Effects of ovarian source, patient age, and menstrual cycle phase on in vitro maturation of immature human oocytes.

OBJECTIVE: To determine the efficiency of in vitro maturation, expressed by nuclear maturation, of oocytes aspirated during gynecologic surgeries or collected from excised ovaries. To assess the effect of patient age and cycle phase at collection on the oocyte's ability to mature in vitro. To examine the time course of oocyte maturation in vitro. DESIGN: Nuclear maturation based on patient criteria compared. SETTING: University-based IVF program and research center. PATIENT(S): Consented patients undergoing gynecologic surgeries or patients undergoing oophorectomy. INTERVENTION(S): Oocytes were maintained in culture for 48 hours and evaluated for maturation. MAIN OUTCOME MEASURE(S): Nuclear maturation evaluated as germinal vesicle breakdown (GVBD) or progression to the metaphase II (MII) stage. RESULT(S): A significantly higher percentage of oocytes collected during the follicular phase of the menstrual cycle underwent GVBD than did oocytes collected during the luteal phase (60% versus 48%, respectively). The percentage of oocytes reaching the MII stage, from these two groups, was not different. No statistically significant differences in maturation were observed in oocytes from different ovarian sources or from patients >40 or <40 years of age. CONCLUSION(S): These data suggest that oocytes collected during the follicular phase are more likely to undergo GVBD than oocytes collected during the luteal phase. In this study, ovarian source, age, or cycle phase did not influence the final meiotic maturation of oocytes to metaphase II.

Activin A stimulates meiotic maturation of human oocytes and modulates granulosa cell steroidogenesis in vitro.

OBJECTIVE: To test the hypothesis that activin A promotes in vitro human oocyte meiotic maturation while inhibiting steroid secretion by nonluteinized antral granulosa cells. DESIGN: Prospective randomized controlled study. SETTING: A university medical center. PATIENT(S): Nine women ranging in age from 31-44 years who were undergoing oophorectomy for nonovarian pathology. INTERVENTION(S): Analysis of meiotic maturation of oocytes and steroid secretion by granulosa cells cultured in the presence or absence of activin A. MAIN OUTCOME MEASURE(S): Germinal vesicle breakdown (GVBD) and attainment of metaphase II (MII) in oocytes, and progesterone and E2 secretion by granulosa cells. RESULT(S): Activin A significantly enhanced GVBD (91% vs. 65%) for control and maturation to MII (56% vs. 35% for control) of immature oocytes. Activin A significantly suppressed basal, and inhibin A-and FSH-stimulated progesterone and E2 secretion by nonluteinized granulosa cells. CONCLUSION(S): Activin A is a promoter of oocyte maturation in vitro and a modulator of granulosa cell steroidogenesis in culture.

Elevated level of follicular fluid vascular endothelial growth factor is a marker of diminished pregnancy potential.

OBJECTIVE: To evaluate whether differences in follicular fluid vascular endothelial growth factor (FF VEGF) concentrations are observed between women achieving a clinical pregnancy and those failing to conceive. DESIGN: Retrospective chart review and analysis of FF VEGF concentrations. SETTING: University teaching center. PATIENT(S): Fifty-seven women < or =42 years of age undergoing follicular aspiration in preparation for IVF or GIFT. INTERVENTION(S): Analysis of FF VEGF concentrations and chart review of a single IVF or GIFT cycle. MAIN OUTCOME MEASURE(S): Follicular fluid VEGF concentrations, clinical pregnancy rate, age, ampules of gonadotropins used, oocytes retrieved, peak estradiol serum concentrations, day 3 FSH levels, and fertilization rate. RESULT(S): Women who did not conceive had higher FF VEGF concentrations than women achieving a clinical pregnancy (4.409 + 2,387 versus 2.793 +/- 1,180 pg/mL: P < .001). A negative correlation was observed between FF VEGF concentrations and peak estradiol levels and number of oocytes retrieved. A positive correlation was found for FF VEGF and patient's age and ampules of gonadotropins used. CONCLUSION(S): Elevated FF VEGF concentrations are associated with poor conception rates after IVF or GIFT.

Follicular fluid vascular endothelial growth factor concentrations are elevated in women of advanced reproductive age undergoing ovulation induction.

OBJECTIVE(S): To determine whether follicular fluid (FF) from women of advanced reproductive age had a relative deficiency of the angiogenic cytokine vascular endothelial growth factor/vascular permeability factor. Furthermore, we sought to determine whether luteinized granulosa cells secrete vascular endothelial growth factor/vascular permeability factor in response to hypoxia. DESIGN: Retrospective cohort study. SETTING: University teaching hospital. PATIENTS: Women undergoing follicular aspiration after superovulation in preparation for IVF-ET. Women of advanced reproductive age consisted of 21 women > or = 38 years old (range, 38 to 46 years); 15 subjects < or = 30 years served as the control population. INTERVENTION(S): Granulosa cells and FF were collected by transvaginal aspiration 35 hours after hCG. Granulosa cells from two women were cultured for 24 and 48 hours in M199 + 10% fetal bovine serum in 1% O2-5% CO2-94% N2 (hypoxic) or 95% air-5% CO2 (normoxic) without or with 0.1 mol/L cobalt chloride. MAIN OUTCOME MEASURE(S): Pooled FF vascular endothelial growth factor/vascular permeability factor concentrations and media vascular endothelial growth factor/vascular permeability factor accumulation at 24 and 48 hours were determined. RESULT(S): Follicular fluid vascular endothelial growth factor/vascular permeability factor concentrations were higher in advanced reproductive age women compared with younger women (3,735 +/- 2,155 versus 2,205 +/- 952 pg/mL, mean +/- SD). Accumulation of vascular endothelial growth factor/vascular permeability factor at 24 and 48 hours was 391 +/- 54 and 744 +/- 2 pg/mL in media maintained in 5% CO2 and air. Cobalt chloride induced a marked increase in vascular endothelial growth factor/vascular permeability factor (2,008 +/- 52 pg/mL at 24 hours and 3,630 +/- 519 pg/mL at 48 hours). An intermediate but significant increase in vascular endothelial growth factor/vascular permeability factor (733 +/- 35 pg/mL at 24 hours and 2,675 +/- 864 pg/mL at 48 hours) was observed with 1% O2 compared with normoxic controls. CONCLUSION(S): After hMG and hCG administration the FF from women of advanced reproductive age showed increased vascular endothelial growth factor/vascular permeability factor concentrations compared with younger women. Increased vascular endothelial growth factor/vascular permeability factor concentrations could be consistent with a hypoxic environment within follicles of older women.

Enhancement of primate oocyte maturation and fertilization in vitro by inhibin A and activin A.

OBJECTIVE: A role for inhibin and activin in primate oocyte maturation was investigated. DESIGN: The maturation and fertilization of rhesus monkey oocytes recovered from the excised ovaries of nine regularly cycling animals was compared for untreated germinal vesicle (GV)-intact controls versus oocytes cultured in the presence of inhibin, activin, inhibin + activin, or in a combination with follistatin. SETTING: Nonhuman primates in a research institute environment. INTERVENTIONS: Bilateral oophorectomy. MAIN OUTCOME MEASURE: Oocyte maturation from germinal vesicle breakdown (GVBD) to metaphase II (MII) and fertilization. RESULTS: Germinal vesicle breakdown, progression to MII and fertilization was monitored in oocytes cultured for 48 hours. Activin alone, at an optimum concentration of 100 ng/mL, stimulated GVBD whereas both GVBD and MII development was enhanced in the presence of inhibin + activin. The latter also accelerated the rate of maturation to MII. All treatment groups exhibited a higher incidence of GVBD compared with controls. When follistatin was added, the stimulatory effect of activin or activin + inhibin was abolished. Exposure to medium containing inhibin + activin significantly increased the percentage of MII oocytes that fertilized compared with controls (68% versus 25%, respectively). CONCLUSIONS: Inhibin and activin are potent stimulators of primate oocyte maturation, producing mature oocytes in vitro that fertilize with high efficiency.

Regulation of cumulus cell steroidogenesis by the porcine oocyte and preliminary characterization of oocyte-produced factor(s).

This study was designed to investigate whether porcine oocytes produce a factor(s) that influences cumulus and mural granulosa cell steroid production and to characterize the biochemical nature and mode of action of a such factor(s). Porcine cumulus-oocyte complexes (COC) were collected from 2-5 mm follicles and cultured either intact or after oocytectomy for 48 h. Steroid levels were then measured in the culture media. Conditioned media, obtained by culturing denuded oocytes for 48 h, were subjected to heat treatment of charcoal extraction and utilized to culture intact and oocytectomized COC. FSH-stimulated progesterone, 20 alpha-OH-progesterone, and estradiol were significantly higher in oocytectomized vs. intact COC cultures. Denuded oocytes cultured with granulosa cells significantly inhibited progesterone production compared to control. Also, media conditioned with different numbers of denuded oocytes (0 to 300) significantly inhibited progesterone production by oocytectomized COC in a manner dependent on oocyte number. Charcoal extraction, but not heat treatment, significantly removed the inhibitory effect of the conditioned media on progesterone production by oocytectomized COC. Increased progesterone production by oocytectomized COC was not accompanied by a similar increase in cAMP formation. Heptanol, a gap junction blocker, did not alter progesterone production by intact COC. In conclusion, porcine oocytes secrete a factor(s) that inhibits cumulus and mural granulosa cell steroidogenesis. This factor(s) is heat stable but extractable by charcoal. The factor(s) appears not to be transferred to somatic cells via gap junctions, and its effect is downstream of cAMP formation.

Rhesus monkey oocyte maturation and fertilization in vitro: roles of the menstrual cycle phase and of exogenous gonadotropins.

Our objectives were to improve the efficiency of rhesus monkey oocyte maturation and fertilization in vitro and to determine the influence of menstrual cycle phase and exogenous gonadotropins on these processes. Immature oocytes were recovered from antral follicles of ovaries excised from unstimulated animals (n = 14). The highest yield and quality was associated with oocytes obtained from early follicular phase ovaries, and more of these oocytes underwent germinal vesicle breakdown (GVBD; 55%) and matured to metaphase II (MII; 40%) than did oocytes from late follicular (GVBD, 28%; MII, 9%) or luteal (GVBD, 13%; MII, 10%) phase ovaries. Exogenous human (h) FSH and hLH improved GVBD and MII levels for oocytes recovered from late follicular phase ovaries and increased GVBD, but not MII, for oocytes from luteal phase ovaries. MII levels for oocytes from early follicular phase ovaries were adversely affected. Early follicular phase oocytes underwent GVBD faster and attained MII sooner than did those from either late follicular or luteal phase ovaries. Whereas exogenous gonadotropins did not significantly affect oocyte fertilization, follicular phase oocytes tended to be fertilized at a higher rate (p = 0.07). These results demonstrate that higher maturation levels are obtained with oocytes from early follicular phase ovaries and that gonadotropins influence oocyte performance in a cycle phase-dependent manner.