Pulsatile pituitary gonadotropin secretion during maturation of the dominant follicle in monkeys: estrogen positive feedback enhances the biological activity of LH.

In the follicular phase of the menstrual cycle, pulsatile patterns of LH and FSH secretion in monkeys change during maturation of the dominant follicle. At the preovulatory surge, the most striking event is the prodigious elevations of bioassayable LH, rising up to 50-fold within 24 h. Principally, establishment of the surge is due to marked enhancement of the amplitude of LH secretory pulses. In contrast, LH and FSH measured by RIA enter, in parallel, the surge modes of secretion approximately 5 h later than bioassayable LH and rise more slowly; the B:I ratio may reach 10:1. This same disparity between bioassayable versus immunoassayable LH was induced in castrate monkeys under estrogen positive feedback stimulation. We conclude that the preovulatory estrogen surge promotes the secretion of an LH molecule(s) having enhanced biological activity.

Partial characterization of a uteroglobin-like protein in the human uterus and its temporal relationship to prostaglandin levels in this organ.

During the past decade several corticosteroid-dependent, low mol wt proteins with phospholipase-A2 (PLA2) inhibitory activity have been described. This family of proteins is collectively known as lipocortins. Blastokinin or uteroglobin (utg), a progesterone-induced protein, first discovered in the pregnant rabbit uterus, is also a potent PLA2 inhibitor, but genetically distinct from lipocortins. Although utg has been found in rabbits, its presence in humans has not been well established. Here, we present biochemical, immunological, and immunohistological evidence for the detection of a utg-like protein in the human uterus. Since inhibition of PLA2 may modulate tissue eicosanoid levels and since rabbit utg has been reported to be a potent PLA2 inhibitor, we also studied the temporal relationship between utg and tissue prostaglandin E2 and F2 alpha levels in estrogen- and progesterone-dominated endometrial tissue. We found an inverse temporal relationship between utg-like protein and eicosanoid levels in this organ. Since some eicosanoids (e.g. prostaglandins, leukotrienes, etc.) are known to be involved in smooth muscle contractility and inflammatory processes, our findings may help to understand the pathogenesis of some human disorders in which abnormal eicosanoid production occurs.

Suppression of lymphocyte proliferation in vitro by macromolecules in the vesicle fluid and tissue extracts of hydatidiform mole.

This study examines the effects of vesicle fluid and tissue extracts from hydatidiform mole trophoblast on lymphocyte proliferation in vitro. Samples were obtained by direct aspiration of vesicles (hydatidiform mole vesicle fluid (HMF] or homogenization of molar tissues (hydatidiform mole extract (HME] following therapeutic uterine evacuation of hydatidiform mole. Dialyzed and lyophylized HMF pooled from two patients exhibited a 30% suppression (P less than 0.05) of mitogen-induced lymphocyte proliferation at a concentration of 12.5 micrograms protein/ml. Similarly, lymphocyte transformation was significantly suppressed (P less than 0.05) by HME at concentrations of 500 and 250 micrograms/ml. Molecular weight chromatography of HME resolved 4 protein fractions. Fraction 3 (35--50 kDa) and fraction 4 (less than 35 kDa) significantly suppressed mitogen-induced lymphocyte transformation while fractions 1 and 2 demonstrated no immunosuppression. Heat treatment (56 degrees C, 30 min) abolished the immunosuppressive activity of HME as well as fractions 3 and 4. These results suggest that hydatidiform mole trophoblast contains heat-labile macromolecules which suppress mitogen-mediated lymphocyte transformation. Such trophoblast-derived factors may interfere with maternal rejection of the allograft.

Expression of Clara cell 10-kD gene in the human endometrium and its relationship to ovarian menstrual cycle.

Clara cell 10-kD (cc10-kD) protein has been suggested to be the human counterpart of rabbit uteroglobin (UG). Like UG, this protein is also a potent inhibitor of phospholipase A2 (PLA2) and a substrate of transglutaminase. Although it has been established that UG gene expression in the rabbit endometrium is stimulated by progesterone, the expression of cc10-kD gene in the human endometrium is not clearly understood. The present study was undertaken to determine whether the cc10-kD gene is expressed in the human endometrium and whether its level of expression changes in relation to the ovarian menstrual cycle. Using reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence, we demonstrate that the cc10-kD gene is expressed in different stages of the menstrual cycle and that the highest level of expression is reached during the luteal phase. These results suggest that like rabbit UG, cc10-kD gene expression in the human endometrium may be stimulated by progesterone. Since cc10-kD is a potent inhibitor of PLA2 activity, this protein may play an important physiological role in regulating eicosanoid levels in the human uterus.

Treatment of persistent ectopic pregnancy with methotrexate and leukovorum rescue: a case report.

The incidence of ectopic pregnancy is increasing in the Western world, and this reproductive complication has had an adverse impact on subsequent fertility. Advances in the surgical treatment of ectopic pregnancies have been designed to preserve future reproductive potential, but conservative tubal surgery may fail to completely remove the trophoblast. Described is a case of persistent ectopic pregnancy successfully treated with methotrexate.

Life-threatening neutropenia following methotrexate treatment of ectopic pregnancy: a report of two cases.

BACKGROUND: Medical treatment of ectopic pregnancy with methotrexate is an increasingly common alternative to surgical management. Initial reports of methotrexate therapy described a very low incidence of complications. We report our experience with two patients who developed profound toxicity following methotrexate treatment of ectopic pregnancy. CASE: The first patient received a single dose of methotrexate (50 mg/m2 intramuscularly) for a confirmed ectopic pregnancy. The second patient received three doses of methotrexate (1 mg/kg). Both patients developed life-threatening neutropenia and febrile morbidity requiring hospitalization and supportive care. CONCLUSION: To our knowledge, this is the first description of significant morbidity secondary to bone marrow suppression following methotrexate treatment of ectopic pregnancy. Most patients with ectopic pregnancy who are treated with methotrexate can expect resolution of their symptoms and a low risk of mild complications. However, serious complications after this therapy are possible and may occur even with the single-dose regimen.

Gonadotropin-releasing hormone agonist (leuprolide acetate) induced ovarian hyperstimulation syndrome in a woman undergoing intermittent hemodialysis.

Moderate ovarian hyperstimulation syndrome occurred after LA was administered to control menorrhagia in an anephric woman who required hemodialysis. We postulate that women who require dialysis may be at special risk for the development of this syndrome.

Relative operating characteristic analysis in reproductive medicine: comparison of progesterone and human chorionic gonadotropin doubling time as predictors of early gestational normalcy.

OBJECTIVE: To directly compare the ability of serum P and hCG doubling time to predict early gestational complications. DESIGN: We analyzed differences in P concentrations and hCG doubling times between spontaneously conceived normal and abnormal pregnancies (abortions and tubal pregnancies) during the first 49 days of pregnancy. Multiple 2 x 2 contingency tables were constructed that compared pregnancy outcome within discriminatory serum P concentrations or hCG doubling time. From these tables relative operating characteristic curves were generated for P concentration and hCG doubling times. The area under the curves were estimated and compared by the critical z score. RESULTS: Calculation of the areas under the curve for hCG doubling time and P concentration yielded values of 0.799 +/- 0.056 and 0.780 +/- 0.051 (mean +/- SEM), respectively. Critical z testing did not reveal a significant difference between the two curves. CONCLUSION: Serum P and hCG doubling times equally predict early gestational normalcy. Thus, a single serum P may be just as valuable in estimating risks of early pregnancy complications as repeated hCG measurements.

Extended clomiphene citrate (CC) and prednisone for the treatment of chronic anovulation resistant to CC alone.

OBJECTIVE: To evaluate effectiveness and safety of a regimen of extended clomiphene citrate (CC) and prednisone for patients who fail treatment with CC alone. DESIGN: Retrospective observational analysis. SETTING: University-based tertiary infertility center. PATIENT(S): Twenty-four anovulatory patients who failed to ovulate after CC 150 mg administered for 5 days. INTERVENTION(S): Treatment consisted of CC given on cycle days 3 through 9 (extended) at a starting dose of 100 to 150 mg/d. Additionally, patients were given prednisone 5 mg orally each night throughout the cycle. MAIN OUTCOME MEASURE(S): Ovulation was confirmed by luteal serum P. Pregnancy was confirmed by rising hCG levels and transvaginal ultrasound. RESULT(S): A total of 60 cycles were available for review. Forty-four of these cycles were ovulatory (73%) and 11 patients (46%) conceived on this therapy. Logistic (two-parameter) pregnancy occurrence over time (cycles) revealed a maximum pregnancy probability of 0.66 and a cycle fecundity of 0.36. No complications of therapy were noted. CONCLUSION(S): Clomiphene citrate-resistant anovulatory patients have high rates of ovulation and pregnancy after treatment with extended CC and prednisone. This therapy offers a potential reduction in cost and risk and should be considered in this group of patients before gonadotropin stimulation or surgery.

The egg-embryo chamber for intraabdominal culture.

We have developed a novel semipermeable chamber for intraabdominal culture of primate gametes and embryos. Application of the egg-embryo chamber offers a unique opportunity to study fertilization and preimplantation embryogenesis. Intrinsically normal monkey embryos displayed timely cleavage and symmetric form when cultured in this intraabdominal chamber for up to 72 hours. The birth of the first normal live monkey infant derived from the egg-embryo chamber is reported.

Cumulative pregnancy analysis of one-tube versus two-tube tubal anastomosis.

OBJECTIVE: To determine the prognosis for pregnancy when tubal anastomosis can be performed on only one tube. DESIGN: Prospective collection of demographic and clinical data. SETTING: University medical center. PATIENT(S): One hundred twenty-six women undergoing elective sterilization reversal. INTERVENTION(S): Sterilization reversal was performed by five reproductive endocrine surgeons. In 35 cases, only one tube could be treated surgically. In all cases, this was because of a previous salpingectomy or inadequate distal tubal segment. MAIN OUTCOME MEASURE(S): Cumulative probability of pregnancy in the two groups (one tube or two tubes) was analyzed by survival analysis and logistic regression. Survival curves were compared by log-rank testing. RESULT(S): Comparison of survival curves revealed no difference between the logistic regression curves for probability of conception after anastomosis of one tube versus two tubes. Logistic regression revealed a similar cumulative probability of conception with a two-tube (0.76) and a one-tube anastomosis (0.63). The monthly fecundity rates were 0.032 and 0.034 for a two-tube and one-tube anastomosis, respectively. There were no differences between the groups with respect to age, gravidity, or parity. Virtually all pregnancies occurred within 2 years of surgery. CONCLUSION(S): The prognosis for conception after one-tube anastomosis was the same as for two-tube anastomosis.

Luteal phase consequences of low-dose gonadotropin-releasing hormone agonist therapy in nonluteal-supported in vitro fertilization cycles.

OBJECTIVE: To determine the follicular and luteal phase impact of low-dose GnRH agonist (GnRH-a) treatment during follicular stimulation for IVF. DESIGN: A randomized prospective study compared patients receiving low-dose GnRH-a and hMG therapy to clomiphene citrate (CC) and hMG cycles. SETTING: Patients were treated through a university-based IVF-ET program. PATIENTS: Thirty-six patients underwent follicular stimulation with low-dose GnRH-a and hMG and were compared with 34 patients undergoing ovulation induction with CC and hMG. RESULTS: Significantly shorter luteal phase length occurred with GnRH-a and hMG therapy; however, there was no statistically significant difference in luteal P levels. Follicular parameters were the same (peak E2, number of follicles, and number of oocytes), suggesting that folliculogenesis was not altered. There were no statistical differences in pregnancy rates. CONCLUSIONS: Sustained low-dose GnRH-a therapy during follicular stimulation does not have a clinical effect on luteal function.

Regulation of early gestational corpus luteum function in spontaneous and follicular stimulated conceptions.

OBJECTIVE: To determine the regulatory role of hCG on P secretion in normal and abnormal (abortive and ectopic) first trimester pregnancies. STUDY DESIGN: The number of doublings of hCG per day (1/DT; reciprocal of hCG doubling time) was correlated with serum P using linear and nonlinear models in normal intrauterine pregnancies, spontaneous abortions, and ectopic pregnancies (EPs) conceived spontaneously or after clomiphene citrate (CC). RESULTS: Linear correlations between P and 1/DTs of hCG were poor. In contrast, nonlinear modeling with a hyperbolic curve fit the data well and allowed all of the data to be included for analysis regardless of the pregnancy type. Furthermore, this model could be used to explain the differential P concentrations seen in normal and abnormal first trimester pregnancies. The nonlinear hyperbolic model demonstrated that follicular events determine the maximal P production during early gestation. Human chorionic gonadotropin 1/DTs approximately equal to 0.5 sustain maximal P production. Progesterone production is then sustained by the rate of change of hCG. CONCLUSION: Nonlinear correlation analysis suggests that P production in early gestations is regulated by prior follicular events and the rate of hCG production.

Serum progesterone predicts abnormal gestations in clomiphene citrate conception cycles as well as in spontaneous conception cycles.

OBJECTIVE: To determine if a "discriminatory" P concentration could be established that predicted abnormal early pregnancies in clomiphene citrate (CC)-conceived cycles. DESIGN: Progesterone concentrations of gestations between 28 and 49 days from last menstrual period in both spontaneously conceived and CC-stimulated cycles were analyzed using a relative-operating characteristic (ROC) curve. INTERVENTIONS: Serum P concentrations were measured in 222 pregnant patients from the first 49 days of gestation. One hundred sixteen patients conceived in a spontaneous cycle and 106 patients conceived in CC-treated cycles. Two by two contingency tables were used to calculate the true-positive (sensitivity) and false-positive rates at 20 specific P at 20 specific P concentrations. A ROC curve was then generated by plotting the sensitivity of the test against the percent of normal patients incorrectly classified (false positive) at each P level. The best discriminatory value was estimated in each curve at a point of high sensitivity associated with a minimal false-positive value. The areas under the curve and SE were calculated for each group and compared by the critical ratio z-test. RESULTS: The best discriminatory P concentration was 10 ng/mL (32 nmol/L) for spontaneously conceived pregnancies and 30 ng/mL (95 nmol/L) for CC-treated pregnancies. The area under each ROC curve was significantly predictive. Comparison of the two curves indicated that the ability of P measurements to predict gestational complications was independent of follicular stimulation. CONCLUSIONS: Follicular stimulation with CC increases the discriminatory P value that predicts gestational normalcy but does not alter the clinical utility of the test.

Elevated luteinizing hormone on the day of human chorionic gonadotropin administration does not reduce cycle fecundity in a low-dose flare-up in vitro fertilization protocol.

OBJECTIVE: To determine if elevated LH at the time of hCG administration occurs and adversely affects success in a low-dose gonadotropin-releasing hormone analogue (GnRH-a) flare-up protocol in hMG-stimulated IVF cycles. DESIGN: Pearson correlation matrix analysis of hormonal, gamete, and clinical data derived from 203 consecutive IVF cycles was performed. All patients were treated with low-dose GnRH-a (250 micrograms SC leuprolide acetate) and hMG. In 203 consecutive IVF cases, serum was obtained on the day of hCG administration and assayed for E2, LH, and P. These data were correlated with peak E2, number of follicles, oocytes, embryos, and conceptions. Additionally, patients with elevated LH were compared with the nonelevated LH group. RESULTS: Twenty six women had LH > 35 mIU/mL (mean +/- SEM; 51.1 +/- 1.9) and five pregnancies (cycle fecundity 19.2% per retrieval). One hundred seventy-seven patients had LH < 35 mIU/mL (16.3 +/- 0.5) and 25 pregnancies (cycle fecundity 14.1%). There were no differences in the mean P (1.0 +/- 0.1 ng/mL, conversion factor to SI unit, 3.81) and E2 (1,672 +/- 144 pg/mL, conversion factor to SI unit, 3.671) of the former group compared with the P (1.1 +/- 0.07 ng/mL) and E2 (1,456 +/- 69 pg/mL) of the latter group. There was no correlation with the number of follicles, oocytes, embryos, pregnancies, E2, or P to LH concentration (rmax = 0.132). CONCLUSION: In a low-dose, GnRH-suppression, IVF induction protocol, elevated LH occurs in a small subset (13%) of women at the time of hCG administration. This event does not appear to alter cycle fecundity nor induce premature luteinization.

Progesterone and 17 alpha-hydroxyprogesterone advance the estrogen-induced bioassayable luteinizing hormone surge in castrate monkeys.

Recent studies have demonstrated marked disparities between bioassayable (BIO) versus radioimmunoassayable concentrations of luteinizing hormone (LH) at midcycle. Other studies in intact women and monkeys have indicated that progesterone (P) may facilitate estrogen induction of the preovulatory gonadotropin surges. We have combined the study of these observations as follows: long-term castrate female monkeys were given estradiol benzoate with and without subsequent P or 17 alpha-hydroxyprogesterone (17-OHP) injections. Plasma was collected during a selected 24-hour interval via a chronic indwelling femoral vein cannula. Initially, estrogen-negative feedback decreased the pulsatility and circulating levels of gonadotropins. P and 17-OHP treatment (after estradiol) hastened initiation of the BIO-LH surge by up to 8 hours. Although estrogen-positive feedback alone enhanced the biologic activity of LH, P and 17-OHP expedited the midcycle-like BIO-LH surges.

Bromocriptine inhibition of anesthesia-induced hyperprolactinemia: effect on serum and follicular fluid hormones, oocyte fertilization, and embryo cleavage rates during in vitro fertilization.

Thirty-two patients undergoing in vitro fertilization (IVF) were given bromocriptine either 1 or 12 hours before anesthesia or received no drug to determine what effect suppression of transient, anesthesia-induced hyperprolactinemia would have on peripheral and follicular fluid hormones, fertilization and cleavage rates, and pregnancy. Thirty minutes after anesthesia, there was a 120-ng/mL rise in serum prolactin (PRL) in control patients versus an insignificant change in women given bromocriptine. Levels of PRL in follicular fluid were significantly less, and estradiol (E2) levels were higher (P less than 0.05) in all bromocriptine-treated patients compared with controls, whereas follicular fluid levels of progesterone (P), inhibin activity, and midluteal serum P were unaffected. Although fertilization and pregnancy rates were similar, a greater proportion of fertilized oocytes from bromocriptine-treated patients advanced to cleaving embryos compared with controls (95% versus 63%, respectively; P less than 0.001). We conclude that bromocriptine, given before anesthesia, can suppress transient, anesthesia-induced hyperprolactinemia and dramatically alter follicular fluid concentrations of PRL and E2. Although these changes in hormonal milieu affected neither oocyte fertilization nor pregnancy rate in our IVF patients, they seemed to have a positive influence on embryonic development after IVF.

Identification of a uteroglobin-like antigen in human uterine washings.

A uteroglobin-like molecule was detected with a sensitive radioimmunoassay for rabbit uteroglobin in the uterine washings of women. This antigen was detectable only in small amounts in the uterine washings obtained from women in the late luteal phase or proliferative phase, but was detected in large amounts in uterine washings of women collected during the early and midluteal phases of the ovarian-menstrual cycle. Our findings suggest that antigenically similar portions of rabbit uteroglobin have been phylogenetically conserved in women, and that endometrial production and secretion of this human uteroglobin-like antigen may be hormonally regulated.

Secretory component in human amniotic fluid and gestational tissues: a potential endogenous phospholipase A2 inhibitor.

OBJECTIVES: Prostaglandins (PGs) are essential mediators of labor during human pregnancy. Phospholipase A2 (PLA2) provides the essential substrate for PG synthesis through the liberation of arachidonic acid from membrane phospholipid stores. Nonlaboring amniotic fluid (NL-AF) contains secretory component (SC)-like protein(s) that suppress in vitro PLA2 activity. This study characterizes the biologic activity, identity, and tissue distribution of these protein(s) in NL-AF and gestational tissues. METHODS: Third-trimester NL-AF was collected by amniocentesis, fractionated by ammonium sulfate precipitation, and submitted to an in vitro PLA2 assay. Identity of the PLA2 inhibitor in NL-AF was confirmed by Western blot and antibody neutralization studies. Secretory component-immunoreactive proteins were purified by immunoaffinity chromatography and visualized by sodium dodecyl sulfate-gel electrophoresis. Tissue distribution of SC in gestational tissues was determined by immunohistochemistry. RESULTS: The 100% pellet and supernatant fractions of NL-AF suppressed PLA2 activity, and this activity was neutralized by a polyclonal antibody to SC. Western blot studies revealed an SC-reactive protein in the 70-80-kD range in the 100% pellet fraction of NL-AF. Two SC-reactive proteins were detected in the 60-80-kD range in the eluate from the SC immunoaffinity column, along with minor proteins of 30 and greater than 100 kD. Immunohistochemical studies revealed SC in placental trophoblast, amniotic membranes, and decidual epithelium. CONCLUSIONS: These results demonstrate that proteins homologous to SC are present in human gestational tissues and possess anti-PLA2 activity. These proteins may contribute to the maintenance of pregnancy by suppressing local PG production.

Hydatidiform mole macromolecules inhibit interleukin-2-mediated murine lymphocyte proliferation in vitro.

Macromolecules extracted from hydatidiform mole trophoblast inhibit mitogen-induced lymphocyte proliferation. To characterize the mechanism of this immunomodulation, we determined the effects of hydatidiform mole vesicle fluid (HMF) and tissue extracts (HME) on lymphokine function in vitro. Utilization of interleukin-1 (IL-1) and interleukin-2 (IL-2) were determined by using a lymphoma cell line (LBRM-33-1A5) and a murine T cell line (CTLL2), respectively. HMF suppressed (P less than .05) IL-2-dependent CTLL2 cell proliferation at 500 (36.4% of controls) and 50 (74.9% of controls) micrograms/ml. HME also suppressed CTLL2 proliferation (P less than .05) at 500 (46.0% of controls), 100 (67.2% of controls), 50 (71.5% of controls), and 10 (85.4% of controls) micrograms/culture ml. In contrast, HMF exhibited no effect on IL-1-stimulated LBRM-33-1A5 production of IL-2. However, 500 micrograms/ml of HME inhibited (P less than .05) IL-2 production (63.0% of controls) in the IL-1 utilization assay. This suppressive effect was probably due to a carry over of HME from the LBRM-33-1A5 culture to the target cells (CTLL2) used to measure IL-2 production. Molecular weight chromatography of an HME sample eluted an IL-2 inhibitor in a low molecular weight (35-50 kd) and high molecular weight (greater than 250 kd) fraction. These data suggest that one way in which macromolecules derived from hydatidiform mole could interfere with in vitro immunologic responses is by modulating interleukin-2 function.