Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus).

The structural features of the chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus) were examined by thin-section and freeze-fracture electron microscopy, with particular emphasis on the morphological appearance of occluding junctions. This epithelium is a flat sheet consisting predominantly of groups of mitochondriarich chloride cells with their apices associated to form apical crypts. These multicellular groups are interspersed in an otherwise continuous pavement cell epithelial lining. The epithelium may be mounted in Ussing-type chambers, which allow ready access to mucosal and serosal solutions and measurement of electrocal properties. The mean short-circuit current, potential difference (mucosal-side negative), and DC resistance for 19 opercular epithelia were, respectively, 120.0 +/- 18.2 microA/cm2, 12.3 +/- 1.7 mV, and 132.5 +/- 26.4 omega cm2. Short-circuit current, a direct measure of Cl- transport, was inhibited by ouabain (5 micron) when introduced on the serosal side, but not when applied to the mucosal side alone. Autoradiographic analysis of [3H]-ouabain-binding sites demonstrated that Na+,K+-ATPase was localized exclusively to basolateral membranes of chloride cells; pavement cells were unlabeled. Occluding junctions between adjacent chloride cells were remarkably shallow (20-25 nm), consisting of two parallel and juxtaposed junctional strands. Junctional interactions between pavement cells or between pavement cells and chloride cells were considerably more elaborate, extending 0.3-0.5 micron in depth and consisting of five or more interlocking junctional strands. Chloride cells at the lateral margins of crypts make simple junctional contacts with neighboring chloride cells and extensive junctions with contiguous pavement cells. Accordingly, in this heterogeneous epithelium, only junctions between Na+,K+-ATPase-rich chloride cells are shallow. Apical crypts may serve, therefore, as focal areas of high cation conductivity across the junctional route. This view is consistent with the electrical data showing that transmural resistance across the opercular eptihelium is low, and with recent studies demonstrating that transepithelial Na+ fluxes are passive. The simplicity of these junctions parallels that described recently for secretory cells of avian salt gland (Riddle and Ernst, 1979, J. Membr. Biol., 45:21-35) and elasmobranch rectal gland (Ernst et al., 1979, J. Cell Biol., 83:(2, Pt. 2):83 a[Abstr.]) and lends morphological support to the concept that paracellular ion permeation plays a central role in ouabain-sensitive transepithelial NaCl secretion.

The effect of transforming growth factor-beta on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells.

Growth factors have been shown to modulate differentiation of cultured ovarian granulosa cells. Transforming growth factors (TGFs) constitute a family of polypeptide growth factors capable of reversibly inducing anchorage-independent growth in normal cells. Epidermal growth factor (EGF), which has significant structural homology with TGF alpha, has been shown to modulate differentiation of granulosa cells in vitro. Similarly, TGF beta (TGFB) has been found to have significant structural homology with ovarian follicular fluid inhibin. To examine whether TGFB might affect granulosa cell growth or differentiation, rat granulosa cells were cultured in serum-free medium containing insulin for up to 3 days with varying concentrations of TGFB in the presence or absence of FSH. TGFB caused a dose-dependent increase in FSH-stimulated LH/hCG receptor binding, but had no effect on binding in the absence of FSH; TGFB (10.0 ng/ml) further increased FSH-stimulated LH/hCG receptor binding by 48 +/- 8% (P less than 0.02). Similarly, FSH-stimulated progesterone production was increased by TGFB in a dose-dependent manner; TGFB (1.0-10.0 ng/ml) increased FSH-stimulated progesterone production 2- to 3-fold (P less than 0.02). In contrast, EGF (10.0 ng/ml) decreased FSH-stimulated LH/hCG receptor binding by 93 +/- 1% (P less than 0.02). Neither FSH-stimulated intracellular nor extracellular cAMP accumulations were affected by TGFB treatment. However, EGF (10.0 ng/ml) diminished extracellular and intracellular FSH-stimulated cAMP accumulation at 48 and 72 h of culture. Culture protein and DNA content were not significantly affected by TGFB. These results suggest that TGFB may enhance FSH-stimulated LH receptor induction and steroidogenesis by mechanisms that do not further increase net cellular cAMP accumulation; TGFB and EGF can have opposite effects on gonadotropin-dependent differentiation; and products of the TGFB/inhibin gene family may have a capacity for autocrine or paracrine modulation of granulosa cell differentiation.

Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: a prospective, controlled study in 254 affected women.

Women with polycystic ovary syndrome (PCOS) are insulin resistant, have insulin secretory defects, and are at high risk for glucose intolerance. We performed this study to determine the prevalence of glucose intolerance and parameters associated with risk for this in PCOS women. Two-hundred and fifty-four PCOS women, aged 14-44 yr, were prospectively evaluated at 2 centers, 1 urban and ethnically diverse (n = 110) and 1 rural and ethnically homogeneous (n = 144). The rural PCOS women were compared to 80 control women of similar weight, ethnicity, and age. A 75-g oral glucose challenge was administered after a 3-day 300-g carbohydrate diet and an overnight fast with 0 and 2 h blood samples for glucose levels. Diabetes was categorized according to WHO criteria. The prevalence of glucose intolerance was 31.1% impaired glucose intolerance (IGT) and 7.5% diabetes. In nonobese PCOS women (body mass index, <27 kg/m2), 10.3% IGT and 1.5% diabetes were found. The prevalence of glucose intolerance was significantly higher in PCOS vs. control women (chi2 = 7.0; P = 0.01; odds ratio = 2.76; 95% confidence interval = 1.23-6.57). Variables most associated with postchallenge glucose levels were fasting glucose levels (P < 0.0001), PCOS status (P = 0.002), waist/hip ratio (P = 0.01), and body mass index (P = 0.021). The American Diabetes Association criteria applied to fasting glucose significantly underdiagnosed diabetes compared to the WHO criteria (3.2% vs. 7.5%; chi2 = 4.7; P = 0.046; odds ratio = 2.48; 95% confidence interval = 1.01-6.69). We conclude that 1) PCOS women are at significantly increased risk for IGT and type 2 diabetes mellitus at all weights and at a young age; 2) these prevalence rates are similar in 2 different populations of PCOS women, suggesting that PCOS may be a more important risk factor than ethnicity or race for glucose intolerance in young women; and 3) the American Diabetes Association diabetes diagnostic criteria failed to detect a significant number of PCOS women with diabetes by postchallenge glucose values.

Effect of age on response to human menopausal gonadotropin stimulation.

Conception rates decline in the latter part of the reproductive years. To examine which ovarian parameters are altered with aging, 486 cycles from 225 ovulatory infertile women undergoing human menopausal gonadotropin (hMG) superovulation and washed intrauterine insemination were analyzed. Infertility factors included endometriosis (68%), unexplained infertility (8.4%), male factor (12.9%), and ovulatory dysfunction (10.7%). Parameters that demonstrated a linear relationship with increasing age included numbers of ampules of hMG required per cycle (r = 0.79; P less than 0.05), days of stimulation (r = 0.73; P less than 0.01), estradiol level at the time of hCG (r = -0.92; P less than 0.0001), number of follicles larger than 15 mm (r = -0.61; P less than 0.05), and rate of rise of estradiol (r = -0.92; P less than 0.0001). These same age-dependent changes were observed in women receiving a standard stimulation protocol (3 ampules hMG beginning on cycle day 2). When standard cycles were limited to the first cycle only, the preovulatory estradiol (r = -0.92; P less than 0.005), slope of estradiol rise (r = -0.92; P less than 0.005), and number of preovulatory follicles (r = -0.92; P less than 0.005) still showed a significant decrease with age. Although the mean estradiol level per preovulatory follicle showed a slight decrease with maternal age, no statistically significant trend was noted. In addition, the cycle day of hCG administration was unaffected by age. With advancing age, there appears to be a decreased ovarian response to an increased amount of stimulation, as measured by steroidogenesis and follicular recruitment; yet the estradiol/follicle remains unaltered, indicating continued health of the follicle. These observations may explain in part the observed decrease in fecundity in older women.

The effect of leuprolide acetate on ovulation induction with human menopausal gonadotropins in polycystic ovary syndrome.

The use of exogenous gonadotropins for treatment of clomiphene-resistant chronic anovulation in women with the polycystic ovary syndrome (PCO) is hazardous and often ineffective, possibly because of the abnormal endogenous gonadotropin secretion characteristic of PCO. We evaluated the effect of leuprolide acetate, a long-acting GnRH agonist, on serum gonadotropin and sex steroid concentrations before and during human menopausal gonadotropin (hMG) induction of ovulation in women with PCO. In this controlled prospective randomized study, leuprolide was administered daily for 4 weeks, followed by concomitant hMG administration. Gonadotropin and steroid hormone concentrations were compared with those during ovulation induction cycles in women with PCO receiving hMG only. Daily administration of leuprolide for 4 weeks resulted in significantly decreased serum LH, estradiol, and testosterone concentrations, but no change in serum progesterone, FSH, and dehydroepiandrosterone sulfate. Compared to ovulation induction using hMG alone, leuprolide administration before and during hMG treatment prevented preovulatory rises in serum LH and P concentrations, while having no effect on serum FSH, testosterone, estradiol, and dehydroepiandrosterone sulfate. We conclude that leuprolide administered to women with PCO decreases gonadal steroid production and is capable of preventing premature luteinization during hMG induction of ovulation.

An experimental model for the endometriosis in athymic mice.

Endometriosis is an adhesion disorder characterized by the presence of endometrial tissue in ectopic sites outside the uterus. The disease is associated with dysmenorrhea, pelvic pain and infertility. Although endometriosis is the most common gynecologic disorder, relatively little is known regarding its etiology, pathogenesis and the course of the disease. This situation is primarily due to the absence of experimental systems to examine the mechanism of endometrial cell adhesion, role of inflammatory cells and the interactions of epithelial, and stromal cells with the peritoneum and ovarian tissue leading to the development of this disorder. Dissociated human endometrial cells were suspended in peritoneal fluids of individuals with and without endometriosis and were injected into the peritoneal cavity of athymic mice. This led to development of ectopic adhesions of endometrial cells at the peritoneal and ovarian surfaces. Endometrial cells which were marked with fluorescent lipophylic dyes, prior to intraperitoneal injection, could be visualized without surgery at such sites. The studies demonstrate a model for endometriosis in athymic mice.

Accuracy of ultrasound in fetal femur length determination. Ultrasound phantom study.

An ultrasound phantom was constructed simulating fetal femurs in amniotic fluid. Bones of 26, 36, 50, 58, and 70 mm, representative of gestational ages ranging from 17 to 36 weeks, were scanned with mechanical sector, phased sector, and linear array systems (ATL, Diasonics, Acuson, and GE). Measurements were made with the bone in both a horizontal and nearly vertical orientation at 5, 10, and 15 cm from the transducer. The ultrasound measurements were compared with the true bone length. With bones in a nearly vertical orientation (parallel to the ultrasound beam) the ultrasound measurements corresponded more closely to the true bone length regardless of the type of equipment or distance from the transducer. The wide aperture linear system was most accurate with no measurable difference from the actual bone length and a mechanical sector scanner had the largest error which was 6 mm. In the horizontal position (perpendicular to the beam) the smallest errors occurred when the bone was in the focal zone. This ranged from no error for the wide aperture linear array to 8 mm for the mechanical sector scanner. When the bone was not in the focal zone the error ranged from 8 to 26 mm for the mechanical sector scanner. Errors in ultrasound-measured femur lengths can be shown to result from the focal characteristics of the equipment as well as the orientation and distance of the bone from the transducer. These differences can produce errors in estimation of gestational age as large as ten weeks.

Adjunctive leuprolide therapy does not improve cycle fecundity in controlled ovarian hyperstimulation and intrauterine insemination of subfertile women.

Problems arising from controlled ovarian hyperstimulation for intrauterine insemination, such as premature luteinization and asynchronous ovarian follicular development, are identical to those encountered with controlled ovarian hyperstimulation for in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT). It has been suggested that the adjunctive use of GnRH agonists for controlled ovarian hyperstimulation improves the efficiency of GIFT and IVF cycles. We hypothesized that adjunctive use of leuprolide acetate, a GnRH agonist, would have a similarly beneficial effect on cycle quality and cycle fecundity in subfertile women treated with controlled ovarian hyperstimulation and intrauterine insemination. We randomly assigned the first cycle of controlled ovarian hyperstimulation and intrauterine insemination for each of 97 subfertile women to include either human menopausal gonadotropins (hMGs) alone or hMGs following midluteal pre-treatment with leuprolide. If a pregnancy did not occur in the first cycle, the woman was given the other treatment in the second cycle. Although the cycles that included leuprolide required a larger amount of hMGs and more days of stimulation per cycle, the mean estradiol concentrations and numbers of follicles were not different. Despite prevention of premature luteinization with leuprolide, the cycle fecundity was not different between groups (0.11 with adjunctive leuprolide treatment and 0.22 with hMGs alone). We conclude that in unselected subfertile patients, the adjunctive use of leuprolide for controlled ovarian hyperstimulation and intrauterine insemination does not improve cycle fecundity compared with treatment cycles that do not include adjunctive leuprolide therapy.

Controlled ovarian hyperstimulation and intrauterine insemination for treatment of infertility.

Empirical therapy for subfertility using assisted reproductive technologies recently has gained popularity; however, the cost-effectiveness of these therapies, compared with an untreated control group, has not been established. Similarly, there has been no comparative cost analysis of the utility of controlled ovarian hyperstimulation and IUI in the management of the same condition. Significant PRs in untreated couples with subfertility mandate the design and execution of controlled trials to ascertain the role of controlled ovarian hyperstimulation and IUI in infertility therapy. Various disorders of subfertility have been treated with controlled ovarian hyperstimulation and IUI. The rationale for this therapy is the increase in gamete density at the site of fertilization, as with GIFT and IVF when used for management of the same problems. The live birth rate per initiated cycle and risk of complications are similar to results recently reported for GIFT and IVF. The utility of controlled ovarian hyperstimulation and IUI still remains controversial. When the relatively low direct and indirect costs of controlled ovarian hyperstimulation and IUI are considered, acknowledging the lack of prospective, controlled studies, this procedure appears to be at least as cost-effective as GIFT and IVF.

The quality of human embryo growth is improved when embryos are cultured in groups rather than separately.

OBJECTIVE: To determine the effect of culturing human embryos in groups on cleavage rates, morphology grades, and embryo scores when compared with embryos cultured singly. DESIGN: Prospective. SETTING: The IVF-ET program of the Pennsylvania State University, Department of Obstetrics and Gynecology, Hershey Medical Center, Hershey, Pennsylvania. PATIENTS: Fifty-five infertile women who each had at least five zygotes underwent IVF-ET. INTERVENTIONS: Zygotes from each patient were allocated to be cultured singly and in groups. MAIN OUTCOME MEASURES: Cleavage rate, morphology grade, and embryo score. RESULTS: Grouping embryos significantly enhanced cleavage rates and embryo scores but not morphology grade as compared with embryos grown singly. Additionally, the size of the groups correlated positively with cell number and embryo score but not the morphology grade. CONCLUSION: Culturing human embryos in groups enhances the quality of their growth by increasing the cleavage rates and embryo scores. Because pregnancy rates are improved by transferring embryos with higher embryo scores, coculturing human embryos may be a way of enhancing pregnancy rates.

Leuprolide acetate: serum and follicular fluid concentrations and effects on human fertilization, embryo growth, and granulosa-lutein cell progesterone accumulation in vitro.

Data from various animal models have demonstrated significant extrapituitary effects of gonadotropin-releasing hormone agonists. The purpose of this study was to determine the effects of therapeutic concentrations of leuprolide acetate on human granulosa-lutein cell steroidogenesis, fertilization, and embryo growth rates in vitro. During leuprolide administration, mean serum concentrations of leuprolide were less than 50 ng/ml and were undetectable 48 hours after cessation of administration. There was no effect of leuprolide on progesterone (P) secretion by granulosa-lutein cells cultured in the presence or absence of human chorionic gonadotropin. The effect of leuprolide on embryo growth rates was evaluated with the mouse two-cell embryo culture model and a retrospective review of early embryo growth rates in humans receiving adjunctive leuprolide therapy. There was no measurable effect of leuprolide on early embryo growth in either species. These data indicate that (1) serum and follicular and peritoneal fluid concentrations are undetectable 2 days after discontinuation of leuprolide; (2) there is no measurable effect of leuprolide on human or murine embryo growth rates up to the 8 cell stage in vitro; and (3) there is no measurable effect of leuprolide on granulosa-lutein cell P accumulation.

Ovarian hyperstimulation in polycystic ovary syndrome during therapy with leuprolide acetate.

Continuous exposure to GnRH eliminates the pituitary as a source of gonadotropins and may have direct suppressive effects on the ovary. A woman with PCO syndrome received leuprolide acetate (1 mg/d SC) for 4 weeks before and simultaneously with hMG stimulation. Human chorionic gonadotropin (5,000 IU) was administered IM on the 8th day of hMG therapy. There were 10 follicles greater than 15 mm and a polycystic appearance to the ovaries with 25 follicles measuring less than 10 mm. The serum E2 concentration was 2,280 pg/mL. She developed severe ovarian hyperstimulation and required hospitalization for 12 days for fluid management. A viable intrauterine pregnancy was present. Four weeks of pretreatment with leuprolide did not prevent hyperstimulation in the presence of an intrauterine pregnancy.

The luteal phase in polycystic ovary syndrome during ovulation induction with human menopausal gonadotropin with and without leuprolide acetate.

Little data exist on the effects of adjunctive therapy with leuprolide acetate (LA) in the luteal phase of women with polycystic ovary syndrome (PCOS) undergoing ovulation induction with human menopausal gonadotropin (hMG). Additionally, it is not known whether gonadal steroid concentrations in the luteal phase of induced cycles in PCOS are predictive of pregnancy. In this prospective, randomized study comparing cycles using hMG alone (n = 26) with cycles using hMG with LA (n = 33), no differences were noted between treatment groups in progesterone (P), estradiol (E2), and P:E2 ratios on luteal days 3, 6, and 9. When all treatment cycles were pooled, there were no differences in P, E2, or P:E2 ratios, comparing conception and nonconception cycles. We conclude that adjunctive therapy with LA in PCOS patients undergoing ovulation induction with hMG does not alter the luteal phase concentrations of P, E2, and P:E2. Furthermore, no correlation was found between the serum concentrations of these luteal phase steroids and cycle fecundity.

Clinical characteristics of ovulation induction with human menopausal gonadotropins with and without leuprolide acetate in polycystic ovary syndrome.

Ovulation induction in polycystic ovary syndrome (PCOS) with human menopausal gonadotropins (hMG) results in suboptimal cycle fecundity and frequently is complicated by ovarian hyperstimulation. The use of a gonadotropin releasing-hormone agonist (Gn-RH-a) with hMG induction of ovulation may improve the therapeutic outcome. In this prospective, randomized trial, 27 women with PCOS underwent a total of 25 cycles of hMG alone and 33 cycles with adjunctive GnRH-a (leuprolide) treatment. Premature luteinization was seen less frequently in the leuprolide-treated cycles than in cycles treated with hMG alone. There were no differences between the treatments in ovarian sensitivity to hMG. Cycle fecundity was 0.16 for hMG alone cycles, and 0.27 for leuprolide with hMG cycles, which were not statistically different. We conclude that the sensitivity of the PCOS ovary to hMG is not affected by 4 weeks of leuprolide pretreatment.

Treatment-independent, treatment-associated, and pregnancies after additional therapy in a program of in vitro fertilization and embryo transfer.

Although the technique of in vitro fertilization and embryo transfer (IVF-ET) was developed for couples with untreatable tubal factor infertility, IVF-ET is now being applied to women with other causes of infertility and normal pelvic anatomy. In an effort to determine the treatment-independent pregnancy rate, we retrospectively reviewed the first 245 couples enrolled in the IVF-ET program at Duke University Medical Center. There were 19 treatment-independent pregnancies in 18 women and 3 treatment-associated pregnancies in cycles in which the oocyte retrieval was canceled (in 2 women washed intrauterine insemination was substituted for oocyte retrieval). Six pregnancies were established after an unsuccessful attempt at IVF-ET with additional non-IVF-ET therapy, including washed intrauterine insemination in three couples, and donor insemination in two couples. These observations suggest that a significant number of treatment-independent pregnancies will occur in couples clinically deemed appropriate for IVF-ET, pregnancies can be established in cycles of controlled hyperstimulation without oocyte retrieval, and additional non-IVF-ET therapy can result in pregnancy despite failure of IVF-ET in selected couples.

Superovulation with intrauterine insemination in the treatment of infertility: a possible alternative to gamete intrafallopian transfer and in vitro fertilization.

In vitro fertilization and embryo transfer (IVF-ET) and gamete intrafallopian transfer (GIFT) are used to treat intractable infertility in women with no distortion of the pelvic viscera, despite the lack of controlled trials demonstrating efficacy. The mechanism of any purportedly enhanced cycle fecundity in ovulatory women without significant distortion of the pelvic viscera is unclear, but both GIFT and IVF-ET increase the number of male and female gametes at the site of fertilization. Intrauterine insemination (IUI) during human menopausal gonadotropin (hMG)-stimulated superovulatory cycles has similar potential but does not require oocyte retrieval. To evaluate the possibility that simply increasing the number of gametes at the site of fertilization might account for pregnancies attributed to IVF-ET and GIFT, the authors retrospectively analyzed the outcome of couples undergoing IUI during hMG cycles between 1983 and 1986 in women with normal pelvic anatomy. IUI during hMG-stimulated cycles yielded a cycle fecundity (f) of 0.17 for endometriosis, 0.29 for cervical factor, and 0.19 for idiopathic infertility, which approaches the fecundity of normal women and equals or exceeds that reported for IVF-ET and GIFT. The authors conclude that treatment with IUI in hMG cycles, alleviating the need for invasive oocyte retrieval, should be considered for inclusion in a randomized, controlled trial in comparison with IVF-ET and GIFT.

Spontaneous conception in the presence of stage IIIC endometrioid ovarian cancer.

OBJECTIVE: To describe a rare case of spontaneous conception in a patient with a preexisting metastatic ovarian cancer. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 39-year-old Asian woman who conceived while undergoing an evaluation for primary infertility and newly detected bilateral adnexal masses. INTERVENTION(S): Staging laparotomy and total abdominal hysterectomy and bilateral salpingo-oophorectomy. MAIN OUTCOME MEASURE(S): Anatomic pathology diagnosis. RESULT(S): Blighted ovum and stage IIIC endometrioid adenocarcinoma of ovary. CONCLUSION(S): Metastatic ovarian cancer does not prevent either spontaneous ovulation or spontaneous conception.

A randomized comparison of the methods of sperm preparation for intrauterine insemination.

OBJECTIVE: To compare the effectiveness of three methods of sperm preparation for IUI during superovulation of infertile women. DESIGN: Randomized assignment of one of three sperm preparation methods. SETTING: University infertility practice. PATIENT(S): Infertile couples undergoing superovulation and IUI. INTERVENTION(S): The method of preparation of sperm for IUI during superovulation was assigned randomly to double centrifugation, multiple-tube swim-up, or Percoll density gradient. MAIN OUTCOME MEASURE(S): Total number and percent recovery of motile sperm, percent of recovered sperm with normal morphology, and cycle fecundity. RESULT(S): No method of sperm preparation provided better cycle fecundity than the others despite differences in sperm recovery. CONCLUSION(S): Double centrifugation, multiple-tube swim-up, and Percoll density gradient sperm preparation for IUI yield similar cycle fecundity rates.