 and Table 2. No differences in maternal thyroid hormones (total T3, total T4, and free T4), glucose, insulin, and IGF1 were evident between control and testosterone-treated mothers. Maternal leptin concentrations were lower in the testosterone-treated group compared with controls (P , 0.05), but no differences among the differentiation factors were evident. Total T3 and T4, as well as free T4, were similar between control and testosterone-treated female or male fetuses. Ir-Inh concentrations were lower in control and testosterone-treated female fetuses compared with their male counterparts (P , 0.01). Testosterone treatment decreased ActA concentrations (P , 0.05) and increased FS concentrations (P , 0.05) in female but not male fetuses. ActA concentrations were also


respectively). Fetal testosterone concentrations were close to assay sensitivity in control females at all gestational ages studied. Testosterone concentrations in control male fetuses were higher at D65 compared with later ages (P , 0.01), with levels near detection limit of the assay at gestational D90 and D140. Testosterone concentrations in testosterone-treated female fetuses were higher during the testosterone treatment period (D65 and D90) compared with controls (P , 0.001). Testosterone-treated male fetuses had higher concentrations of testosterone compared with control males at D90 (P , 0.01), but not at D65. Testosterone concentrations differed between control females and their male counterparts (P , 0.05) only on D65. Overall, a treatment-by-age interaction in testosterone was found in male and females fetuses (P , 0.001 and P , 0.01, respectively). Fetal E2 concentrations in controls increased at D140 compared with earlier ages, with E2 concentrations being higher in male than female fetuses at D140 (P , 0.05). Testosterone-treated female fetuses had higher E2 concentrations on D65 and D90 (during testosterone treatment period) relative to controls. Testosterone-treated males had higher E2 concentrations at D65 (P ¼ 0.07, tendency) and D90 (P , 0.05), but lower concentrations at D140 (P , 0.05), compared with their control male counterparts. There was no age effect on estrone concentrations in control male fetuses. Testosterone treatment increased estrone concentrations in D65 and D90 female fetuses relative to controls (P , 0.05). Estrone concentrations were higher only at D65 in testosterone-treated male fetuses compared with control males (P , 0.05). Overall, a treatment-by-age interaction in E2 concentration was found in both male and females fetuses (P , 0.01 and P , 0.001, respectively). Such an interaction in estrone concentrations was

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DISCUSSION Findings from this study demonstrate that maternal testosterone treatment from Days 30–90 of gestation increases fetal exposure, not only to androgens, but also to estrogens, disrupts maternal and fetal endocrine milieu, and results in IUGR at term. The implication of these findings as they relate to the development of adult reproductive and metabolic disruptions is discussed below. Body Growth Measures

FIG. 5. Mean 6 SEM concentrations activin A (ng/ml), immunoreactive (Ir)-inhibin (pg/ml), and follistatin (ng/ml) in maternal uterine artery (Mat, left) and umbilical artery of female (F, middle) and male (M, right) fetuses at gestational D140. Controls are shown as white bars, and testosterone treated as black bars. *Significant differences (P , 0.05) between treatment groups within gender; #significant gender differences (P , 0.05) within treatment group and developmental time point. Number of fetuses assessed was as follows: Control females, 6; control males, 4; T females, 7; and T males, 7 (for D140). Number of maternal samples assessed was as follows: C, 9 and T, 9 (for D140).

Maternal Endocrine Milieu Maternal testosterone concentrations achieved following testosterone treatment are in the range found in adult males [41]. Maternal testosterone levels were reported to be higher in women with PCOS [42]. In contrast, levels of E2 achieved were similar between control and testosterone-treated dams. In the absence of measures of conjugated E2, aromatization of administered testosterone at the maternal level cannot be ruled out. An increase in conjugated E2 in the maternal circulation, supportive of increased aromatization, has been reported in prenatal testosterone-treated monkeys [25]. The progressive increase with advancing pregnancy in maternal E2 in both

lower in testosterone-treated female fetuses compared with their male counterparts (P , 0.05). No differences were found in glucose, insulin, IGF1 and leptin between control and testosterone-treated male or female fetuses. However, an effect of offspring number was evident for female fetuses in umbilical measures of leptin at D140.

TABLE 2. IGF1, glucose, and other hormone concentration measurements in Control (C) and Testosterone-treated (T) maternal uterine artery and umbilical artery of female (F) and male (M) fetuses at Gestational Day 140. Female umbilical arteryz

Maternal uterine artery Measure* Total T3 (nmol/L) Total T4 (nmol/L) Free T4 (pmol/L) Glucose (mg/dl) Insulin (lU/L) IGF1 (pg/ml) Leptin (ng/ml)

C 0.4 30.0 9.0 49.6 6.7 69.6 0.8

6 6 6 6 6 6 6

T 0.09 4.6 0.8 6.5 0.9 6.3 0.04a

0.3 23.3 9.3 52.4 5.5 82.2 0.7

6 6 6 6 6 6 6

C 0.06 4.7 1.5 2.2 0.5 8.2 0.05b

0.5 121.0 88.5 11.7 11.0 72.0 0.68

6 6 6 6 6 6 6

Male umbilical arteryz

T 0.1 12.7 16.4 5.7 2.0 3.7 0.03

0.3 93.0 70.6 5.2 10.5 67.2 0.65

6 6 6 6 6 6 6

C 0.1 8.7 7.6 2.1 1.6 5.0 0.05

0.4 119.0 81.7 5.8 12.8 65.1 0.61

6 6 6 6 6 6 6

T 0.1 2.0 9.2 2.2 1.8 8.2 0.03

0.2 107.9 79.4 7.2 12.3 87.3 0.64

6 6 6 6 6 6 6

0.1 7.9 5.3 1.2 2.0 15.3 0.04

* Total T3 (nmol/L), total 3,5,3 0 triiodothyronine; Total T4 (nmol/L), total thyroxine; free T4 (pmol/L), free-thyroxine. Number of maternal samples: C, 9; T, 9. z Number of fetuses: Female: C, 6; T, 7; Male: C, 4; T, 7. a,b Different letters denote significant differences (P , 0.05) between treatment groups. Note the statistical significance shown is based on adjusted means controlling for litter size, while data presented represents absolute means. All data are mean 6 SEM.


Current findings of IUGR in female fetuses exposed to excess testosterone are consistent with our previous findings [20, 21, 34]. The lack of IUGR in male fetuses in this study, in contrast to previous findings from the larger cohort (18 control and 27 testosterone-treated males) that documented IUGR [21], may relate to the small sample size. Another study using a different testosterone treatment paradigm found no IUGR in males [35]. Differences in growth responses of male and female fetuses, as in other forms of prenatal insult [17, 18], are likely the result of gender-specific susceptibility of growth signaling pathways. Adverse fetal outcomes in human observational studies appear also to be sex specific [36] and involve epigenetic influences [37]. The decreased testes-tofetal weight ratio of the D90 male fetuses exposed to excess testosterone, similar to other prenatally manipulated models [38, 39], suggests that the developing male gonad is extremely sensitive to changes in the steroidal milieu. Differences in testes weight in D90, but not in D140 testosterone-treated fetuses, suggests a developmental delay in progression of testes differentiation and its potential contribution to the disrupted sperm motility and counts found in prenatal testosteronetreated offspring [40].


was not reflected as an increase in E2 in maternal circulation. One possibility is that the aromatized E2 from administered testosterone undergoes rapid conjugation in the maternal circulation. Estrone levels in control fetuses increased from D65 to D90 of gestation, and then remained constant. The sulfoconjugated form of estrone, which can be actively deconjugated by the fetus [60], was not measured in this study. The finding of decreased ActA levels and increased FS levels in female fetuses is consistent with decreased ActA availability [61]; FS is a binding neutralizer of activin activity [62]. Considering that ActA has a role in numerous processes, including growth and immune function [63, 64], reduced activin availability would have an impact in the differentiation of other organs/systems as well. This decrease could also have an effect on follicular proliferation and germ cell survival [65]. Contributory Role of Androgens and Estrogens from Gestational Testosterone Excess The finding that gestational testosterone treatment results in increased fetal exposure to testosterone and E2 provides the fist direct evidence that the developmental trajectory of the fetus is subject to both androgenic and estrogenic modulation. Comparison of adult phenotype of prenatal T, DHT, and T plus androgen antagonist-treated females are consistent with androgenic and estrogenic programming at the neuroendocrine and ovarian levels [4, 5, 11–13]. While androgens are involved in Wolffian duct development and spermatogenesis in the male [66], estrogens can also influence testis weight and spermatogenesis [67]. An increase in unconjugated E2 was not evident in the prenatal testosterone-treated monkey model of PCOS [25], in spite of the similarity in adult reproductive/metabolic disruptions shared between the sheep and monkey PCOS models. Translational Relevance

Fetal Steroid Measures Changes in testosterone concentrations in control male and female fetuses across gestation are consistent with previous findings [53, 54]. Physiologically, in sheep, concentrations of testosterone in male fetuses are elevated from Days 35–70, decline between Days 70 and 90 gestation [53], and remain low and stable until term [54], as in other domestic species [55] and humans [56, 57]. Importantly, the concentration of testosterone achieved in D65 female fetuses is in the physiologic range of control male fetuses. Elevated levels of testosterone in D90 testosteronetreated male and female fetuses, but not in control males, suggest that the fall in testosterone concentrations in control males must have occurred before D90. The changes in testosterone in control males correspond to the timing of testicular differentiation [57]. Conceivably, exposure of male fetuses to higher levels of testosterone during this differentiation period had a negative impact on gonadal ontogeny, as supported by the finding of decreased sperm count and motility in prenatal testosterone-treated male offspring [40]. The finding that E2 levels increase toward term in both control male and female fetuses is in agreement with previous studies [58]. Similar magnitude of E2 increases in both male and female fetuses during testosterone treatment (D65 and D90) points to the placenta, and not the fetal gonad, as the primary site of this aromatization. While aromatase is expressed by the fetal ovary beginning around fetal D35 [59], the fetal contribution to this process is likely to be minimal. Paradoxically, this increase in placental aromatization

Considering that the adult phenotype of prenatal testosterone-treated female sheep recapitulates the reproductive and metabolic phenotype of women with PCOS, namely oligoanovulation, functional hyperandrogenism, LH excess, disrupted neuroendocrine feedback systems, polycystic ovarian morphology, and insulin resistance [3, 4, 7–10], findings from this study have translational relevance relative to the developmental origins of PCOS phenotype. It has been hypothesized that androgen excess early in life may contribute to the manifestation of PCOS phenotype during adulthood [68, 69]. Measurements of testosterone in cord blood [70], amniotic fluid measures [71], hirsutism, ovarian theca-lutein cysts, and thecal cell hyperplasia seen in female stillbirth offspring from diabetic pregnancies [72] provide support in favor of exposure of female fetuses to excess testosterone. Does evidence exist in humans to relate elevated fetal androgen excess to development of PCOS phenotype? The only documentable evidence supportive of this premise comes from prevalence of PCOS in women with classical congenital adrenal hyperplasia and with congenital adrenal virilizing tumors [73, 74]. The few studies that relate offspring outcomes to fetal steroid exposure rely heavily on mixed blood samples [48, 75] collected from umbilical vein [76] at the time of birth, a time point well past the critical period of ovarian differentiation in precocial species [77]. These studies have focused only on maternal androgen levels and failed to include estrogen measures [42, 75]. In one study, maternal testosterone and DHEA levels at 18 wk of gestation were found to be similar in mothers of adolescent daughters manifesting ‘PCOS’


control and prenatal testosterone-treated groups is consistent with placental contribution of E2 [22, 23]; in sheep, more than 85% of the E2 found in maternal circulation at D90 of gestation is of placental origin [43]. In this species, aromatase is expressed throughout gestation, but peaks once around midgestation, and again close to term [23], in contrast to humans and primates, where aromatase is highly expressed throughout gestation [44, 45]. The increase in maternal estrone levels toward term is also consistent with previous studies [46, 47]. The decrease in E2 and estrone in testosterone-treated mothers at D140 of gestation is reflective of compromised placental development, and is supported by the advanced placental differentiation in testosterone-treated females (Astapova and Padmanabhan, unpublished results). Reduced E2 levels in mixed cord blood at term found in offspring of women with PCOS is also supportive of disrupted placental steroidogenesis in those women [48]. The transient increase in circulating A4 found in maternal circulation of testosterone dams at gestational D65 may reflect reduced expression of 17b-hydroxy-steroid dehydrogenases, specifically 17b-HSD-5, which is ubiquitously expressed [49] at the maternal or placental level, leading to precursor (A4) buildup or, alternatively, conversion of administered testosterone to A4. The decrease in maternal DHEA levels at term is likely a placental adjustment to account for the increased estrogen precursors supplied by the fetus. While maternal DHEA is the main source of estrogen precursor to the placenta during early and midgestation [50], 50% of DHEA at term is of fetal adrenal origin in humans [51]. Placental insufficiency may also be the underlying cause for the reduced leptin levels in the testosterone females, since the placenta is also a source of leptin [52]. Considering the role leptin plays in controlling appetite and energy balance, the reduced maternal leptin levels may lead to energy-deficient pregnancies in the testosteronetreated females, thus contributing to the development of IUGR.

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ACKNOWLEDGMENTS We are grateful to Mr. Douglas Doop for providing quality care and maintenance of animals used in this study, and Ms. Olga Astapova, Mr. Jonathan Flak, Ms. Carol Herkimer, Dr. Leslie M. Jackson, Ms. Erica Lavire, Mr. James S Lee, Dr. Mohan Manikkam, Dr. Sheba Mohankumar, Ms. Eila Roberts, and Mr. Michael Zakalic for their participation and help with prenatal testosterone treatments and/or collection and processing of fetal/ maternal samples. We thank Mr. Steve Jacoris in Assay Services of WNPRC for assistance with LC/MS-determined steroid values.

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and ‘non-PCOS’ phenotype [75]. In contrast, another study found higher circulating levels of androgens in maternal circulation [42] and hyperandrogenism and insulin defects in offspring of PCOS women [78]. A recent study found testosterone levels in umbilical veins of PCOS women to be elevated in PCOS pregnancies compared with control pregnancies, with levels in umbilical vein of PCOS women carrying female babies to be in the same range as control women carrying male babies [76]. Assuming that changes in maternal testosterone levels in the human studies actually translate to parallel changes in fetal testosterone, the time points when these measures are undertaken need to be evaluated relative to the fetal reproductive organ differentiation. In sheep, testosterone exposure that includes the onset of gonadal differentiation beginning around D30 results in PCOS phenotype. In humans, primordial follicles are already evident by Week 16 of gestation [79, 80]. If so, Week 18 of gestation, when measures were undertaken in the human study [75], would have missed the critical period of early ovarian differentiation. It is of interest, however, that the fall in maternal and cord blood (male fetuses only) E2 levels seen at term in this study parallel the fall in mixed cord blood E2 levels seen at the time of birth in offspring of PCOS women [48]. A possibility to consider is that this fall in E2 in cord blood of PCOS mothers may be a function of an early increase in negative feedback in the fetus stemming from increased androgens/estrogens during the first two trimesters, accompanied by a reduction in gonadotropins in the fetus, leading to reduced E2 production at term. As such, the jury is still out in proving or disproving the prenatal androgen and/or estrogen hypothesis in the development the PCOS phenotype. In conclusion, findings of increased fetal androgen and estrogen exposure provide the first direct evidence in support of both androgenic and estrogenic programming of the adult PCOS-like reproductive and metabolic phenotype resulting from prenatal testosterone treatment and implicate involvement of activin in the process. These longitudinal studies, encompassing three gestational time points, also stress the importance of measuring both fetal androgen and estrogen levels in delineating the potential steroid mediaries of adult dysfunction in humans. In doing so, it is important to target such measurements to time points of relevance to the developmental trajectory of the organ system being studied.


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