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Context: Vascular endothelial growth factor A (VEGF-A) is a potent cytokine that promotes angiogenesis and vascular permeability. After controlled ovarian stimulation (COS) for in vitro fertilization (IVF), excessive VEGF-A production can occur, particularly in women with polycystic ovarian syndrome (PCOS); however, it is unclear whether the regulation of VEGF-A production is different between PCOS and non-PCOS women., Objective: The aim of this study was to determine whether there were differences in the dose- and time-dependent effects of insulin and IGFs on VEGF-A production by luteinized granulosa cells (LGCs) from women with and without PCOS., Design and Setting: A prospective comparative experimental study was conducted at an institutional practice., Patients: Patients included six PCOS and six non-PCOS women undergoing COS and IVF., Interventions: Interventions included COS for IVF., Main Outcome Measures: VEGF-A levels in culture media were collected daily for 3 d from LGCs after incubation with variable doses of insulin, IGF-I, and IGF-II in the presence and absence of LH., Results: In both study groups, exposure to LH alone did not alter VEGF-A levels. However, insulin or IGF increased VEGF-A levels within 1 d and appeared to synergize with LH at 3 d. VEGF-A production by non-PCOS LGCs was more sensitive to IGF exposure, whereas PCOS cells were more sensitive to insulin. Although an increase in DNA content (P < 0.05) was noted in cultures of PCOS cells, progesterone levels were lower compared with non-PCOS LGCs., Conclusion: Insulin and IGFs promote VEGF-A production in LGCs, but the response patterns are different when cells from PCOS and non-PCOS women are compared.

Experiments were designed to investigate the expression and regulation of vascular endothelial growth factor (VEGF) in the primate corpus luteum (CL) throughout the luteal life span in the natural menstrual cycle. Corpora lutea were collected during the early (ECL; Days 3-5 post-LH surge), mid (MCL; Day 6-8 post-LH surge), mid-late (MLCL; Days 10-12 post-LH surge), late (LCL; Days 14-16 post-LH surge), and very late (Days 17- 18 post-LH surge) luteal phase. Specific primers were designed to amplify mRNAs encoding VEGF isoforms 206, 189, 183, 165, 145, and 121. Only two cDNA products were obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends; cloning and sequencing confirmed their 98% homology to the corresponding human VEGF 165 and 121 sequences. Semiquantitative RT-PCR assays indicated that VEGF 165 mRNA levels increased (P < 0.05) from ECL to MLCL but then declined (P < 0.05) by LCL. Although VEGF 121 mRNA levels were limited in ECL, they increased significantly in MCL (P < 0.05). Levels of VEGF protein, as measured by Western blot analysis, were two- to fourfold higher for VEGF 165 versus VEGF 121. Also, VEGF 165 levels were higher (P < 0.05) in ECL and MCL compared to those at later stages. During 2-day culture, preparations of dispersed luteal cells secreted VEGF into the media; the highest levels were observed in ECL and declined (P < 0.05) by LCL. Regardless of luteal stage, hypoxic conditions increased (P < 0.05) VEGF levels, whereas LH exposure increased (P < 0.05) progesterone, but not VEGF, in the media. These results are consistent with a dynamic, local regulation of VEGF production during the life span of the primate CL that is not directly controlled by LH.

During the periovulatory interval, intrafollicular progesterone (P) prevents follicular atresia and promotes ovulation. Whether P influences oocyte quality or maturation and follicle rupture independent of the midcycle gonadotropin surge was examined. Rhesus monkeys underwent controlled ovarian stimulation with recombinant human gonadotropins followed by a) experiment 1: an ovulatory bolus of hCG alone or with a steroid synthesis inhibitor (trilostane, TRL), or TRL + the progestin R5020; or b) no hCG, but rather sesame oil (vehicle), R5020, or dihydrotestosterone (DHT). In experiment 1, the majority of oocytes remained immature (65% +/- 20%) by 12 h post-hCG. However, the percentage of degenerating oocytes increased (P < 0.05) with TRL (42% +/- 22% vs. 0% controls), but was reduced (P < 0.05) by progestin replacement (15% +/- 7%). By 36 h post-hCG, the majority of oocytes in all three groups reached metaphase II (MI). In experiment 2, no evidence of follicle rupture was observed in the vehicle, R5020, or DHT groups. Despite the absence of hCG, a significant (P < 0.05) percentage of oocytes resumed meiosis to metaphase I in R5020- (41 +/- 9) and DHT- (36 +/- 15) but not vehicle- (4 +/- 4) treated animals. Only oocytes from R5020-treated animals continued meiosis in vivo to MII. More (P < 0.05) oocytes fertilized in vitro with R5020 (40%) than with vehicle (20%) or DHT (22%). Thus, P is unable to elicit ovulation in the absence of an ovulatory gonadotropin surge; however, P and/or androgens may prevent oocyte atresia and promote oocyte nuclear maturation in primate follicles.

Continual administration of low doses of the antiprogestin ZK137316 permits ovarian/menstrual cyclicity, but prevents pregnancy in female rhesus monkeys. The sites of contraceptive action remain unknown. This study determined whether chronic, low-dose antiprogestin exposure during follicular development impairs oocyte maturation in vivo, as well as fertilization and preimplantation embryogenesis in vitro. Adult, female rhesus monkeys exhibiting normal menstrual cycles received vehicle (n=9) or 0.03 mg ZK137316 (n=8)/kg body weight i.m. daily for 3 months. Controlled ovarian stimulation with recombinant gonadotropins was initiated in the 3rd month. Oocytes collected from preovulatory follicles were evaluated for nuclear maturity and inseminated in vitro. Preimplantation embryonic development was monitored in vitro. The total number of oocytes and percentage collected at each nuclear stage were similar in both groups. More (P<0.05) atretic oocytes were recovered following antiprogestin relative to vehicle treatment. Fertilization rates and percentages of embryos that progressed to the morula stage were similar between groups, but antiprogestin-treated females exhibited less (P<0.05) normal cleavage. Embryonic development was accelerated by 1 day (P<0.05) from the 16-cell to the morula stage in the antiprogestin group relative to vehicle. Despite this, the majority of embryos became blastocysts within 6 days in vitro in the antiprogestin group, but fewer expanded (P=0.09) and hatched (P<0.05) compared to vehicle. During in vivo treatment with chronic, low-dose antiprogestin, oocytes retained their ability to resume and complete meiosis as well as fertilize following insemination in vitro. However, preimplantation embryogenesis in vitro was impaired, particularly during the later stages of blastocyst development. Thus, antiprogestin exposure during follicular development altered oocyte functions that are critical for normal preimplantation embryogenesis; this may contribute to pregnancy prevention.

Background: Administration of low doses of an antiprogestin to rhesus monkeys permits ovarian/menstrual cyclicity, suppresses endometrial proliferation and prevents pregnancy without adverse or toxic side-effects after 5-6 months of daily treatment. The purpose of this study was to test the reversibility with respect to restoration of fertility after 1 year of low-dose antiprogestin treatment., Methods: This experiment included a daily 1 year vehicle- or antiprogestin-treatment interval followed by a 9 month post-treatment interval for adult, female rhesus monkeys (n = 5/group) of proven fertility and exhibiting regular menstrual cycles. Co-habitation occurred with a male of proven fertility and vaginal swabs were taken to identify the presence of sperm during the treatment (antiprogestin females) and post-treatment intervals (vehicle and antiprogestin females)., Results: Mating and vaginal sperm were evident in all antiprogestin females during, and, in both groups, after treatment. Based on ultrasonography, none of the antiprogestin-treated females became pregnant during the treatment interval. However, upon cessation of treatment, pregnancy rates were similar between antiprogestin-treated (3/5) relative to vehicle-treated (4/5) females with live, healthy infants born in both groups. There were no differences between groups in fetal measurements, gestation lengths, live birth rates and infant weights., Conclusions: The reversal of the anti-fertility effects of chronic, low-dose antiprogestin treatment supports the clinical feasibility of potent and selective antiprogestins as potential contraceptives for women.

Extensive destruction of primordial follicles by exposure to ovarian toxicants can cause early menopause in women. Primordial follicle destruction is known to result from dosing of mice and rats with three polycyclic aromatic hydrocarbons (PAHs), contaminants commonly found in cigarette smoke. Therefore, the purpose of this study was to compare relative ovotoxicity in mice and rats using the PAHs, 9, 10-dimethylbenzanthracene (DMBA), 3-methylcholanthrene (3-MC), and benzo[a]pyrene (BaP). Female B6C3F(1) mice and Fischer 344 rats (age 28 days) were dosed daily (ip) with vehicle control or a range of doses of the PAHs. Two groups were dosed with the occupational chemicals 4-vinylcyclohexene (VCH; 500 mg/kg ip) or its diepoxide metabolite (VCD; 80 mg/kg ip), other known ovotoxicants. After 15 days, ovaries were collected, histologically prepared, and follicles were microscopically classified (primordial, primary, or secondary) and counted. The dose of each chemical that produced 50% loss of primordial follicles (p < 0.05) was determined (ED50) and used to calculate an ovotoxic index (OI) in mice and rats (ED50 x 15 days). Thus, a chemical with a lower OI is more toxic. Primordial follicles in mice displayed a lower OI than rats to all chemicals tested (mouse: DMBA, 0.0012; 3-MC, 0.003; BaP, 0.18; VCD, 6.8; VCH, 69; rat: DMBA, 0.45; 3-MC, >3.4; BaP, >3.6; VCD, 8.6; VCH, >69). In mice, DMBA targeted primordial follicles at a 10-fold lower concentration than primary and secondary follicles, whereas 3-MC exposure targeted primordial and primary follicles to a similar degree. BaP exposure targeted primordial and primary follicles at a 100-fold higher concentration than DMBA or 3-MC. Although BaP and 3-MC did not target secondary follicles in mice, secondary follicles in rats were most susceptible to 3-MC. Furthermore, all three PAHs were more ovotoxic (lower OI) with repeated low-dose exposure compared with OIs calculated from other studies using single high-dose exposures. The earliest day of impending primordial follicle loss (increase in percentage of unhealthy follicles, p < 0.05) in mice was factored into the OI (ED50 x first day of damage, p < 0.05 x % healthy follicles remaining, relative to control). The revised OI became DMBA d15, 0.0006; 3-MC d12, 0.0008; BaP d15, 0.132; and VCD d8, 2.96. These results predict that DMBA is the most potent ovarian toxicant (lower OI) in both species but VCD damages primordial follicles after shorter exposures. Calculation of the OI in mice and rats represents a method for comparing the relative potential risk of a variety of chemicals that produce ovarian damage at low levels following repeated exposures. The results also demonstrate that low-dose repeated exposures are substantially more toxic to the ovary than a single high-dose exposure. This finding is particularly important in view of the implications for chronic low-dose exposures of women to environmental chemicals., (Copyright 2000 Academic Press.)

Repeated dosing of rats with the ovotoxic chemical, 4-vinylcyclohexene diepoxide (VCD), destroys primordial and primary ovarian follicles via apoptosis (physiological cell death) by accelerating the normal rate of atresia. The present study investigated the effect of a single dose (1x) of VCD. Immature (d28) female Fischer 344 rats were dosed 1x or 15x with VCD (80 mg/kg ip). Ovaries were collected 24 h or 15 days following 1x VCD or after 15x for classification and evaluation. Following 1x VCD the number of healthy primary follicles was greater (p < 0.05) than control 24 h and 15 days later. This effect reflected a slowing of the normal rate of atresia seen in control ovaries. There was no effect of a single dose on primordial or growing follicles at any time. Expression of mRNA encoding the cell death gene bax was reduced (p < 0.05) on d1 after 1x VCD in isolated primordial and primary follicles. These observations were in contrast to a decreased (p < 0. 05) number of healthy primary and primordial follicles in ovaries and increased (p < 0.05) bax mRNA in isolated follicles from rats dosed 15x for 15 days. Immunofluorescence staining revealed that, the distribution of Bax protein was similar between ovaries from controls and 1x or 15x VCD-treated rats. These data provide evidence for a "protective" response against the normal rate of atresia in primary ovarian follicles following exposure to 1x VCD. Additionally, changes in expression of bax mRNA paralleled alterations in the rate of atresia., (Copyright 1999 Academic Press.)