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.

Endometriosis occurring in leiomyomatosis peritonealis disseminata: ultrastructural study and histogenetic consideration.

Leiomyomatosis peritonealis disseminata is a pseudomalignant condition which mimics disseminated abdominal carcinoma. A new case is reported and compared to the 11 well-documented cases in the literature. All reported patients have experienced a benign clinical course. The peritoneal nodules are composed of histologically benign-appearing smooth-muscle cells, substantiated by electron-microscopic study. The histogenesis of this entity is discussed in the light of our ultrastructural findings and the observation of concurrent endometriosis. The theory of metaplasia of the subcoelomic mesenchyme in the pathogenesis of the leiomyomatous lesions is favored. The serum estrogen levels determined in our patient did not reveal any abnormal change. The inducing factor(s) remain to be identified. Also, an unusual sensitivity of the coelomic tissues in these patients, in response to metaplastic stimuli, is postulated as a possible contributing factor.

Unusual mullerian anomalies associated with distal extremity abnormalities.

A 13-year-old girl was evaluated with nonfamilial mullerian anomalies consisting of bilateral blind uterine horns, a separate cervical remnant, and total vaginal agenesis. The observed musculoskeletal abnormalities of the distal extremities differed from those usually associated with both nonfamilial mullerian agenesis (Rokitansky-Küster-Hauser syndrome) and the familial syndromes associated with mullerian anomalies. The pattern of mullerian dysgenesis is unusual in that the entire vagina is absent and a cervical remnant separate from the two blind uterine horns is present in the midline in the normal course of the paramesonephric ducts.

Reevaluation of hysterosalpingography in infertility investigation.

The hysterosalpingogram has been extensively used in infertility investigations to assess tubal patency, however, the diagnostic reliability of this technique is not known. Two hundred thirty-one consecutive hysterosalpingograms were retrospectively evaluated. Sixty-two percent (143) of the patients subsequently underwent laparoscopy. Comparison of hysterosalpingogram and laparoscopic findings revealed a 15.9% false positive tubal patency rate and a 14.9% false negative tubal patency rate. Seventy-six percent of laparoscopies revealed previously undiagnosed intraperitoneal disease. Seventeen percent of hysterosalpingograms demonstrated intrauterine pathology. There was a 0.9% major complication rate with hysterosalpingograms due to two cases of acute pelvic inflammatory disease. No significant laparoscopic complications were noted. The results suggest that laparoscopy provides a more accurate assessment of tubal patency and peritoneal factors than hysterosalpingogram in the investigation of infertility.

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.

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.

Diverse humoral and cell-mediated effects of antisperm antibodies on reproduction.

No single test to detect the presence of antisperm antibodies has correlated precisely with subsequent fertility. The purpose of this study was to determine whether heterogeneous effects of antibodies could potentially explain this observation. The effects of serum on sperm motility, complement-mediated sperm lysis, mouse macrophage-mediated sperm phagocytosis, and sperm IgG opsonization were assessed in several patients with known antisperm antibodies. Each patient's serum produced its own unique profile. Motility ranged from normal (70% to 85%) to 10% using different subjects' serum. Antibody-dependent complement-mediated sperm lysis ranged from 35% to 65%. Normal sperm incubated with normal serum had approximately 200 molecules of IgG per sperm, whereas normal sperm incubated with patient sera had 546 to 900 molecules of IgG per sperm. In all cases where serum enhanced IgG sperm opsonization, there was enhanced mouse macrophage-mediated phagocytosis of the opsonized sperm (a three- to fourfold increase). These data suggest that antisperm antibodies may affect reproduction by different mechanisms, including direct humoral effects (immunoglobulin and/or complement) and indirect cell-mediated effects (macrophage-mediated sperm phagocytosis). However, the mechanism(s) involved are unique to each individual's antibody. The heterogeneity of these potential mechanisms may explain why the presence of antisperm antibodies as measured by a single assay correlate poorly with infertility.

Macrophages and infertility: enhancement of human macrophage-mediated sperm killing by antisperm antibodies.

The mechanism by which antisperm antibodies inhibit fertility is not completely understood. Macrophages may play a role in mediating infertility by interacting with sperm and destroying gametes. Experiments were conducted evaluating the effect of antisperm antibody on the phagocytosis and lysis of sperm by human peritoneal macrophages in vitro. Sperm from a fertile man treated with sera from normal men and women or medium alone had 5 to 280 molecules of IgG/sperm, as determined by a 125I-labeled anti-human IgG monoclonal antibody assay. By contrast, sperm treated with sera containing antisperm antibodies had 310 to 1240 molecules of IgG/sperm. Peritoneal macrophages harvested from infertile women with tubal/adhesive problems mediated phagocytosis and lysis of 111In-labeled sperm which was enhanced by treatment of the sperm with sera containing antisperm antibodies (39.0% +/- 1.5% versus 76.3% +/- 3.2% phagocytosis, and 3.3% +/- 0.3% versus 23.3% +/- 2.3% lysis of sperm [control versus antibody-treated]). The likelihood of fertilization in couples with antisperm antibody may be determined not only by the antibody but also by the presence of genital tract macrophages capable of destroying the antibody-coated sperm.

Ovulation induction with pulsatile gonadotropin-releasing hormone administration in patients with polycystic ovarian syndrome.

Gonadotropin-releasing hormone (0.025 microgram/kg) was administered intravenously in a pulsatile fashion to four subjects with polycystic ovary syndrome for a total of six cycles. Five of the six cycles culminated in ovulation, although in one course the response occurred too early to be attributed to the therapy alone. No pregnancies resulted. All luteal phases were of normal duration, but progesterone production as manifested by serum progesterone determination was deficient in some. If additional investigation confirms these preliminary findings, this form of therapy may offer a safe and economic alternative for anovulatory patients refractory to clomiphene citrate therapy. The response of the four subjects suggests that pulsatile gonadotropin-releasing hormone administration may override hypothalamic-pituitary dysfunction and result in ovulatory menstrual cycles.

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.

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.

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 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).

Morphometric assessment of the murine ovarian toxicity of 7,12-dimethylbenz(a)anthracene.

7,12-Dimethylbenz(a)anthracene (DMBA) is a polycyclic aromatic hydrocarbon and a component of cigarette smoke that has been identified as a murine reproductive toxicant. The morphometric parameters of total ovarian volume, individual corpus luteum volumes, and total corpora lutea volume were measured in C57BL/6N mice treated with DMBA. Each group received single intraperitoneal injections of 0, 0.1, 1.0, or 10 mg/kg and were sacrificed at 1, 2, 3, or 4 weeks after treatment. DMBA produced a dose-dependent decrease in ovarian volume and number of corpora lutea in each ovary. The observed reduction in total corpora lutea volume did not fully account for the loss in total ovarian volume. This is consistent with previous descriptions of a toxic effect on all ovarian components including growing and resting follicles. Growing follicles that escaped the toxic effects of DMBA and achieved ovulation resulted in a corpus luteum that appeared histologically normal. Morphometric analysis of this animal model further defines the dynamic changes in the mouse ovary in response to DMBA.

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.

In vitro fertilization: an overview.

The advanced reproductive technologies are an acceptably safe but invasive method to treat infertility. The decision to proceed with IVF/ET should be based on 1) a complete infertility investigation, 2) an understanding by the couple of the emotional, physical and financial aspects of the process and 3) an understanding by physicians of the real advantages and disadvantages of the procedures.