Granulosa cell aromatase activity in women undergoing IVF: a comparison of good and poor responders.

OBJECTIVE: We wished to investigate the aromatase activity (AA) of granulosa cells (GCs) in women undergoing ovarian follicular stimulation for in vitro fertilization (IVF). METHODS: Granulosa cells were harvested from follicular fluid aspirated at the time of oocyte retrieval in women undergoing IVF. Data related to the follicular stimulation and IVF were collected by chart review. We conducted a retrospective analysis of the relation between the response to stimulation and the AA of GCs obtained from IVF patients. We assessed the response to stimulation by calculation of the area under the curve (AUC) of the monitored serum estradiol levels, and divided patients into "poor responders" and "good responders." RESULTS: There was no difference in AA between women with a poor response to stimulation and women with a good response. Implantation rates and pregnancy rates were significantly lower in poor responders (5.3% and 9.1% respectively) than in good responders (19.7% and 54.8% respectively), even though embryo quality was similar in each group. CONCLUSIONS: Women who have a poor response to ovarian follicular stimulation preceding IVF have lower pregnancy rates than women with a good response. The lower pregnancy rates do not appear to be a consequence of an abnormal follicular environment, because AA and the ratio of serum estradiol AUC to oocytes retrieved was similar in both groups of women.

Perspectives: the possible influence of assisted reproductive technologies on transgenerational reproductive effects of environmental endocrine disruptors.

Demasculinization by environmental endocrine-disrupting chemicals (EDCs) is observed in many animal species but less evident in humans. Rodent studies with gestational exposure to either the fungicide vinclozolin or the insecticide methoxychlor demonstrate impaired male fertility with abnormal DNA methylation patterns in spermatozoa. Once established, these epigenetic changes may be permanent and thus paternally passed to subsequent generations. Conclusive evidence of a similar phenomenon in humans has not been established, but several observations bring up the possibility. Some, but not all, studies show an increase in male genital abnormalities after prenatal EDC exposure. Other studies demonstrate sperm abnormalities in males with EDC contact, although it is unclear as to whether this is due to prenatal or postnatal exposure. Although not examined in males with EDC exposure, one study shows gamete DNA methylation abnormalities in males with severe oligospermia. A subsequent study failed to corroborate these findings. The use of assisted reproductive techniques including intracytoplasmic sperm injection has removed natural selection barriers thus enabling reproduction in males that would otherwise be sterile. This review explores the hypothesis that prenatal EDC exposure results in transgenerational male reproductive abnormalities propagated by the use of assisted reproductive technologies.

Binding of progesterone to cell surfaces of human granulosa-lutein cells.

Progesterone is produced by granulosa cells under the influence of luteinizing hormone. Nuclear progesterone receptors have been found in rat granulosa cells. Human granulosa-lutein cells rapidly respond to progesterone with an increase in intracellular calcium suggesting the existence of a nongenomic mechanism. This study was conducted to determine whether binding of progesterone to granulosa cells could occur at the membrane. Granulosa cells were obtained from an in vitro fertilization program and examined immunohistochemically with an antiserum to membrane progesterone receptors. Approximately 14-70% of freshly harvested or cultured granulosa cells of six patients showed a positive reaction to the antiserum, limited to the cell membrane. Western blot analysis of homogenates of granulosa cells and a granulosa cell tumour confirmed the presence of progesterone receptors A, B and C and low amounts of a putative membrane receptor. These results demonstrate that the plasma membranes of human granulosa cells possess binding components for progesterone which may be involved in its nongenomic mechanism of action.

A simplified method for preparing IVF granulosa cells for culture.

PURPOSE: To validate an expedited method for the removal of erythrocytes when preparing IVF granulosa-luteal cells for culture. METHODS: Granulosa cells were enriched for culturing from follicular aspirates by density gradient centrifugation and by hypo-osmotic lysis treatments. RESULTS: Cells prepared by either method showed the same cell viability and produced progesterone in similar quantities. CONCLUSIONS: Using hypo-osmotic lysis to remove erythrocytes does not impair granulosa cell viability or steroidogenesis. It avoids multiple density gradient centrifugations and washings, and yields IVF granulosa cells ready for culture efficiently.

Rapid effects of pesticides on human granulosa-lutein cells.

Following our previous demonstration that p,p'-DDE (dichlorodiphenylchloroethylene), at environmentally relevant concentrations, can rapidly increase intracellular calcium [Ca2+]i concentrations in human granulosa-lutein cells, we examined whether other pesticides, such as Kepone, o,p-DDE and methoxychlor, have similar effects. Cultured human granulosa-lutein cells were loaded with Fura-2 AM, and changes in [Ca2+]i concentrations within small areas of single cells were studied with a dynamic digital Ca2+ imaging system. Kepone, at concentrations of 0.2-2 nmol/ml, consistently increased [Ca2+]i concentrations 2-6 times higher than baseline values within minutes of exposure. Methoxychlor at concentrations of 2.8-280 nmol/ml failed to alter [Ca2+]i levels consistently in cells from 10 patients. However, at 0.28 and 1.4 nmol/ml, increases in [Ca2+]i concentrations could be elicited by methoxychlor. The isomer o,p-DDE at 3 nmol/ml increased [Ca2+]i in granulosa cells of 11/20 patients. Pertussis toxin treatment inhibited the [Ca2+]i increases induced by estradiol, p,p'-DDE, o,p-DDE and methoxychlor, but not by Kepone or progesterone, indicating that Kepone and progesterone may act through an insensitive G protein-coupled receptor. The [Ca2+]i increases induced by Kepone also occurred in Ca2+-free medium, suggesting that [Ca2+]i mobilization occurred from the smooth endoplasmic reticulum. Thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca2+ pump, also stimulated [Ca2+]i increases but did not inhibit the Ca2+ response to all the pesticides. These results demonstrate that pesticides can have a rapid effect on human granulosa-lutein cells, and a nongenomic mechanism of action is suggested.

Rapid action of pesticides on cytosolic calcium concentrations in cultures of human umbilical vein endothelial cells.

Persistent metabolites of pesticides such as p,p'-DDE, at environmentally relevant concentrations, have been shown to have a rapid effect on intracellular calcium [Ca2+]i concentrations in human granulosa-lutein cells. Since endocrine disrupting substances can be transferred from the maternal circulation to the fetus the present study examined whether the pesticides, kepone, o,p-DDE, p,p'-DDE and methoxychlor, could alter cytoplasmic calcium [Ca2+]cyt concentrations in human umbilical vein endothelial (HUVE) cells. Cultured HUVE cells were loaded with Fura-2 AM and changes in [Ca2+]cyt of single cells were studied using a dynamic digital Ca2+ imaging system. Kepone and methoxychlor consistently increased [Ca2+]cyt concentrations, similar to the effects of estradiol and progesterone. p,p'-DDE increased [Ca2+]cyt concentrations in 80% of experiments whereas o,p-DDE stimulated its increases in 42%. Estrone, estriol, pregnenolone and cortisol were not effective. These results demonstrate that pesticides can have a rapid effect on HUVE cells probably through a nongenomic mechanism of action.

Environmental and occupational factors affecting fertility and IVF success.

Reproductive function has been shown to be sensitive to changes in the physical, psychosocial and chemical environments. Although reproductive effects of occupational exposure to hazardous chemicals have been well documented in the literature, the potential effects of chemical contaminants at levels representative of contemporary exposures in the general population are much less certain. Evidence for adverse effects of exposure to environmental contaminants is more conclusive among the lower animals than for humans where considerable controversy remains. In addition to potential reproductive hazards of exposure to environmental contaminants, there is also evidence for adverse reproductive effects of the physical and psychosocial environments. In this review we focus on the difficulties involved in linking exposure to putative hazardous substances in environmental and occupational settings to adverse reproductive outcomes, especially success of IVF procedures. We highlight the plausibility of adverse events through animal and cell studies and the application of these results to the interpretation of human data. We consider both the male and female partners since it is essentially their combined contributions of gametes which may be affected by chemicals, which lead to successful outcomes.

Synergistic effects between FSH and 1,1-dichloro-2,2-bis(P-chlorophenyl)ethylene (P,P'-DDE) on human granulosa cell aromatase activity.

BACKGROUND: 1,1-Dichloro-2,2-bis(P-chlorophenyl)ethylene (P,P'-DDE, DDE), a metabolite of DDT, is a persistent hormonally active environmental toxicant which has been found in human serum and follicular fluid. The objective of this study was to investigate the interaction between FSH and ppDDE on aromatase activity in primary cultures of human granulosa cells. METHODS: Granulosa cells were obtained at the time of oocyte retrieval for IVF procedures and cultured in defined medium containing FSH and environmentally relevant concentrations of DDE. Aromatase activity was measured by incubating the cells with 1beta-[(3)H]androstenedione and measuring the release of (3)H(2)O. RESULTS: The granulosa cell response to FSH was highly dependent on the basal level of aromatase activity (r = -0.703, P = 0.001, n = 17) with the highest activity occurring at low basal levels of aromatase activity. Enzyme activity was significantly stimulated at 100 ng DDE/ml. A synergistic effect on aromatizing activity was observed when cells were co-cultured with DDE and FSH. CONCLUSIONS: Concentrations of DDE similar to those present in human follicular fluid enhance basal and FSH-stimulated granulosa cell aromatizing enzyme activity.

Mammary gland morphology in Sprague-Dawley rats following treatment with an organochlorine mixture in utero and neonatal genistein.

In a related reproductive toxicology study designed to investigate the effects of in utero exposure to environmental toxicants and potential interaction with postnatal genistein, gross enlargement of thoracic mammary glands was observed in female offspring at 200 days of age. Therefore, the objective of this study was to analyze the effect of in utero exposure to a mixture of toxicants on mammary gland morphology. Time-mated Sprague-Dawley rats were treated on days 9-16 of gestation with vehicle or a mixture of environmental toxicants at 1x the acceptable daily intake. Furthermore, it is unclear whether postnatal exposure to phytoestrogens in soy formulas poses breast cancer benefit or risk, and potential interactions with environmental toxicants are unknown. Therefore, half the female pups from each treatment group received either subcutaneous vehicle or genistein (10 microg/g body weight [bw]/day) on postnatal days 2-8. Following necropsy at 200 days of age, a pathologist, blinded to treatment groups, examined mammary gland histopathology. Only mild histological changes were found in mammary glands of rats exposed to the mixture in utero while pronounced ductal hyperplasia, lactational changes, and fibrosis were observed in mammary glands from the genistein group and were more prominent in the mixture + genistein group. Mammary glands of the control group were histologically normal. Collectively, our results reveal that postnatal exposure to pharmacological levels of genistein induces profound morphological changes in the mammary glands of adult female rats, and that high levels of phytoestrogens possess the potential to modulate the toxicological effects of toxicant mixtures.

Effects of subchronic exposure to a complex mixture of persistent contaminants in male rats: systemic, immune, and reproductive effects.

Human populations throughout the world are exposed daily to low levels of environmental contaminants. The consequences of potential interactions of these compounds to human endocrine, reproductive, and immune function remain unknown. The current study examines the effects of subchronic oral exposure to a complex mixture of ubiquitous persistent environmental contaminants that have been quantified in human reproductive tissues. The dosing solution used in this study contained organochlorines (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], polychlorinated biphenyls [PCBs],p,p'-dichlorodiphenoxydichloroethylene [p,p'-DDE],p,p-dichlorodiphenoxytrichloroethane [p,p'-DDT], dieldrin, endosulfan, methoxychlor, hexachlorobenzene, and other chlorinated benzenes, hexachlorocyclohexane, mirex and heptachlor) as well as metals (lead and cadmium). Each chemical was included in the mixture at the minimum risk level (MRL) or tolerable daily intake (TDI) as determined by the U.S. EPA or ATSDR or, for TCDD, at the no observable effect level (NOEL) used to calculate the TDI. Sexually mature male rats were exposed to this complex mixture at 1, 10, 100, and 1000 times the estimated safe levels daily for 70 days. On day 71, all animals were sacrificed and a variety of physiological systems assessed for toxic effects. Evidence of hepatotoxicity was seen in the significant enlargement of the liver in the 1000x group, reduced serum LDH activity (100x), and increased serum cholesterol and protein levels (both 1000x). Hepatic EROD activities were elevated in animals exposed to10x and above. The mixture caused decreased proliferation of splenic T cells at the highest dose and had a biphasic effect on natural killer cell lytic activity with an initial increase in activity at 1x followed by a decrease to below control levels in response to 1000x. No treatment-related effects were seen on bone marrow micronuclei, daily sperm production, serum LH, FSH, or prolactin levels or weights of most organs of the reproductive tract. The weights of the whole epididymis and of the caput epididymis were significantly decreased at 10x and higher doses, although no effect was seen on cauda epididymal weight. The sperm content of the cauda epididymis was increased at the 1x level but not significantly different from control at higher dose levels. A slight, but significant, increase in the relative numbers of spermatids was seen in the animals from the 1000x group with a trend towards reduced proportion of diploid cells at the same dose. Only minor, nondose related changes were seen in parameters related to condensation of chromatin, as determined by flow cytometry, in epididymal sperm. We conclude that the mixture induced effects on the liver and kidney and on general metabolism at high doses but caused only minor effects on immune function, reproductive hormone levels, or general indices of reproductive function measures. These data suggest that additive or synergistic effects of exposure to contaminants resulting in residue levels representative of contemporary human tissue levels are unlikely to result in adverse effects on immune function or reproductive physiology in male rats.