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Wisconsin Regional Primate Research Center, Department of Obstetrics and Gynecology, University of Wisconsin, Madison, Wisconsin 53715-1299; and the Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Daniel A. Dumesic, M.D., Division of Reproductive Endocrinology, Department of OB/GYN, Mayo Clinic, Rochester, Minnesota 55905. E-mail: . ddumesic{at}mayo.edu
Abstract
To determine whether prenatal T propionate exposure beginning gestational d 40–44 (early-treated) or 100–115 (late-treated) affects oocyte competence, five early-treated and five late-treated prenatally androgenized and five normal monkeys underwent recombinant human FSH injections with oocyte-retrieval after hCG administration. Serum FSH, LH, estradiol (E2), progesterone (P4), androstenedione (A4), T, and dihydrotestosterone were measured basally, during gonadotropin stimulation and at oocyte-retrieval; fasting serum glucose and insulin also were determined basally and at oocyte-retrieval. Follicle fluid sex steroids were analyzed. Oocyte number, nuclear maturity, and fertilization were comparable among female groups, but the percentage of zygotes developing into blastocysts was reduced in early-treated prenatally androgenized females. The intrafollicular P4/E2 ratio was significantly elevated in early-treated prenatally androgenized females, whereas intrafollicular P4/A4 and T/A4 ratios were significantly increased in all prenatally androgenized females. Early-treated prenatally androgenized females demonstrated persistent LH hypersecretion. They also were unable to suppress circulating insulin levels during gonadotropin stimulation. Circulating sex steroid levels and serum P4/E2, P4/A4, and E2/androgen ratios were similar in all females. Early prenatal androgenization in monkeys receiving gonadotropins impairs oocyte developmental competence and seems to induce premature follicle differentiation in the presence of LH hypersecretion and relative insulin excess.
IMPLANTATION FAILURE AND pregnancy loss after in vitro fertilization (IVF) are common features of polycystic ovary syndrome (PCOS), a reproductive disorder characterized by LH hypersecretion, hyperandrogenism, and insulin resistance. The high miscarriage rate in PCOS women undergoing transfer of morphologically normal embryos into the uterus suggests either impaired developmental competence of oocytes, abnormal endometrial receptivity, or both (1). Unfortunately, any attempts to investigate the mechanisms governing oocyte developmental competence in these women are limited, because of ethical and experimental constraints on the use of human embryos for biomedical research.
Instead, the effects of PCOS-like endocrinopathies on the meiotic and developmental competence of oocytes might be assessable using a previously established nonhuman primate model for PCOS. In female rhesus monkeys exposed prenatally to T propionate (TP), delayed menarche is accompanied by luteal deficiency in adolescence (2). LH hypersecretion and hyperandrogenism in adulthood are associated with anovulation, and multifollicular ovaries are present in 50% of anovulatory prenatally androgenized females (2, 3). Pancreatic ß-cell function also is impaired when prenatal exposure to TP begins on d 40 post conception but not on d 100–115 post conception (4). Perhaps most importantly, however, fecundity has occurred in only 20% of these females, whereas all normal female monkeys have given birth to at least one offspring (5).
In other studies, normal adult female rhesus monkeys received gonadotropin therapy for IVF to investigate the meiotic and developmental competence of oocytes, defined as the ability of oocytes to complete meiosis and undergo fertilization, embryogenesis, and term development (6). In this nonhuman primate model, oocyte competence is associated with a periovulatory shift in steroidogenesis from estrogen and androgen to progesterone (P4) production (7).
To assess the preimplantation development of oocytes cultured in vitro, the present study investigated whether prenatally androgenized adult female rhesus monkeys have impaired meiotic or developmental competence of oocytes after ovarian stimulation for IVF and, if so, whether these impairments are associated with abnormal ovarian steroidogenesis or insulin action. Our results demonstrate that early prenatal androgenization in monkeys undergoing ovarian stimulation for IVF impairs oocyte developmental competence and seems to induce premature follicle differentiation in the presence of LH hypersecretion and relative insulin excess.
Materials and Methods
Experimental animals
The general care and housing of rhesus monkeys (Macaca mulatta) at the Wisconsin Regional Primate Research Center (WRPRC) have been described previously (8, 9). WRPRC is fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care as part of the University of Wisconsin Graduate School. Animal protocols and experiments were approved by the Graduate School Animal Care and Use Committee of the University of Wisconsin, Madison. The animals were maintained according to recommendations of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act (with its subsequent amendments). All animals were studied between September and May to avoid seasonal effects on menstrual cyclicity (10, 11).
The study consisted of 15 sexually mature female rhesus monkeys raised at the WRPRC. The control group consisted of 5 normal adult females. The study group comprised 10 prenatally androgenized females exposed in utero to TP. A detailed description of study design and methodology has been reported previously (8).
Briefly, prenatally androgenized females were produced by injecting (10–15 mg TP for 15–35 d) pregnant rhesus monkeys carrying female fetuses. TP was initiated on either d 40–44 (early-treated, n = 5) or d 100–115 (late-treated, n = 5) post conception. Dams of prenatally androgenized and normal females were similar in age [early-treated, 10.5 ± 1.4; late-treated, 5.4 ± 0.5; normal females, 10.5 ± 2.2 yr (mean ± SEM), P = 0.06] and parity [medians: early-treated, 1 (range, 0–2); late-treated, 1 (range, 0–1); normal females, 1 (range, 0–2), P = 0.8]. Dams of late-treated prenatally androgenized females, however, weighed less than those of normal females [late-treated, 5.6 ± 0.5; normal females, 7.6 ± 0.6 kg (mean ± SEM), P < 0.025] but were similar in weight, compared with early-treated prenatally androgenized females (6.9 ± 0.4 kg, P = 0.09). This weight difference probably occurred because dams of late-treated prenatally androgenized females tended to be younger than dams of the normal females. Sires of prenatally androgenized and normal females were similar in age (early-treated, 15.4 ± 4.1; late-treated, 9.5 ± 2.2; normal females, 10.8 ± 2.5 yr, P = 0.4) and number of offspring [medians: early-treated, 5 (range, 0–11); late-treated, 4 (range, 0–9); normal females, 4 (range, 1–12), P = 0.8]. Sires of all female types also were similar in weight (early-treated, 9.9 ± 1.1; late-treated, 9.6 ± 1.1; normal females, 9.8 ± 0.6 kg, P = 0.9).
The physical and psychosexual consequences of prenatal androgen exposure in female rhesus monkeys have been characterized (12). Prenatal TP treatment, starting before d 60 post conception, induced external genital masculinization and obliteration of the external vaginal orifice. Female offspring exposed to TP beginning after d 110 post conception showed no genital virilization except for clitoromegally. All prenatally androgenized animals exhibited masculine behavior, which occurred independently of genital masculinization in late-treated prenatally androgenized females (12).
Prenatally androgenized and normal females were similar in age (early-treated, 19.9 ± 0.5; late-treated, 17.1 ± 0.9; normal females, 17.8 ± 1.2 yr) and weight (early-treated, 9.5 ± 0.6; late-treated, 8.9 ± 0.7; normal females, 9.0 ± 1.0 kg). No study animals were obese (13). Thirteen animals had ovulatory menstrual cycles, based on two serum P4 levels above 1 ng/ml within 15 d of menses (8), whereas two prenatally androgenized females (one early-treated; one late-treated) were anovulatory.
Experimental design
Gonadotropin stimulation for IVF. Each female received twice-daily im injections of 30 IU recombinant human (rh)FSH for 7–8 d, beginning on d 1–3 of the menstrual cycle (d 1 = the first day of menses) (14, 15) or beginning during a period of anovulation. Blood samples (5 ml) were drawn from the saphenous vein on d 1, 2, 4, and 6 during rhFSH treatment to quantify changes in circulating levels of LH, FSH, estradiol (E2), P4, androstenedione (A4), T, and dihydrotestosterone (DHT). Ovarian follicular sizes were measured on the last day of rhFSH treatment, by transabdominal ultrasonography (7.5-MHz linear probe; EUB-410 scanner; Hitachi Scientific Instruments, Inc., Tarrytown, NY) of sedated (im-administered 10 mg/kg Ketamine HCl) monkeys. One day later, rhCG (1000 IU, im) was administered to induce oocyte maturation, and laparoscopic oocyte retrieval was performed 27 h after rhCG (14, 15). A blood sample (5 ml) was withdrawn immediately before anesthesia, for laparoscopy, to quantify circulating endocrine values on the day of oocyte collection. Blood samples before rhFSH treatment and on the day of oocyte retrieval were taken at 0800–0900 h, after an overnight fast, and used to determine circulating glucose and insulin levels. No animal experienced a spontaneous LH surge.
Laparoscopic ovarian retrieval. All large follicles (5–7 mm) on each ovary were aspirated individually into separate collection tubes with 200 µl protein-free TL-HEPES medium containing 0.1 mg/ml polyvinyl alcohol. Oocytes from each of these large follicles were cultured separately in individual culture drops so that their meiotic and developmental competence could be directly compared with concentrations of hormones in their individual FF samples. The remaining follicles that were more than 2 mm in diameter were aspirated into several collection tubes and pooled. Aspirates uncontaminated by blood [but containing diluted follicular fluid (FF)] were centrifuged, and supernatants were then frozen at -20 C. Each aspirate of FF was assayed for E2, P4, A4, and T; and the values were corrected for total protein concentration of the aspirate to quantitatively reflect the volume of FF present.
IVF and embryo culture. Ooctyes were retrieved from aspirates and were placed into culture within 1 h of collection. Oocytes were cultured for 4–10 h post aspiration in 25-µl drops of modified Conwaught Medical Research Laboratories medium containing 20% bovine calf serum under 3.5 ml of mineral oil in a humidified atmosphere of 5% CO2 in air (16). Oocytes were examined for nuclear maturation every 2 h and were inseminated approximately 2–4 h after extrusion of the first polar body. Metaphase II oocytes possessed one polar body in the perivitelline space and no visible nuclear structure in the cytoplasm. Metaphase I oocytes displayed no polar body in the perivitelline space and no visible nuclear structure in the cytoplasm. Prophase I oocytes displayed no polar body in the perivitelline space and a germinal vesicle in the cytoplasm.
All semen samples were collected by penile electroejaculation from two male monkeys that previously have sired offspring. Spermatozoa capacitation and IVF were achieved as described previously (17). Briefly, 20 x 106 washed motile spermatozoa were resuspended in 2 ml modified Tyrode’s solution with albumin, lactate and pyruvate medium overlaid with 2 ml mineral oil, and incubated at 37 C in 5% CO2 in air for 2–10 h. Spermatozoa were diluted into 100-µl drops (2 x 105/ml) of modified Tyrode’s solution with albumin, lactate and pyruvate medium containing 2% bovine calf serum and 1.0 mM each of caffeine and dibutyryl cAMP to induce hyperactivation (18). Spermatozoa were coincubated with mature oocytes for 12–16 h at 37 C in a humidified atmosphere of 5% CO2 in air. Spermatozoa and remaining cumulus cells were then removed manually by pipetting through a pulled glass pipette, and oocytes were examined for evidence of fertilization. All diploid (two pronuclei) zygotes were cultured in G1/G2 medium (19) in 5% CO2-5% O2-90% N2, at 37 C, for up to 11 d, and placed into fresh culture media every other day, as described previously (14, 15). Embryos were examined daily using Nomarski optics (x200–400 magnification) on an Eclipse TE300 inverted microscope with a heated (37 C) environmental control chamber (Nikon, Tokyo, Japan) (17). After developmental arrest or zona escape, embryos were fixed in 1% formalin and stained with Hoechst 33342 for the determination of the nucleated cell number, by fluorescent microscopy (20).
Hormone assays
FSH, E2, A4, and insulin were measured by RIA in the WRPRC Hormone Assay Services Laboratory as previously described (3). The intraassay coefficients of variation (CVs) were: FSH, 4.0%; E2, 3.4%; A4, 8.6%; and insulin, 3.0%. The interassay CVs were: FSH, 4.6%; E2, 9.8%; A4, 14.4%; and insulin, 5.2%. Bioactive LH was measured by the mouse Leydig cell bioassay using the rhLH-RP1 reference preparation. The intra- and interassay CVs for LH were 5.9% and 9.7%, respectively. P4, T, and DHT were measured by an enzyme immunoassay. The intraassay CVs were: P4, 2.8%; T, 3.4%; and DHT, 3.2%. The interassay CVs were: P4, 12.1%; T, 7.5%; and DHT, 19.3%. Glucose was measured by the glucose oxidase method. The intra- and interassay CVs for glucose were 2.9% and 4.0%, respectively.
Statistical analysis
Log transformation of the hormonal data and arcsine transformation of the oocyte/embryo proportional data were performed to achieve homogeneity of variance and to increase linearity (21). Variables were compared by two-way ANOVA using prenatal androgen exposure and either IVF cycle phase or diploid zygote developmental stage as factors to determine the independent effects of these variables and their possible interaction. When significant statistical interactions were present by ANOVA, post hoc univariate analysis was performed on the variables (Systat, Version 5.2, 1992; Macintosh, Evanston, IL). The effects of prenatal androgen exposure on oocyte number/development and follicle hormone concentration were compared by one-way ANOVA. All hormonal and oocyte/embryo proportional data are expressed as the back transformed means and 95% confidence intervals.
Linear regression analysis was used to determine the correlation between serum LH levels and percentages of zygotes developing to blastocysts. Linear regression analysis also was used to examine the correlation in normal and late-treated prenatally androgenized females between intrafollicular P4/E2 ratios and E2 levels in follicles associated with blastocyst development. The slopes of the regression lines for these two female groups were tested for homogeneity, using the Systat software package.
Results
Oocyte maturation, fertilization, and embryo development
The mean numbers of total oocytes recovered from early-treated [14.6 (2.9–26.3)] and late-treated [21.8 (10.1–33.4)] prenatally androgenized females were similar to that recovered from normal females [17.8 (6.1–29.4), P = 0.7]. The proportion of oocytes completing meiotic maturation [metaphase II: early-treated, 54.6 (37.2–72.0); late-treated prenatally androgenized, 54.9 (37.5–72.3); normal females, 59.1% (41.7–76.5%), P = 0.9] and the incidence of fertilization [early-treated, 64.7 (38.6–90.7); late-treated prenatally androgenized, 77.3 (51.2–100.0); normal females, 59.9% (33.8–86.0%), P = 0.6] also were comparable among the female groups.
A lower proportion of zygotes developed into blastocysts in early-treated (compared with late-treated) prenatally androgenized (P < 0.05) and normal females (P < 0.05; female type-embryo development interaction effect, P < 0.005) (Fig. 1
). When examined by female type, normal females demonstrated a similar proportion of zygotes developing to the 2- to 4-cell (91%), 5- to 8-cell (91%), 9- to 16-cell (88%), morula (68%), and blastocyst (62%) stages. In late-treated prenatally androgenized females, however, less zygotes formed blastocysts than 2- to 4-cell stage (P < 0.01) or 5- to 8-cell stage embryos (P < 0.05), because of a reduction in the proportion of zygotes that developed from the 2- to 4-cell stage (100%) to the 5- to 8-cell (83%), 9- to 16-cell (79%), morula (77%), and blastocyst (41%) stages. In early-treated prenatally androgenized females, fewer zygotes formed morula and blastocysts than 2- to 4-cell or 5- to 8-cell stage embryos (morula, P < 0.05; blastocyst, P < 0.005, both stages) because the proportion of zygotes advancing to the next stage of development decreased from the 2- to 4-cell (100%), 5- to 8-cell (97%), and 9- to 16-cell (59%) stages to the morula (32%) and blastocyst (4%) stages.
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There were no significant effects of IVF cycle phase (P = 0.6) or interactions between female type and IVF cycle phase (P = 0.9) on serum LH. Before and during rhFSH treatment combined, serum LH levels were significantly greater in early-treated prenatally androgenized females than in late-treated prenatally androgenized females (P < 0.001) or normal females (P < 0.001), whereas serum LH levels also were lower in late-treated prenatally androgenized females than in normal females (P < 0.05) (Fig. 2A). Serum LH levels after rhCG were similar in all female groups (female type effect, P = 0.16). Of nine prenatally androgenized (four early-treated, five late-treated) and three normal females producing zygotes, there was a significant negative correlation between serum LH levels before rhFSH treatment and the percentage of zygotes developing into blastocysts (r2 = 0.42; P < 0.025).
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). Therefore, data for endogenous serum FSH from all female types were pooled for IVF cycle analysis. Serum FSH levels rose slightly on d 2 of rhFSH treatment and then declined to basal values on d 4 (P < 0.005, vs. d 2) and d 6 (P < 0.01, vs. d 2) of rhFSH treatment.
There also were no significant effects of female type or interactions between female type and IVF cycle phase on serum E2, P4, A4, T, and DHT levels. Serum E2 levels rose significantly and progressively during rhFSH treatment in all female types combined (P < 0.001, d 1 vs. d 2, d 2 vs. d 4; P < 0.05, d 4 vs. d 6 of rhFSH treatment) and increased further at 27 h after hCG administration (P < 0.05, d 6 of rhFSH treatment) (Fig. 3A). Serum P4 levels remained at basal values throughout rhFSH treatment but increased significantly by 27 h after hCG (P < 0.001, d 6 of rhFSH treatment) (Fig. 3B). Similarly, serum A4, T, and DHT levels were unchanged during rhFSH treatment, and they increased significantly above d-6 rhFSH treatment values by 27 h after hCG (A4 and DHT, P < 0.005; T, P < 0.001) (Figs. 3, C–E).
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). In all female groups combined, serum P4/E2 ratios significantly decreased to lowest levels on d 4 of rhFSH treatment (P < 0.001, d 1 vs. d 2; P < 0.01, d 2 vs. d 4 of rhFSH treatment), after which they remained suppressed for 2 d before significantly increasing at 27 h after hCG administration (P < 0.001, d 6 of rhFSH treatment). Serum P4/A4 ratios decreased below basal values by d 2 of rhFSH treatment (P < 0.05) and remained low throughout ovarian stimulation, before significantly increasing at 27 h after hCG administration (P < 0.001, d 6 of rhFSH treatment). In contrast, serum E2/A4 and E2/T ratios rose progressively to highest levels on d 6 of rhFSH treatment (P < 0.01, d 1 vs. d 2, d 2 vs. d 4; P < 0.05, d 4 vs. d 6 of rhFSH treatment, both ratios) and were unchanged at 27 h after hCG.
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Follicular fluid hormone levels
Intrafollicular E2 levels were lower in early- than in late-treated prenatally androgenized females (P < 0.005) or in normal females (P < 0.05) and were similar in the latter two female groups (P = 0.8) (Fig. 4A). Intrafollicular P4 concentrations tended to be approximately 45% greater in early-treated and late-treated prenatally androgenized females than in normal controls (P = 0.3, Fig. 4B). Consequently, intrafollicular P4/E2 ratios were higher in early- than in late-treated prenatally androgenized females (P < 0.001) or in normal females (P < 0.001), though being similar in the latter two female groups (P = 0.2, Table 2).
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). In contrast, intrafollicular T concentrations were comparable among prenatally androgenized females and normal females (early-treated, P = 0.6; late-treated, P = 0.1) but were significantly greater in late-treated than in early-treated prenatally androgenized females (P < 0.025; Fig. 4D). Compared with normal females, the FF P4/A4 and T/A4 ratios were significantly increased in early-treated (P4/A4, P < 0.001; T/A4, P < 0.05) and late-treated prenatally androgenized females (P4/A4, P < 0.05; T/A4, P < 0.005, Table 2). On the other hand, the ratios of E2/A4 and E2/T in FF were similar in all female groups (E2/A4, P = 0.2; E2/T, P = 0.6). Intrafollicular DHT concentrations and the FF DHT/T ratio also were comparable in early-treated and late-treated prenatally androgenized females, as well as in normal females (DHT, P = 0.7; DHT/T, P = 0.3) (Fig. 4E, Table 2).
Significant negative correlations between the intrafollicular P4/E2 ratio and the E2 concentration existed in normal (r2 = 0.97; P < 0.025) and late-treated prenatally androgenized females (r2 = 0.50; P < 0.05). The slope of the linear regression line for the intrafollicular P4/E2 ratio vs. E2 concentration in normal females (Y = -11.8x + 75.7) differed significantly from that in late-treated prenatally androgenized females (Y = -1.7x + 42.5, P < 0.004; Fig. 5). Controlling for FF E2 concentration, the FF P4/E2 ratio in follicles associated with blastocyst development was higher in late-treated prenatally androgenized than in normal females. The FF P4/E2 ratio also was elevated in two comparable follicles of early-treated prenatally androgenized females, despite blood contamination in the aspirates.
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Previous studies of adult female rhesus monkeys have demonstrated long-term effects of prenatal androgenization on reproduction. Prenatal exposure of female rhesus monkeys to androgen induces luteal deficiency at puberty, followed by anovulation in adulthood, with ovarian dysfunction characterized by multifollicular ovaries in one-half of anovulatory prenatally androgenized females (2). Prenatal androgen exposure also causes LH hypersecretion in adult females, with loss of hypothalamic sensitivity to estrogen-negative feedback (3, 23). In addition, only 20% of prenatally androgenized females (whereas all normal females) have given birth to at least one offspring, implicating prenatal androgen exposure in reduced fecundity (5). The results of the present study demonstrate that prenatal exposure of adult female rhesus monkeys to TP, beginning gestational d 40–44, impairs oocyte developmental competence, as determined by the percentage of zygotes developing into blastocysts.
Most of the serum hormones measured in the present study were unable to predict the impaired oocyte developmental competence observed in early-treated prenatally androgenized females. Similar serum FSH levels and circulating sex steroid concentrations in the three female groups demonstrated the typical periovulatory shift in steroidogenesis after hCG administration from estrogen and androgen to P4 production (7). This hCG-induced shift in ovarian steroidogenesis from estrogen and androgen to P4 production agrees with the observation that hCG administration to similarly-treated normal female monkeys increases the relative expression of granulosa cell 3ß-hydroxysteroid dehydrogenase (3ß-HSD), compared with ovarian cytochrome P450 17
-hydroxylase/17–20 lyase (P450 c17) (24). Prenatal androgenization seems to exaggerate this phenomenon, as evidenced by the elevated intrafollicular P4/A4 ratio observed in all prenatally androgenized females and the reduced intrafollicular A4, but not P4, level found in early-treated prenatally androgenized females (25). Such 3ß-HSD overexpression in the follicles of prenatally androgenized females parallels the up-regulation of thecal cell 3ß-HSD enzyme activity (26) and overproduction of P4 observed in ovaries of PCOS women before (27, 28, 29), but not necessarily after, gonadotropin stimulation (30, 31, 32).
Hyperandrogenemia previously has been shown in our prenatally androgenized females by frequent blood sampling techniques (2); it was undetected in this study, which used only one of several blood samples for basal androgen determinations. Nevertheless, prenatally androgenized females and PCOS women normally exhibit hyperresponsiveness of serum 17-hydroxyprogesterone to hCG, implicating increased intraovarian P450 c17 activity as a cause for androgen excess (33, 34). Therefore hyperandrogenemia may have been ameliorated in our prenatally androgenized females from both an increase in aromatase activity during gonadotropin stimulation (as determined by serum E2/A4 and E2/T ratios) and an exaggerated shift in steroidogenesis from androgen to P4 production after hCG administration (as measured by the intrafollicular P4/A4 ratio). Nevertheless, the increased intrafollicular conversion of A4 to T observed in all prenatally androgenized females resembles the up-regulation of thecal cell 17ß-HSD enzyme activity previously reported in PCOS ovaries, suggesting that a subtle defect in intraovarian androgen biosynthesis persists in these females during ovarian stimulation (26).
Intrafollicular androgens in late-treated prenatally androgenized females were sufficient in amount to maintain E2 synthesis (7), given normal ovarian aromatase activity in all prenatally androgenized females. On the other hand, the significantly reduced intrafollicular A4 and E2 levels in early-treated prenatally androgenized (compared with the other two) female groups raises the possibility that follicular development was different in the former females. In this regard, an inability to significantly suppress serum insulin levels with rising ovarian estrogen production during gonadotropin stimulation was observed in our early-treated prenatally androgenized females and was similar to the hyperinsulinemia noted in insulin-resistant PCOS women undergoing ovarian stimulation for IVF (35). Serum insulin levels decreased from basal values during gonadotropin stimulation, by 67% and 43% in normal and late-treated prenatally androgenized females, respectively, but were unchanged in early-treated prenatally androgenized females. In addition, LH hypersecretion in early-treated prenatally androgenized females, as previously reported, occurred before gonadotropin treatment and persisted during ovarian stimulation (3). This relative excess of circulating insulin, combined with LH hypersecretion in early-treated prenatally androgenized females, parallels the hormone profile of PCOS women, in which hyperinsulinemia from insulin resistance enhances LH-induced follicle differentiation (27, 28, 29). An interesting posit, therefore, is that premature follicular differentiation occurs in early-treated prenatally androgenized females undergoing gonadotropin stimulation (27, 28, 29, 36), a hypothesis consistent with preferential secretion of P4, compared with E2, in the follicles of early-treated, but not late-treated, prenatally androgenized females.
Although low intrafollicular E2 levels in early-treated prenatally androgenized females were associated with normal oocyte maturation and fertilization, they also were accompanied by a reduced percentage of zygotes developing beyond the 9- to 16-cell developmental stage, at which point they completely depend on the embryonic genome (37). Our finding supports the role of estrogen in regulating oocyte developmental competence, perhaps at the level of embryonic genome activation. It also complements previous nonhuman primate studies in which drastic reduction of E2 production by the use of aromatase or 3ß-HSD inhibitors during gonadotropin stimulation impairs oocyte maturation and fertilization (38, 39). In addition, high FF E2 content in women undergoing IVF is positively correlated with oocyte fertilization, cleavage, and implantation (40, 41, 42), whereas low E2 production in IVF patients with 17-hydroxylase deficiency is associated with in vitro embryonic developmental arrest and inability to conceive after embryo transfer (43, 44). Preferential secretion of E2, compared with T, by human oocyte-corona-cumulus complexes also is a feature of fertilized oocytes that cleave and lead to pregnancy (45). These collective observations are supported by the detection of estrogen receptor , P450 c17, and 3ß-HSD mRNA expression in human oocytes (46, 47, 48).
LH hypersecretion also may have preferentially impaired oocyte developmental competence in early-treated prenatally androgenized females, as evidenced by the significant negative correlation between serum LH levels before gonadotropin treatment and the percentage of zygotes developing to blastocysts. LH hypersecretion in women undergoing gonadotropin stimulation for IVF also has been linked with low oocyte fertilization and poor embryonic development (49, 50), whereas LH suppression by pituitary desensitization in some, but not all, PCOS women undergoing gonadotropin stimulation improves pregnancy outcome (51, 52). Moreover, LH hypersecretion with a relative excess of circulating insulin may further impair oocyte quality, because obesity in PCOS women predisposes to miscarriage (53), with postprandial hyperinsulinemia associated with low oocyte fertilization and failure of embryos to implant in the uterus of such individuals or their surrogates (54). Consistent with this hypothesis, in vitro cell culture studies demonstrate that coincubation of insulin and FSH with murine oocyte-cumulus cell complexes accelerates granulosa cell LH receptor mRNA expression and reduces the percentage of fertilized oocytes developing into blastocysts (55).
Even in late-treated prenatally androgenized females without LH hypersecretion, however, excess serum insulin levels, as determined by lowered serum glucose/insulin ratios during gonadotropin stimulation, were accompanied by overproduction of P4, relative to E2, in follicles associated with blastocyst development. Because a similar endocrine abnormality in women undergoing IVF is associated with reduced pregnancy outcome (56, 57), circulating insulin excess in prenatally androgenized females may lead to subtle impairments of postimplantation (rather than preimplantation) embryo development. It is well established that acquisition of the blastocyst stage of development is not an absolute indication of complete developmental normality; and therefore, postimplantation development failure of morphologically normal blastocysts may be analogous to the high miscarriage rate of PCOS women (1).
The present study demonstrates that early prenatal androgenization in monkeys undergoing rhFSH administration for IVF impairs embryo development beyond the time of genome activation. In these female monkeys, it is plausible that LH hypersecretion with hyperinsulinemia up-regulates LH receptors on cumulus and/or mural granulosa cells, causing their premature cytodifferentiation. The consequence of these cellular events, and of the resulting abnormal intrafollicular steroidogenesis, is poor embryo development, perhaps from impaired acquisition of maternally-derived proteins and/or transcripts that are important for embryonic gene activation. It remains to be determined whether prenatal androgenization impairs oocyte developmental competence during intrauterine life or after birth, during either oocyte development or maturation. Future studies on genetic expression in oocytes and embryos may reveal the underlying molecular impairments induced by prenatal androgenization.
Acknowledgments
We thank the following personnel at the WRPRC: D. Wade, S. Maves, S. L. Knowles, M. Shotsko, and M. Brown for assistance with animal procedures; S. G. Eisele and the animal care staff of the WRPRC for maintenance of the animals and computerized records; I. Bolton (D.V.M.) and B. D. Florence (D.V.M.) for veterinary care; and F. W. Wegner, D. J. Wittwer, S. T. Baum, and S. Jacoris for assistance with hormone assays and glucose determinations. We also thank Rebekah R. Herrmann for preparation of the manuscript.
Footnotes
This work was supported by NIH Grants RR-14093, RR-13635, and RR-00167. This is publication number 41-013 of the WRPRC.
Abbreviations: A4, Androstenedione; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; CV, coefficient of variation; DHT, dihydrotestosterone; E2, estradiol; FF, follicular fluid; IVF, in vitro fertilization; PCOS, polycystic ovary syndrome; P4, progesterone; P450 c17, P450 17-hydroxylase/17–20 lyase; rhFSH, recombinant human FSH; TP, T propionate; WRPRC, Wisconsin Regional Primate Research Center.
Received August 2, 2001.
Accepted November 19, 2001.
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  D. H Abbott, D. K Barnett, J. E Levine, V. Padmanabhan, D. A Dumesic, S. Jacoris, and A. F Tarantal Endocrine Antecedents of Polycystic Ovary Syndrome in Fetal and Infant Prenatally Androgenized Female Rhesus Monkeys Biol Reprod, July 1, 2008; 79(1): 154 - 163. [Abstract] [Full Text] [PDF]
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A. Veiga-Lopez, W. Ye, D.J. Phillips, C. Herkimer, P.G. Knight, and V. Padmanabhan Developmental Programming: Deficits in Reproductive Hormone Dynamics and Ovulatory Outcomes in Prenatal, Testosterone-Treated Sheep Biol Reprod, April 1, 2008; 78(4): 636 - 647. [Abstract] [Full Text] [PDF]
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N. Acevedo, J. Ding, and G. D Smith Insulin Signaling in Mouse Oocytes Biol Reprod, November 1, 2007; 77(5): 872 - 879. [Abstract] [Full Text] [PDF]
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 D. A. Dumesic, T. G. Lesnick, and D. H. Abbott Increased Adiposity Enhances Intrafollicular Estradiol Levels in Normoandrogenic Ovulatory Women Receiving Gonadotropin-Releasing Hormone Analog/Recombinant Human Follicle-Stimulating Hormone Therapy for in Vitro Fertilization J. Clin. Endocrinol. Metab., April 1, 2007; 92(4): 1438 - 1441. [Abstract] [Full Text] [PDF]
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 J. Smitz, A.N. Andersen, P. Devroey, J.-C. Arce, and for the MERIT Group Endocrine profile in serum and follicular fluid differs after ovarian stimulation with HP-hMG or recombinant FSH in IVF patients Hum. Reprod., March 1, 2007; 22(3): 676 - 687. [Abstract] [Full Text] [PDF]
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S. C. Foong, D. H. Abbott, M. A. Zschunke, T. G. Lesnick, J. L. Phy, and D. A. Dumesic Follicle Luteinization in Hyperandrogenic Follicles of Polycystic Ovary Syndrome Patients Undergoing Gonadotropin Therapy for in Vitro Fertilization J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2327 - 2333. [Abstract] [Full Text] [PDF]
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 M. Manikkam, T. L. Steckler, K. B. Welch, E. K. Inskeep, and V. Padmanabhan Fetal Programming: Prenatal Testosterone Treatment Leads to Follicular Persistence/Luteal Defects; Partial Restoration of Ovarian Function by Cyclic Progesterone Treatment Endocrinology, April 1, 2006; 147(4): 1997 - 2007. [Abstract] [Full Text] [PDF]
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R. Zhou, I. M. Bird, D. A. Dumesic, and D. H. Abbott Adrenal Hyperandrogenism Is Induced by Fetal Androgen Excess in a Rhesus Monkey Model of Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6630 - 6637. [Abstract] [Full Text] [PDF]
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 D.H. Abbott, D.K. Barnett, C.M. Bruns, and D.A. Dumesic Androgen excess fetal programming of female reproduction: a developmental aetiology for polycystic ovary syndrome? Hum. Reprod. Update, July 1, 2005; 11(4): 357 - 374. [Abstract] [Full Text] [PDF]
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J. L. Zehr, P. E. Van Meter, and K. Wallen Factors Regulating the Timing of Puberty Onset in Female Rhesus Monkeys (Macaca mulatta): Role of Prenatal Androgens, Social Rank, and Adolescent Body Weight Biol Reprod, May 1, 2005; 72(5): 1087 - 1094. [Abstract] [Full Text] [PDF]
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M. Savabieasfahani, J. S. Lee, C. Herkimer, T. P. Sharma, D. L. Foster, and V. Padmanabhan Fetal Programming: Testosterone Exposure of the Female Sheep During Midgestation Disrupts the Dynamics of Its Adult Gonadotropin Secretion During the Periovulatory Period Biol Reprod, January 1, 2005; 72(1): 221 - 229. [Abstract] [Full Text] [PDF]
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J. L. Phy, C. A. Conover, D. H. Abbott, M. A. Zschunke, D. L. Walker, D. R. Session, I. S. Tummon, A. R. Thornhill, T. G. Lesnick, and D. A. Dumesic Insulin and Messenger Ribonucleic Acid Expression of Insulin Receptor Isoforms in Ovarian Follicles from Nonhirsute Ovulatory Women and Polycystic Ovary Syndrome Patients J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3561 - 3566. [Abstract] [Full Text] [PDF]
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 T. Waterhouse, S.-L. Cox, M. Snow, G. Jenkin, and J. Shaw Offspring produced from heterotopic ovarian allografts in male and female recipient mice Reproduction, June 1, 2004; 127(6): 689 - 694. [Abstract] [Full Text] [PDF]
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S. Jonard and D. Dewailly The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest Hum. Reprod. Update, March 1, 2004; 10(2): 107 - 117. [Abstract] [Full Text] [PDF]
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, early-treated prenatally androgenized females (n = 5); 
, late-treated prenatally androgenized females (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.005 vs. 2–4 cell stage. ¶, P < 0.05; ¶¶, P < 0.005 vs. 5–8 cell stage. 
, P < 0.05 vs. the other female groups.
, Normal females (n = 5); 
, early-treated prenatally androgenized females (n = 5); 