Tamoxifen-induced alterations in meiotic maturation and cytogenetic abnormalities in mouse oocytes and 1-cell zygotes.

Alterations in the rate of oocyte meiotic maturation (OM) and the timing of the metaphase-anaphase transition may predispose oocytes to premature centromere separation (PCS) and aneuploidy. Tamoxifen has the potential for perturbing the rate of OM since it can function as a calcium antagonist by binding to calmodulin and inhibiting the formation of a calcium-calmodulin complex which is needed for activating calmodulin-dependent cAMP phosphodiesterase and initiating OM. The objective of this study was to test the hypothesis that tamoxifen alters the rate of OM and predisposes oocytes to PCS and aneuploidy. Different does of tamoxifen were administered by oral gavage to female mice preovulation. Metaphase II oocyte and 1-cell zygote chromosomes were C-banded and cytogenetically analysed. Tamoxifen treatment resulted in a modest, but significant (p < 0.05), increase in oocytes with PCS. Similar frequencies of hyperploidy and oocytes with unpaired, single chromatids (SC) were found. Metaphase I, diploid and premature anaphase (PA) oocytes were not detected. Hyperploidy, polyploidy, PCS, PA and SC were not detected in zygotes. These data indicate that the levels of tamoxifen-induced PCS found in mouse oocytes did not predispose zygotes to aneuploidy. Tamoxifen did, however, reduce the proportion of females exhibiting oestrus.

Centrosome-specific perturbations during in vitro maturation of mouse oocytes exposed to cocaine.

Previous studies indicating that cocaine may perturb meiotic chromosome segregation in mammalian oocytes prompted an analysis of the effects of cocaine on mouse oocytes matured in vitro under defined exposure conditions. Cumulus-enclosed mouse oocytes were matured in vitro in the continuous presence of cocaine and assessed for meiotic cell cycle progression and centrosome-microtubule organization using a combination of cytogenetic and fluorescence microscopic techniques. Both of these approaches demonstrated that cocaine had little effect on meiotic cell cycle progression to metaphase of meiosis-2 except at the highest dose tested (1000 microg/ml) where progression from metaphase-1 to metaphase-2 was inhibited. Cytogenetic analyses further showed that bivalent segregation was moderately affected and the incidence of premature centromere separation was significantly decreased following cocaine treatment. Under conditions of cocaine exposure, striking changes in meiotic spindle structure and cytoplasmic centrosome organization were observed. A 36% reduction in spindle length was associated with a loss of nonacetylated microtubules and fragmentation of spindle pole centrosomes. Moreover, in oocytes exposed to cocaine during maturation, a doubling in cytoplasmic centrosome number was observed. These results are discussed with respect to the relative roles of chromosomes and centrosomes in establishing and maintaining functional microtubule organization during meiosis in oocytes.

Clomiphene citrate-induced perturbations during meiotic maturation and cytogenetic abnormalities in mouse oocytes in vivo and in vitro.

OBJECTIVE: To determine if clomiphene citrate induces temporal perturbations during meiotic maturation and aneuploidy in mouse oocytes. DESIGN: A controlled dose study involving mouse oocytes in vivo and in vitro. SETTING: Clinical and academic research setting in a university medical center. INTERVENTION(S): Oocytes were obtained after superovulation and from mature follicles. MAIN OUTCOME MEASURE(S): Cytogenetic analysis of oocytes for aneuploidy, premature centromere separation, premature anaphase, and single chromatids, and the frequencies of metaphase I and diploid oocytes. RESULT(S): Clomiphene citrate resulted in a decrease in the number of ovulated oocytes and a significant (P<.05) increase in hyperploidy at 100 mg/kg in vivo. In vitro, 5.0 microg/mL of clomiphene citrate significantly (P<.05) increased hyperploidy and reduced the proportion of metaphase I oocytes. CONCLUSION(S): These findings suggest that clomiphene citrate has the potential for inducing aneuploidy in mouse oocytes both in vivo and in vitro and that the rate of oocyte maturation is altered after clomiphene exposure in vitro. Additional data are needed to support the results of this study.

Sensitivity of mouse oocytes to nicotine-induced perturbations during oocyte meiotic maturation and aneuploidy in vivo and in vitro.

Oocyte meiosis is sensitive to endogenous and exogenous perturbations that upset the temporal sequence of biochemical reactions during oocyte maturation (OM) and predispose oocytes to aneuploidy. Nicotine is an alkaloid that has been reported to disrupt the rate of OM, reduce ovulation and fertilization rates, and increase diploidy. The objective of this study was to test the hypothesis that nicotine perturbs the rate of OM and induces aneuploidy in mouse oocytes in vivo and in vitro. Female mice were given 7.5 IU pregnant mare's serum and either 0, 5.0, 7.5, or 10 mg/kg nicotine in vivo at -3, 0, and +3 h relative to a 5 IU injection of HCG. Oocytes were also cultured in vitro in the presence of 0, 1.0, 5.0, or 10.0 mmol/l nicotine. In vivo, significant (P < 0.05) differences in the proportions of oocytes with premature centromere separation and premature anaphase were found at 10.0 mg/kg nicotine suggesting that the rate of OM was advanced. Also, at this dose the proportion of ovulated oocytes was reduced by approximately 50% relative to controls. In vitro, only non-significant differences were found among the parameters measured. Although nicotine reduced the ovulation rate and perturbed the rate of OM in vivo, these data show that the rate of aneuploidy was not significantly elevated.

Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes.

To increase our understanding about the potential risks of chemically-induced aneuploidy, more information about the various mechanisms of aneuploidy induction is needed, particularly in germ cells. Most chemicals that induce aneuploidy inhibit microtubule polymerization. However, taxol alters microtubule dynamics by enhancing polymerization and stabilizing the polymer fraction. We tested the hypothesis that taxol induces meiotic delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Super-ovulated ICR mice received 0 (control), 2.5, 5.0, and 7.5 mg/kg taxol intraperitoneally immediately after HCG. Females were paired (1:1) with males for 17 h after taxol treatment. Mated females were given colchicine 25 h after taxol and their one-cell zygotes were collected 16 h later. Ovulated oocytes from non-mated females were collected 17 h after taxol. Chromosomes were C-banded for cytogenetic analyses. Oocytes were also collected from another group of similarly treated females for in situ chromatin and microtubule analyses. Taxol significantly (p<0.01) enhanced the proportion of oocytes exhibiting parthenogenetic activation, chromosomes displaced from the meiotic spindle, and sister-chromatid separation. Moreover, 7.5 mg/kg taxol significantly (p<0.01) increased the proportions of metaphase I and diploid oocytes and polyploid zygotes. A significant (p<0.01) dose response for taxol-induced hyperploidy in oocytes and zygotes was found. These results support the hypothesis that taxol-induced meiotic delay and spindle defects contribute to aneuploid mouse oocytes and zygotes.

Postovulatory ageing of mouse oocytes in vivo and premature centromere separation and aneuploidy.

Two paramount observations exist regarding aneuploidy in human oocytes: its association with maternal age and its more frequent occurrence during meiosis I. Numerous experimental studies have shown that fertilization of postovulatory aged oocytes is coupled with reproductive failure and cytogenetic aberrations in embryos. However, the basic cytogenetic defect(s) of aged oocytes that causes these abnormalities has not been adequately described. The objective of this study was to test the hypothesis that postovulatory oocyte ageing results in increased frequencies of premature centromere separation (PCS) in metaphase II (MII) oocytes and aneuploidy in zygotes. MII oocytes and one-cell zygotes were collected from superovulated mice at different times after ovulation and fertilization. Chromosomes were C-banded and analyzed for structural and numerical aberrations. The frequencies of PCS in oocytes significantly (p < 0.01) increased with time postovulation: 15 h (15 of 529, 2.8%), 20 h (82 of 627, 13.1%), and 25 h (118 of 502, 23.5%). In zygotes, the frequencies of hyperploidy significantly (p < 0.01) increased with time post-fertilization: 0-4 h (0 of 260), 4-8 h (5 of 212, 2.4%), and 8-12 h (8 of 262, 3.1%). These data support the hypothesis that postovulatory ageing results in elevated levels of PCS in oocytes and of aneuploidy in zygotes. The link between PCS and aneuploidy may be random segregation of sister chromatids during anaphase II.

Electromagnetic fields enhance chemically-induced hyperploidy in mammalian oocytes.

Epidemiological studies suggest that exposure to electromagnetic fields (EMFs) in the environment may be associated with mutagenic changes, but the relation between EMF exposure and aneuploidy has not previously been studied. Environmental EMFs apparently lack the energy necessary to function as aneugens, but the possibility exists that EMFs could influence the incidence of aneuploidy synergistically because EMFs can activate the neuroendocrine system, and ovulation and oocyte meiotic maturation are under neurohormonal control. This hypothesis was tested by examining the effect of EMF exposure on the occurrence of hyperploidy in mouse oocytes induced by vinblastine sulphate (VBS), which was employed as a surrogate for aneugens in the environment. The incidence of hyperploidy in metaphase II oocytes of individual mice following superovulation was determined, and statistical methods were developed to assess whether EMF exposure during oogenesis in the presence of VBS altered the rate of hyperploidy. A significant effect of EMF exposure on VBS-induced hyperploidy was found (P < 0.05). The data suggested that the EMF primarily affected the mice that exhibited a high incidence of VBS-induced hyperploidy. Exposure had no effect on the number of oocytes ovulated nor on the occurrence of hypoploidy. The results support the hypothesis that EMF exposure can promote the occurrence of aneuploidy caused by an aneugen via a mechanism involving the neuroendocrine system.

1,2-propanediol-induced premature centromere separation in mouse oocytes and aneuploidy in one-cell zygotes.

Aneuploidy in germ cells results in reproductive failure and mental and physical disorders in humans. Unfortunately, little is known about the causes and mechanisms of aneuploidy induction. The objective of this study was to test the hypothesis that propylene glycol (1,2-propanediol; PG) induces cytogenetic aberrations in mouse metaphase II (MII) oocytes that predispose zygotes to aneuploidy. Female ICR mice received 7.5 IU eCG and 5.0 IU hCG 48 h later. PG doses of 1300, 2600, and 5200 mg/kg body weight were given 3 h post-hCG; controls received the solvent deionized water. Ovulated oocytes were collected 16 h after administration of PG and processed for cytogenetic analysis. For the one-cell zygote cytogenetic study, females were given PG and paired (1:1) with ICR males for 16 h. Females that mated were given 2 x 10(-3) M colchicine 22 h post-PG, and zygotes were collected 18 h later. PG significantly (p < 0.05) increased both the proportion of MII oocytes with premature centromere separation (PCS) and the proportion of aneuploid one-cell zygotes. These results support the hypothesis that PG-induced PCS in MII oocytes predisposes zygotes to aneuploidy.

Thiabendazole-induced cytogenetic abnormalities in mouse oocytes.

Of the various classes of human genetic disorders, aneuploidy is the most prevalent. Besides its association with maternal age and its predominant origin during maternal meiosis I, little is known about the etiology of aneuploidy. Although various classes of chemicals have been shown to induce aneuploidy in experimental systems, there is no definitive evidence for the role of chemically induced aneuploidy and adverse human health effects, particularly germ cell effects. Thus, it is important to understand the potential of chemicals for inducing aneuploidy in germ cells. There are conflicting data in the literature about the ability of thiabendazole (TBZ) to induce aneuploidy; therefore, we investigated the potential of TBZ for inducing aneuploidy in oocytes. Superovulated ICR female mice were administered 0, 50, 100, or 150 mg/kg TBZ by intraperitoneal injection. The frequencies and percentages of hyperploid oocytes were 0/472 (0), 2/410 (0.5), 6/ 478 (1.3), and 3/427 (0.7) for control, 50, 100, and 150 mg/kg TBZ, respectively. The difference between controls and the 100 mg/kg dose was statistically significant. Also, the proportions of ovulatory mice and the number of oocytes collected per ovulatory female were reduced in the TBZ groups relative to controls. Based on these results, we conclude that TBZ induces a small, but significant increase in the frequency of aneuploid oocytes at toxic doses that also impair ovulation.

Mifepristone (RU486): a review.

OBJECTIVE: To review the literature concerning the mechanism of action and pharmacodynamics of mifepristone (RU486), potential new uses of RU486, and its current use not only as an abortifacient but also as therapy for endometriosis, leiomyoma, breast cancer, and meningioma. DATA IDENTIFICATION AND SELECTION: Studies that relate to RU486 were identified through a MEDLINE search. CONCLUSION(S): RU486 is an 11 beta-dimethyl-amino-phenyl derivative of norethindrone with a high affinity for P and glucocorticoid receptors. The receptor binding is not followed by transcription of P-dependent genes. Mifepristone effectively blocks P receptors in the placenta, resulting in the termination of pregnancy. In addition, it has been used in the treatment of leiomyomata, endometriosis, advanced breast cancer, and meningioma. It is a powerful tool to study the molecular action of P and in the future may be used as an estrogen-free contraceptive.

PIP: Through an online search of MEDLINE, the authors reviewed the literature on the development of mifepristone (RU-486); RU-486's mechanism of action, pharmacodynamics, and distribution; the physiologic action of RU-486; potential new uses for RU-486; and its current use as both an abortifacient and therapy for endometriosis, leiomyoma, breast cancer, and meningioma. RU-486 is an 11beta-dimethyl-amino-phenyl derivative of norethindrone with a high affinity for P and glucocorticoid receptors. Receptor binding is not followed by the transcription of P-dependent genes. RU-486 effectively blocks P receptors in the placenta, resulting in the termination of pregnancy. It has also been used to treat leiomyomata, endometriosis, advanced breast cancer, and meningioma. The following therapeutic uses of RU-486 are discussed: the termination of early pregnancy, treatment with RU-486 in combination with prostaglandins, the termination of second-trimester pregnancy, cervical ripening, labor induction, postcoital contraception, uterine leiomyomata, endometriosis, breast cancer, and meningioma.

Preterm premature ruptured membranes: a randomized trial of steroids after treatment with antibiotics.

OBJECTIVE: To assess the effectiveness of corticosteroids in patients with preterm premature rupture of membranes (PROM) after treatment with a broad-spectrum antibiotic, ampicillin-sulbactam. METHODS: A randomized clinical trial of corticosteroids in patients with preterm PROM was undertaken after treating these patients for a minimum of 12 hours with ampicillin-sulbactam. No digital vaginal examinations were performed on these patients. Antibiotics were continued for 7 days and the steroids were repeated weekly. No tocolytics were used. The primary outcome measure was the incidence of respiratory distress syndrome (RDS). Secondary outcome measures included latency period and neonatal and maternal infectious morbidity. RESULTS: Seventy-seven patients were enrolled and data about their pregnancies were analyzed. No statistically significant difference in latency period was noted (14.7 days in the steroid group, 15.8 days in the no-steroid group). Both neonatal and maternal infectious morbidity were similar. A significant reduction in the incidence of RDS (18.4 versus 43.6%, P = .03) were observed in the steroid group. CONCLUSION: These data suggest that treating preterm PROM patients with a broad-spectrum antibiotic before corticosteroids decreases RDS without apparent adverse sequelae.

Numerical and structural chromosome aberrations induced by etoposide (VP16) during oocyte maturation of mice: transmission to one-cell zygotes and damage to dictyate oocytes.

The antineoplastic drug etoposide (ET) inhibits topoisomerase II (topo II) activity by forming a ternary complex (DNA-ET-topo II). This complex prevents the DNA-strand-rejoining activity of topo II and may result in structural chromosome aberrations. Inhibition of topo II activity may also predispose cells to aneuploidy because this enzyme is needed for removing regions of DNA catenation prior to chromosome segregation. Our objectives were to study the dose response for ET-induced numerical and structural chromosomal aberrations in mouse one-cell zygotes, to compare these data with those obtained from a contemporary metaphase II (MII) oocyte study and to evaluate the sensitivity of dictyate oocytes to ET-induced aneuploidy. ICR female mice were superovulated and injected i.p. with either 6% dimethylsulphoxide (controls) or 20, 40 or 60 mg/kg ET 2 h after human chorionic gonadotrophin (HCG). ICR males were paired (1:1) with females immediately after treatment. After 17 h the males were removed, and after 24 h the females with a vaginal plug were given colchicine. One-cell zygotes were harvested for cytogenetic analysis 17 h after colchicine. The percentages of hyperploid zygotes were 1.1, 5.7, 13.8 and 20.7 and of zygotes with structural aberrations were 2.5, 16.3, 37.7 and 64.7, for control, 20, 40 and 60 mg/kg ET respectively. The differences between each succeeding dose for both structural and numerical aberrations were statistically significant (P < 0.01). When the ET dose response aneuploidy data from zygotes were compared with similar data from a contemporary study involving metaphase II oocytes, the frequencies of hyperploidy were greater in zygotes than in oocytes. We conclude that when ET is administered during the preovulatory phase of meiosis, it is both an aneugen and a clastogen in mouse one-cell zygotes.

Cytogenetic effects of caffeine during in vivo mouse oocyte maturation.

Numerous investigators have studied the reproductive and genetic toxicity of caffeine. Caffeine has also been reported to retard meiotic progression and induce aneuploidy in hamster oocytes in vitro. However, the ability of caffeine to induce aneuploidy in mammalian oocytes in vivo has not been reported. The objective of this study was to test the hypothesis that chemical-induced perturbations during in vivo oocyte meiotic maturation (OM) predispose oocytes to chromosome missegregation. Caffeine inhibits cAMP phosphodiesterase, which is needed for dephosphorylating p34(cdc2) kinase and initiating OM. Following superovulation, a dose of 150 mg/kg caffeine was administered to Institute of Cancer Research (ICR) female mice at various times prior to metaphase I (MI). Ovulated oocytes were collected from the oviducts and processed for cytogenetic analysis. Statistical analyses of the frequencies of hyperploid, MI, diploid, premature centromere separation and single chromatids revealed nonsignificant (P > 0.05) differences between the controls and each of the caffeine groups. Structural chromosome aberrations were not found. Under our experimental conditions, we rejected the hypothesis and concluded that caffeine neither retarded the rate of OM nor increased the incidence of aneuploidy in mouse oocytes. The factors responsible for the different in vivo and in vitro responses require investigation.

Dose-response study and threshold estimation of griseofulvin-induced aneuploidy during female mouse meiosis I and II.

The relative sensitivity of the two meiotic divisions of mouse oogenesis to griseofulvin (GF)-induced aneuploidy was investigated. The first meiotic division was studied by administering GF 4 h after human chorionic gonadotrophin (HCG) injection and analyzing metaphase II (MII) oocytes, whereas study of the second meiotic division involved treating the females 10 h after HCG and analyzing one-cell (1-Cl) zygotes. Data from previous studies have shown that these treatment times represented the most sensitive times for aneuploidy induction during meioses I and II. The statistical analyses of the data showed that the dose-response curves for aneuploidy induction did not differ quantitatively or qualitatively between the two meiotic divisions. The percentages of hyperploid MII oocytes and 1-Cl zygotes were significantly higher (P < 0.001) than in the controls for all doses except 125 mg/kg GF. The highest percentages of hyperploid cells were found after administering 1500 mg/kg GF. However, these percentages were not different (P > 0.05) from those observed after 500 or 1000 mg/kg GF, suggesting saturation of the GF aneuploid target(s). These results suggest that the relative sensitivity to GF-induced aneuploidy between the two meiotic divisions of oogenesis are similar. They also suggest the presence of a lower (125 mg/kg) and an upper 500 mg/kg) threshold for GF-induced aneuploidy.

Galanin: a novel intraovarian regulatory peptide.

Galanin is a 29-amino acid peptide that acts as a neuropeptide in many tissues. To date, galanin action and the hormonal regulation of galanin gene expression have not been described in the ovary of any species. To study possible ovarian expression and regulation of galanin, immature gonadotropin-primed rats were given hCG (10 IU), and their ovaries were collected 0, 4, 8, 12, and 20 h after hCG treatment for determination of galanin messenger RNA (mRNA) concentration by solution hybridization. Galanin mRNA levels progressively increased after hCG administration, peaking at 12 h (2.4-fold increase vs. 0 h), with a subsequent return to 0 h levels at 20 h. To determine a possible ovarian role for galanin, rats were killed 48 h after gonadotropin administration, their ovaries were removed, and granulosa cells were harvested. These cells and the ovarian tissue remaining after granulosa cell collection (i.e. "shells") were each cultured for 24 h with increasing concentrations of galanin (0, 10, 100, and 1000 nM) in the presence or absence of LH. The medium was examined for steroid production and metalloproteinase inhibitor activity. In granulosa cell cultures, galanin increased the levels of estradiol by 26% and had no effect on progesterone, but decreased metalloproteinase inhibitor activity by 61% in the conditioned medium. In the shell cultures, galanin increased estradiol, progesterone, and androstenedione in the medium, suggesting that galanin acts on cells other than granulosa cells or that galanin action requires a paracrine interaction between granulosa and thecal cells. Our data demonstrate that galanin message is increased by hCG, and that galanin acts to amplify ovarian steroidogenesis while decreasing metalloproteinase inhibitor activity. These findings establish that ovarian galanin mRNA is hormonally stimulated and that galanin acts as an intraovarian regulatory peptide.

The effects of cyclic adenosine monophosphate, forskolin, and theophylline on motility parameters in gossypol-treated human sperm.

OBJECTIVE: To examine the effect of gossypol on human sperm in vitro and the mechanism for the effect. DESIGN: Fresh sperm ejaculates obtained from normal donors to the University of Kentucky Andrology Donor Program were exposed to gossypol. Motility was studied manually and using computer-assisted sperm analysis. In subsequent experiments, the effects of forskolin, theophylline, and cyclic adenosine monophosphate (cAMP) on sperm motion were measured. SETTING: University of Kentucky Department of Obstetrics and Gynecology Andrology Laboratory. MAIN OUTCOME MEASURES: Manual and computer-assisted measurements of sperm motility and motion characteristics. RESULTS: Gossypol inhibited sperm motility, which could be reversed partially by increasing cAMP. CONCLUSION: Gossypol exposure in vitro adversely affects sperm motility in a dose- and time-dependent manner by a cAMP-dependent mechanism.

PIP: The antimotility effect of gossypol on human sperm has been documented. Gossypol is commonly derived from cottonseed oil. The exact mechanism by which this effect occurs is unknown. This paper reports research on the effects of gossypol on sperm motility. Elucidation of the site(s) of action and whether the effects are reversible are also discussed. Fresh human sperm was collected, centrifuged into pellets of equal numbers, washed, and then either overlayered with Ham's F-10 media or treated with gossypol. Gossypol solutions contained 10, 12.5, 20, 25 or 50 mcg/mcl of gossypol. These concentrations were used in measuring the sperm dose-response. Forskolin, theophylline, or cyclic adenosine monophosphate (cAMP) was added to gossypol-treated sperm and motility levels were measured. Motility measurements were conducted manually or via computer-assisted readings of sperm motion characteristics and their actual ability to move. Antimotility effects of gossypol on sperm are related to both dose and exposure time. This study supports the hypothesis that gossypol inhibits cAMP formation. At lower doses of gossypol (10 mcg/mcl), both theophylline and forskolin reversed gossypol's antimotility effect. At higher concentrations (20 mcg/mcl), the effect of gossypol appeared to be rapid and was irreversible. This latter finding has implications for its use as a vaginal contraceptive agent.

Antiprogesterone (RU486) effects on metalloproteinase inhibitor activity in human and rat granulosa cells.

OBJECTIVE: To determine if granulosa cells are a source of metalloproteinase inhibitor activity and whether P regulates this activity. DESIGN: Inhibitor activity was measured in media from human and rat granulosa cells cultured with the antiprogesterone mifepristone (RU486). Human granulosa cells were obtained at the time of oocyte retrieval from gonadotropin-stimulated patients after hCG administration. Rat cells were collected from gonadotropin-primed animals before the LH surge. Human and rat cells were cultured for 24 hours in the absence or presence of LH (100 ng/mL or 3.3 nmol/L) and/or RU486 (5 microM or 50 microM). Inhibitor activity and P were measured in the media. SETTING: Reproductive Endocrinology Laboratories, University of Kentucky. RESULTS: Media from human granulosa cells contained metalloproteinase inhibitor activity and the addition of LH did not change this activity. RU486 at 50 microM decreased inhibitor activity in cells cultured in the absence or presence of LH (0.59 +/- 0.12- and 0.24 +/- 0.18-fold change, respectively, versus control cultures; mean +/- SEM). In rat granulosa cells, inhibitor activity increased with LH treatment (1.97 +/- 0.12-fold change). RU486 decreased the activity present in cells cultured in the presence of LH. Progesterone production was stimulated by LH in both human and rat granulosa cells (3.71 +/- 0.90- and 7.18 +/- 0.24-fold change, respectively). In the human cells, RU486 inhibited P production whereas RU486 stimulated P production in the rat cells. CONCLUSIONS: These findings demonstrate for the first time that human granulosa cells are a source of metalloproteinase inhibitor activity. The decrease in granulosa cell-derived inhibitor activity by RU486 suggests that P stimulates inhibitor activity. Thus, P may regulate proteolysis associated with follicular rupture via its ability to stimulate granulosa cell production of metalloproteinase inhibitors. Differences in P production between the human and rat cells may be due to differences in hormonal stimulation regimens (i.e., hCG exposure).

Detection of a unique 32-kd protein in the peritoneal fluid of women with endometriosis.

OBJECTIVE: To test the hypothesis that endometriotic tissue secretes endometriotic-specific proteins into the peritoneal fluid (PF) of women with endometriosis. DESIGN: A prospective design was utilized in this study. SETTING: Tertiary care, university-based center and reproductive endocrinology laboratory. PARTICIPANTS: Women of reproductive age who were infertile with endometriosis (n = 19), as well as without endometriosis (n = 7), and fertile women undergoing tubal ligation (n = 6). INTERVENTIONS: Collection of PF fluid via laparoscopy. MAIN OUTCOME MEASURES: Peritoneal fluid proteins were isolated and assessed by two-dimensional polyacrylamide gel electrophoresis. RESULTS: Two-dimensional electrophoresis of PF proteins isolated a group of proteins (M(r) = 32 to 40 kd, pI = 4.5 to 5.2) in all PF samples that was similar to the rat endometriotic implant-specific protein, Endo-1. This group of proteins consisted of 5 to 12 individual proteins with endometriosis PF containing a significantly higher number of proteins (median = 11) compared with either PF from infertile women without endometriosis (median = 8) or from women undergoing tubal ligation (median = 7). In addition, one protein (M(r) = 32 kd, pI = 5.8), termed EPF-32, was detected predominantly (18 of 19 samples analyzed) in PF from women with endometriosis. This protein was also detected in PF from infertile women without endometriosis (2 of 7 samples) but not in the PF of fertile women undergoing tubal ligation (0 of 6 samples). The appearance of this protein was not associated with the severity of endometriosis. CONCLUSION: It is concluded from this study that PF from women with endometriosis predominantly contains a 32-kd protein (EPF-32) compared with the PF of women without the disease. The role of EPF-32 in the pathophysiology of endometriosis is not established but this protein may function as a diagnostic marker for endometriosis.

The presence of luteinized unruptured follicle syndrome and altered folliculogenesis in rats with surgically induced endometriosis.

OBJECTIVE: Our purpose was to determine in the rat model whether endometriosis could influence ovarian function by altering oocyte release or folliculogenesis. STUDY DESIGN: We histologically examined the ovaries of reproductively cycling rats with (n = 16) and without (n = 10) surgically induced endometriosis. The rats in these two groups were further subdivided into unilaterally ovariectomized or ovarian-intact groups. Serial sections of ovaries were examined, and follicular development and frequency of luteinized unruptured follicles were determined. RESULTS: A significant tenfold increase in the number of luteinized unruptured follicles was observed in the ovaries from rats with endometriosis (2.7 per rat) compared with unoperated and sham-operated control groups (overall mean 0.26 per rat, p < 0.05). Additionally, ovaries from unilateral ovariectomized animals with endometriosis contained four times as many luteinized unruptured follicles (four per rat) as did the ovaries from bilaterally ovarian-intact rats with endometriosis (1.40 per rat, p < 0.01). Fewer follicles were present in rats with endometriosis (180 follicles per ovary) than in control rats (231 follicles per ovary, p < 0.05). CONCLUSION: In the rat model the presence of ectopic endometrium is associated with an increased frequency of luteinized unruptured follicles and altered follicular development.

Serial transvaginal ultrasound scans and beta-human chorionic gonadotropin levels in early singleton and multiple pregnancies.

OBJECTIVE: To determine if serum beta-hCG levels are higher in multiple gestation than in singleton pregnancy at the time of intrauterine sac visualization and the first appearance of fetal heart activity as documented by serial transvaginal ultrasound (US). DESIGN: Prospective analysis of serial transvaginal US findings in 19 pregnancies correlated with serum hCG levels during early gestation. SETTING: Reproductive endocrinology division of the University of Arkansas for Medical Sciences, Little Rock, Arkansas. PATIENTS: Nineteen infertility patients were studied after conceiving. Thirteen underwent IVF or GIFT, 4 received hMG therapy, 1 was treated with clomiphene citrate, and 1 pregnancy followed spontaneous ovulation. INTERVENTIONS: Transvaginal US and hCG levels were obtained every Monday, Wednesday, and Friday from 20 to 22 days after ovulation until the appearance of fetal heart activity. RESULTS: Initial sac visualization occurred at lower serum hCG levels in singleton versus multiple pregnancies (2,180 +/- 1,170 versus 7,028 +/- 4,280 mIU/mL, mean +/- SD). Sacs were always seen when the serum hCG level (mIU/mL) was > or = 1,161 in singleton, 1,556 in twin, 3,372 in triplet, and 9,399 in quadruplet pregnancies. CONCLUSION: Failure to observe an intrauterine sac by transvaginal US in the presence of serum hCG levels in the 1,000 to 2,000 mIU/mL range is not pathognomonic for an ectopic gestation. Clinical symptomatology, risk of multiple pregnancies, and gestational age must also be considered.