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Reproductive Technology |
Departments of Obstetrics and Gynecology and Internal Medicine,3 Mayo Clinic, Rochester, Minnesota 55905
Wisconsin Primate Research Center 4
and
Department of Obstetrics and Gynecology,5 University of Wisconsin-Madison, Madison, Wisconsin 53715
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxyprogesterone (17 OHP), androstenedione (A4), testosterone, and dihydrotestosterone were measured basally during rhFSH therapy and at oocyte retrieval. Follicle fluid (FF) sex steroids, oocyte fertilization, and embryo development were analyzed. Circulating FSH, E2, 17 OHP, A4, and dihydrotestosterone levels increased similarly in all females. Serum LH levels decreased from basal levels in normal and late-treated prenatally androgenized females but were unchanged in early-treated prenatally androgenized females. Serum P levels at oocyte retrieval were comparable with those before FSH treatment in all females. All prenatally androgenized females showed reduced FF levels of A4 and E2 but not P or dihydrotestosterone. Intrafollicular T concentrations also were significantly lower in late-treated compared with early-treated prenatally androgenized females or normal females. In early-treated prenatally androgenized females, but not the other female groups, intrafollicular A4 and E2 levels were reduced in follicles containing oocytes that failed fertilization or produced zygotes with cleavage arrest before or at the five- to eight-cell embryo stage. Therefore, in monkeys receiving rhFSH therapy alone without human chorionic gonadotropin administration, early prenatal androgenization reduced FF concentrations of E2 and A4 in association with abnormal oocyte development, without having an effect on P, testosterone, or dihydrotestosterone concentrations.
estradiol, follicular development, granulosa cells, in vitro fertilization, insulin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase/17–20 lyase (P450c17) compared with that of granulosa cell 3ß-hydroxysteroid dehydrogenase [3]. The endocrinological dynamics of follicle differentiation are perturbed in prenatally androgenized female rhesus monkeys with a polycystic ovary syndrome (PCOS)-like disorder characterized by LH hypersecretion, hyperandrogenism, and anovulation accompanied by hyperinsulinemia [4–8]. When prenatally androgenized female rhesus monkeys receive FSH therapy for IVF, an exaggerated shift in ovarian steroidogenesis from androstenedione (A4) and estradiol (E2) to P production after human chorionic gonadotropin administration is associated with a decreased percentage of zygotes developing into blastocysts [9]. In such female rhesus monkeys exposed prenatally to testosterone propionate (TP) toward the end of the first third (and including a portion of the second one-third) of gestation, high P:E2 and P:A4 ratios, as well as low A4 and E2 levels, occur in follicles containing mature oocytes that fertilize but develop poorly beyond the eight-cell stage [9]. These endocrine changes within prematurely differentiated follicles of early-treated prenatally androgenized females after FSH therapy with human chorionic gonadotropin administration implicate abnormal signaling between the oocyte and granulosa cells as a cause for impaired embryo development.
Although the previous study establishes that developmental competence of in vivo matured oocytes is impaired in early-treated prenatally androgenized females, it is unclear as to when the endocrine abnormalities occur during folliculogenesis. Therefore, the aim of the present study was to determine whether prenatal androgenization alters intrafollicular steroidogenesis during development of the oocyte before resumption of meiosis. Sex steroid levels were measured in the circulation and follicular fluid (FF) of normal and prenatally androgenized female rhesus monkeys receiving FSH therapy, without human chorionic gonadotropin administration, for IVF. Additionally, fasting serum glucose and insulin levels were measured before and after FSH therapy, because insulin excess in prenatally androgenized female rhesus monkeys receiving FSH therapy appears to alter granulosa cell differentiation [9]. The percentage of zygotes developing into blastocysts also was recorded.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The general care and housing of rhesus monkeys (Macaca mulatta) at the Wisconsin Primate Research Center (WPRC) have been described [10, 11]. The WPRC is fully accredited by Association for the 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 [12, 13].
The study consisted of 14 sexually mature female rhesus monkeys raised at the WPRC. The control group consisted of five normal adult females. The study group consisted of nine prenatally androgenized females exposed in utero to TP. A detailed description of study design and methodology has been reported previously [9, 10].
Briefly, prenatally androgenized females were produced by injecting pregnant rhesus monkeys carrying female fetuses with 10–15 mg TP for 15–35 days. TP was initiated on either Days 40–44 (early-treated, n = 5) or Days 100–115 (late-treated, n = 4) postconception. The physical and psychosexual consequences of prenatal androgen exposure in female rhesus monkeys have been characterized [14]. Prenatal TP treatment starting before Day 60 postconception induces external genital masculinization and obliteration of the external vaginal orifice. Female offspring exposed to TP beginning after Day 110 postconception show no genital virilization except for clitoromegaly. All prenatally androgenized animals exhibit masculine behavior that occurs independently of genital masculinization in late-treated prenatally androgenized females [14].
Prenatally androgenized and normal females were similar in age (early-treated, 21.0 ± 0.4; late-treated, 19.3 ± 1.0; normal females, 19.7 ± 0.9 yr) and weight (early-treated, 8.4 ± 0.4; late-treated, 9.5 ± 0.6; normal females, 9.7 ± 0.9 kg). No study animals were obese [15]. Twelve animals had ovulatory menstrual cycles, on the basis of two serum P levels above 1 ng/ml within 15 days of menses [10], whereas two early-treated prenatally androgenized females were anovulatory.
Study Design
Gonadotropin stimulation for IVF
All females were observed twice daily for vaginal bleeding with swabs as necessary to detect the onset of menses as a sign of the early follicular phase [4]. Each female received twice-daily intramuscular injections of 30 IU recombinant human FSH (rhFSH), beginning on Days 1–3 of the menstrual cycle (Day 1 = the first day of menses [9, 16, 17]) or beginning during a period of anovulation. Recombinant human FSH was administered for 7–9 days until at least 1 follicle measuring 
5 mm in diameter was detected by transabdominal ultrasonography (7.5 MHz convex probe, Aloka SSD-1400 scanner, Wallingford, CT). Blood samples (5 ml) were drawn from the saphenous vein on Days 1, 2, 4, and 6 during rhFSH treatment to quantify changes in circulating levels of FSH, LH, E2, P, 17-hydroxyprogesterone (17 OHP), A4, T, and dihydrotestosterone (DHT). The blood sample obtained on Day 1 (before rhFSH treatment) was used to measure rhesus FSH, whereas blood samples obtained during rhFSH treatment were used to measure human FSH. Ovarian follicular sizes were measured on the last day of rhFSH treatment by transabdominal ultrasonography of sedated (intramuscularly administered 10 mg/kg ketamine HCl) monkeys. Laparoscopic oocyte retrieval was performed 24 h later without administration of human chorionic gonadotropin [9, 16, 17]. 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 0630–0800 h after an overnight fast and were used to determine circulating glucose and insulin levels. No animal experienced a spontaneous LH surge, as determined by serum LH and P measurements.
Laparoscopic ovarian retrieval All dominant follicles (i.e., 5–7 mm in diameter) on each ovary were counted and 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 dominant 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. Aspirates uncontaminated by blood (but containing diluted FF) were collected only from dominant follicles produced after a similar number of rhFSH treatment days in all three female groups (see below). All aspirates were centrifuged, and supernatants were frozen at -20°C. Each aspirate of FF was assayed for E2, P, A4, T, and DHT, and the values were corrected for total protein content according to Coomassie Blue protein-dye binding (Pierce Biotechnology, Rockford, IL) to quantitatively reflect the volume of FF present [18].
IVF and embryo culture Oocytes were retrieved from aspirates and placed into culture within 1 h of collection. Oocytes were cultured for 28 h postaspiration in 25-µl drops of modified CMRL-1066 medium [19] containing 20% bovine calf serum under 3.5 ml of mineral oil in a humidified atmosphere of 5% CO2 in air. Oocytes were examined for evidence of nuclear maturation before insemination and were classified as metaphase II, metaphase I, or prophase I oocytes on the basis of nuclear maturation, as previously described [9].
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 [20]. Briefly, 20 x 106 washed motile spermatozoa were resuspended in 2 ml TALP medium overlaid with 2 ml mineral oil and incubated at 37°C in 5% CO2 in air for 2–6 h. Spermatozoa were diluted into 100-µl drops (2 x 105/ml) of TALP medium containing 1.0 mM each of caffeine and dibutyryl cAMP to induce hyperactivation [21]. Spermatozoa were co-incubated 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 [22] in 5% CO2, 5% O2, and 90% N2 at 37°C for up to 11 days and placed into fresh culture media every other day, as described previously [16, 17]. Embryos were examined daily with Nomarski optics (200–400x magnification) on a Nikon Eclipse TE300 inverted microscope with a heated (37°C) environmental control chamber [20]. 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 [23]. Oocyte developmental competence was classified as normal if the oocyte fertilized and the resulting zygote cleaved beyond the five- to eight-cell embryo stage, at which point the zygote completely depends on the embryonic genome [24]. Oocyte developmental competence was classified as abnormal if the oocyte failed to fertilize or if zygote cleavage arrested before or at the five- to eight-cell embryo stage.
Hormone Assays
Rhesus and human FSH, E2, 17 OHP, A4, and insulin were measured by RIA in the WPRC Hormone Assay Services Laboratory as previously described [4, 9]. Blood samples for all FSH and insulin determinations were batched in a single assay to eliminate interassay variability. The intraassay coefficients of variation (CVs) were rhesus FSH, 1.6%; human FSH, 6.5%; E2, 5.3%; 17 OHP, 4.9%; A4, 1.7%; and insulin, 3.6%. The interassay CVs were 17 OHP, 8.3%; E2, 11.1%; and A4, 3.1%. Bioactive LH was measured in a single assay by the mouse Leydig cell bioassay with the rhLH-RP1 reference preparation. The intraassay CV for LH was 8.1%. P, T, and DHT were measured by enzyme immunoassay. The intraassay CVs were P, 6.2%; T, 2.0%; and DHT, 2.6%. The interassay CVs were P, 8.7%; T, 8.2%; and DHT, 7.1%. Glucose was measured in a single assay by the glucose oxidase method. The intraassay CV for glucose was 1.5%.
Statistical Analysis
Log transformation of the hormonal data was performed to achieve homogeneity of variance and to increase linearity [25]. Variables were compared by two-way ANOVA with a repeated measures design by using prenatal androgen exposure and IVF cycle day, defined as the time interval from immediately before rhFSH treatment to the day of oocyte retrieval, 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, Macintosh Version 5.2, 1992, Evanston, IL). The effects of prenatal androgen exposure and rhFSH treatment duration on follicle development were examined by one-way ANOVA without a repeated measures design. The effect of prenatal androgen exposure on basal serum follicle hormone concentration and the degree of change in serum gonadotropin levels during rhFSH treatment also were examined by one-way ANOVA without a repeated measures design. Intrafollicular steroid levels were compared by using regression models with female type (i.e., normal, early-treated prenatally androgenized, and late-treated prenatally androgenized) or female type and oocyte development (i.e., normal, abnormal) nested within female type. These models were fit using generalized estimating equations to adjust for intrafollicular hormone correlations within a female. All hormonal data are expressed as the back transformed means and 95% confidence intervals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant human FSH was administered for 7 days (four normal female cycles, three early-treated prenatally androgenized female cycles), 8 days (one normal female cycle, two late-treated prenatally androgenized female cycles), or 9 days (two early-treated, two late-treated prenatally androgenized female cycles). The number of rhFSH treatment days did not affect the number of dominant follicles noted at laparoscopy (7 days, 4.0 [0.6–7.4]; 8 days, 4.7 [0.0–9.9]; 9 days, 5.5 [0.9–10.1] follicles; P = 0.9). Moreover, prenatal androgen exposure did not affect the number of rhFSH treatment days required to induce a dominant follicle (control, 7.2 [6.5–7.9]; early-treated, 7.8 [7.1–8.5]; late-treated, 8.5 [7.7–9.3] days; P = 0.08) or the number of dominant follicles noted at laparoscopy (control, 3.0 [0.0–6.4]; early-treated, 3.2 [0.0–6.6]; late-treated. 8.2 [4.4–12.1] follicles; P = 0.1). Prenatal androgen exposure also did not affect the number of dominant follicles contributing FF for hormone analyses (control, 1.8 [0.0–3.8]; early-treated, 1.4 [0.0–3.4]; late-treated, 3.3 [0.7–6.0] follicles (P = 0.5). Approximately 65% (27%–100%), 42% (8%–75%), and 62% (19%–100%) of all dominant follicles from normal, early-treated prenatally androgenized, and late-treated prenatally androgenized females, respectively, contributed FF for hormone analysis (P = 0.6).
Serum Hormone Levels
Serum rhesus FSH levels on Day 1 of rhFSH treatment were similar in prenatally androgenized (early-treated, 2.5 [1.6–4.0]; late-treated, 1.8 [1.2–2.7]) and normal females (2.0 [1.3–3.0 ng/ml]; P = 0.6). Serum human FSH levels in all female types combined rose significantly through Day 6 of rhFSH treatment but did not increase further on the day of oocyte retrieval (female type effect, P = 0.7; female type-IVF cycle day interaction, P = 0.2, Fig. 1, top). The mean percent increase in serum human FSH levels during rhFSH treatment was similar in all female groups (normal, 137.7% [119.2%–159.1%]; early-treated prenatally androgenized 156.0% [135.0%–180.2%]; late-treated prenatally androgenized, 152.4% [129.6%–179.3%] all days combined; P = 0.5). Serum LH levels before and during rhFSH treatment combined were lower in late-treated prenatally androgenized females than in early-treated prenatally androgenized females or normal females (Fig. 1, bottom, P 
0.001), and were unaffected by IVF cycle day (P = 0.2) or interactions between female type and IVF cycle day (P = 0.5). There was a significant female type effect, however, in the degree to which serum LH levels declined during rhFSH treatment (P 0.05). During all rhFSH treatment days combined, serum LH levels decreased from basal levels in normal (58% [41%–82%]) and in late-treated prenatally androgenized females (60% [41%–87%]) but were unchanged in early-treated prenatally androgenized females (107% [76%–150%]).
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Serum E2 and P levels were unaffected by female type (P = 0.8) or by interactions between female type and IVF cycle day (P = 0.4). Serum E2 levels in all female types combined rose significantly during rhFSH treatment and increased further on the day of oocyte retrieval (Fig. 2A). Serum P levels in all female types combined decreased on Day 2 of rhFSH treatment, then returned to basal values during the remaining days of rhFSH treatment (Fig. 2B).
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Serum 17 OHP, A4, T, and DHT levels were similar among female types (P > 0.3). Serum 17 OHP levels in all female types combined were unchanged during rhFSH treatment but significantly increased at oocyte retrieval (Fig. 2C). Serum A4 levels in all female types combined significantly increased on Day 6 of rhFSH therapy, reaching maximal levels at oocyte retrieval (Fig. 2D). Serum T and DHT levels in all female types combined gradually rose during rhFSH treatment to reach levels greater than those of Day 1 on Day 6 of rhFSH treatment (P 0.005) and at oocyte retrieval (P 0.01; Fig. 2, E and F).
Serum glucose levels were unaffected by female type (P = 0.8), IVF cycle day (P = 0.1), or female type-IVF cycle day interactions (P = 0.2). There was a trend for an interaction between female type and IVF cycle day on serum insulin levels (P = 0.08). On the day of oocyte retrieval, serum insulin levels in normal females (398.2 [180.2–890.7]) and late-treated prenatally androgenized females (268.4 [109.8–656.0] pmol/L) were lower than those before rhFSH administration (normal females, 531.9 [230.8–1225.8]; late-treated prenatally androgenized, 688.4 [270.5–1752.0] pmol/L). Serum insulin levels in early-treated prenatally androgenized females, however, did not change between Day 1 of rhFSH treatment (402.5 [158.1–1024.5] pmol/L) and the day of oocyte retrieval (403.5 [165.1–986.1 pmol/L). Serum insulin levels decreased from basal values during rhFSH treatment in normal females to 75.3% (46.9%–121.0%) and in late-treated prenatally androgenized females to 39.0% (23.0%–66.1%) but were unchanged over the same time interval in early-treated prenatally androgenized females (100% [59.1%–170%], P = 0.08).
FF Hormone Levels
Intrafollicular E2 levels were significantly decreased in early-treated prenatally androgenized females (P 0.01) and in late-treated prenatally androgenized females (P 0.001) compared with normal females (Fig. 3, left top), whereas FF P concentrations were similar between all prenatally androgenized females and normal females (P = 0.7, early-treated; P = 0.4, late-treated; Fig. 3, left bottom). Intrafollicular A4 concentrations also were lower in early-treated prenatally androgenized females (P 0.05) and in late-treated prenatally androgenized females (P 0.005) than in normal females (Fig. 3, right top). Intrafollicular T concentrations were significantly lower in late-treated compared with either early-treated prenatally androgenized females (P 0.005) or normal females (P 0.001; Fig. 3, right center). Intrafollicular DHT concentrations were comparable between the female groups (P = 0.2; Fig. 3, right bottom).
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Intrafollicular E2:T ratios were significantly lower in early-treated prenatally androgenized females (1.2 x 102 [0.6–2.3 x 102]) than in late-treated prenatally androgenized females (10 x 102 [5.5–18.5 x 102]; P 0.001) or in normal females (6.1 x 102 [3.5–10.4 x 102]; P 0.005). Intrafollicular DHT:T ratios were significantly higher in all prenatally androgenized females (early-treated, 0.5 [0.2–1.3]; late-treated, 1.4 [0.6–3.8]) than in normal females (0.2 [0.1–0.3]; P 0.001 versus early-treated and late-treated).
Oocyte Developmental Competence
The mean numbers of total oocytes recovered from early-treated (6.2 [0.0–15.9]) and late-treated (20.0 [7.5–32.5]) prenatally androgenized females were similar to those recovered from normal females (14.8 [5.1–24.5], P = 0.2) despite an inability to retrieve oocytes from one early-treated prenatally androgenized female with severe pelvic adhesions. The incidence of fertilization (early-treated prenatally androgenized, 83.6% [59.9%–100.0%]; late-treated prenatally androgenized, 75.0% [51.3%–98.7%]; normal females, 94.4% [76.7%–100.0%], P = 0.9) was comparable among the female groups. The percentage of fertilized oocytes that cleaved in normal females and in all prenatally androgenized females combined was 99.2% and 97.7%, respectively, although the small number of zygotes did not allow for meaningful comparison of the rates for embryo development between female groups. Eight percent of zygotes from normal females and no prenatally androgenized females developed into blastocysts.
Reduced FF E2 and A4 levels accompanied abnormal oocyte development in early-treated prenatally androgenized females (E2, P 0.005; A4, P 0.001) but not in normal females (E2, P = 0.7; A4, P = 0.2) or in late-treated prenatally androgenized females (E2, P = 0.3; A4, P = 0.2) (Fig. 4, A and B). Intrafollicular P, T, and DHT, however, did not significantly differ based upon oocyte development in normal females (P, P = 0.3; T, P = 0.9; DHT, P = 0.4) or early-treated (P, P = 0.9; T, P = 0.06; DHT, P = 0.9) and late-treated prenatally androgenized females (P, P = 0.3; T, P = 0.5; DHT, P = 0.7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Low FF E2 and A4 levels in prenatally androgenized females were unassociated with similar changes in circulating steroid levels. This discrepancy is relevant because FF E2 reflects net production (i.e., synthesis minus metabolism and secretion) by a single follicle, whereas circulating E2 reflects the size of the cohort of all dominant follicles, which was similar among females groups. Moreover, FF steroids are less likely than serum steroids to be affected by amounts of steroids entering the blood from ovarian or nonovarian sources and leaving the blood through clearance. Therefore, intrafollicular steroid measurements must be used to understand the steroid environment of the developing follicle and how it relates to the outcome of the oocyte it contains.
Because aromatase is a potential "rate-limiting step" in intrafollicular E2 synthesis, the FF E2:T ratio was used as an index of aromatase expression, nullifying to some degree the number of cells in the follicle giving rise to the steroids. A low FF E2:T ratio in early-treated prenatally androgenized females receiving rhFSH treatment suggests decreased intrafollicular aromatase activity as a factor contributing to reduced E2 production. Moreover, reduced intrafollicular aromatase activity in early-treated prenatally androgenized females was accompanied by a high FF DHT:T ratio as a marker of elevated 5-reductase activity. This reciprocity between reduced aromatase and elevated 5-reductase activities emphasizes the inhibitory role of 5a-reduced androgens on aromatase, as demonstrated by the ability of 5-androstane-3,17-dione, in amounts present in follicles of women with PCOS but not of normal women, to suppress E2 secretion by cultured human granulosa cells [26]. Our data suggest that reduced FF E2 levels in early-treated prenatally androgenized females receiving rhFSH therapy alone represent an arrest in transition from 5-reductase to aromatase activity during folliculogenesis [27–30].
Although reduced E2 production may result from decreased aromatase activity, our data also suggest it may be further limited in prenatally androgenized females by the availability of aromatizable androgen, as evidenced by reduced FF A4 levels versus normal females. Although the regulation of A4 production in the dominant follicle is uncertain, it is unlikely that the reduced FF A4 levels in prenatally androgenized females represent loss of A4 to the circulation because serum A4 levels were similar in all female groups. It is also unlikely that reduced FF A4 levels in early-treated prenatally androgenized females represent increased metabolism by steroidogenic enzymes because aromatase activity is low, whereas FF T levels are normal. Because low FF A4 levels were confined to follicles containing oocytes that developed abnormally, a more plausible explanation is that reduced FF A4 levels in early-treated prenatally androgenized females represent paracrine dysregulation of thecal cell A4 production from disordered folliculogenesis. In this regard, LH-stimulated thecal cell A4 production by way of P450c17 activity is stimulated by granulosa cell-derived paracrine factors [31], which, like E2 production, is impaired in early-treated prenatally androgenized females. Rodent models also have shown increased LH-induced androgen responsiveness of thecal/interstitial cells cultured in vitro with recombinant inhibin-A or medium exposed to granulosa cells and enhanced LH-induced androgen release with P450c17 mRNA upregulation in thecal/interstitial cells exposed in vivo to rhFSH [32]. Moreover, low serum androgen responsiveness to human chorionic gonadotropin in a woman with FSH deficiency and endogenous LH hypersecretion has been shown to increase in parallel with serum inhibin B concentration after FSH administration [33, 34].
The present study did not demonstrate LH hypersecretion in early-treated prenatally androgenized females, as previously shown in these females with or without rhFSH treatment [4, 9]. Nevertheless, the mean percent decrease in serum LH levels with normally rising serum E2 levels during rhFSH therapy was significantly less in early-treated prenatally androgenized females than in normal or late-treated prenatally androgenized females. Loss of LH responsiveness to E2 replacement therapy is a characteristic of both orchidectomized male monkeys and ovariectomized prenatally androgenized female monkeys and represents loss of hypothalamic sensitivity to estrogen-negative feedback [35]. This finding is biologically relevant because a similar loss of hypothalamic sensitivity to hormone negative feedback also has been shown in women with PCOS [36]. If such estrogen-negative feedback on LH release is entrained during fetal life, our present inability to identify LH hypersecretion in early-treated prenatally androgenized females, despite apparent loss of LH responsiveness to E2, might represent variations in this trait within and between such females, some of which are different from those previously investigated [9].
Early-treated prenatally androgenized females also were unable to suppress serum insulin levels with normally rising serum E2 levels during rhFSH treatment, as previously described [9]. This finding has biological relevance because a changing pattern, rather than a fixed pattern, of insulin delivery to target tissue is optimal for stimulating insulin action in vivo [37]. In other words, inability of early-treated prenatally androgenized females to suppress serum insulin levels may create relative insulin excess at target tissues because of negligible declines in circulating insulin levels during rhFSH treatment. Although such interactions between insulin and its target tissues are beyond the scope of this study, co-incubation of insulin and FSH with murine oocyte-cumulus cell complexes in vitro accelerates granulosa cell LH receptor mRNA expression [38] while reducing the percentage of fertilized oocytes developing into blastocysts. These findings agree with our previous observations that high FF P:E2 and P:A4 ratios, as well as low FF A4 and E2 levels, are associated with poor embryo development in early-treated prenatally androgenized female rhesus monkeys receiving rhFSH therapy followed by human chorionic gonadotropin administration [9]. The present study further demonstrates that early prenatal androgenization in monkeys receiving rhFSH therapy alone also reduces FF A4 and E2 concentrations in association with abnormal oocyte development, without having an effect on P, testosterone, or dihydrotestosterone concentrations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Daniel A. Dumesic, Department of OB/GYN, Department of Internal Medicine, Mayo Clinic Rochester, 200 First St. SW, Charlton 3A, Rochester, MN 55905. FAX: 507 284 1774; ddumesic{at}mayo.edu
Received: 31 December 2002.
First decision: 21 January 2003.
Accepted: 21 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Reductase activity in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1999 84:2414-2418-reductase and aromatase pathways of androgen metabolism during controlled ovarian hyperstimulation with human menopausal gonadotropins. J Assist Reprod Genet 2001 18:527-533[CrossRef][Medline]This article has been cited by other articles:
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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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 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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 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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 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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