Ovarian responses in women to recombinant follicle-stimulating hormone and luteinizing hormone (LH): a role for LH in the final stages of follicular maturation.

During the follicular phase of the menstrual cycle, FSH stimulates follicular growth, granulosa cell aromatase activity, induction of LH receptors on the granulosa cell membrane, and estradiol secretion. As a result of negative feedback of estradiol on the pituitary, serum FSH concentrations decline. Despite the fall in FSH concentrations, the maturing follicle continues to develop to the preovulatory stage. In a prospective randomized trial, we tested the hypothesis that a key mechanism by which the dominant follicle continues to develop in the face of decreasing concentration of FSH is by acquiring LH responsiveness. In 24 women, pituitary gonadotropin secretion was down-regulated with a GnRH agonist. Follicular growth was then stimulated with recombinant human FSH (r-hFSH) until a 14-mm follicle was identified by ultrasound. The women were then randomized to 1 of 4 groups for a 2-day period: continued r-hFSH treatment, substitution of r-hFSH with saline, low dose r-hLH (150 IU, twice daily), or high dose r-hLH (375 IU, twice daily). Serum estradiol concentrations in the women receiving saline declined by the end of the 2-day randomization period. In contrast, serum estradiol concentrations continued to rise in women receiving either r-hFSH or r-hLH compared with those in the saline-treated group (P < 0.05). Pregnancies occurred in each of the gonadotropin treatment groups. These findings indicate that once FSH initiates follicular growth, either FSH or LH is capable of sustaining follicular estradiol production. Extrapolating these findings to the normal menstrual cycle suggests that the maturing follicle may continue to develop in the presence of diminishing FSH concentrations by acquiring the capacity to respond to LH.

Regional specialization of the cell membrane-associated, polymorphic mucin (MUC1) in human uterine epithelia.

The cell membrane-associated, polymorphic mucin, MUC1, has been proposed to hinder implantation by virtue of its anti-adhesive properties. Consistent with this proposal is the observation of a dramatic decrease in MUC1 protein and mRNA expression in the uterine epithelium of several species at the time of implantation. In contrast, little change in glandular epithelial expression of MUC1 protein or its mRNA during the peri-implantation period has been detected in humans. However, expression in the luminal epithelium, i.e. the epithelium involved in embryo attachment, has not been reported. Using tissue samples with a clearly defined luminal epithelium and antibodies directed against the cytoplasmic domain found in all cell-associated MUC1 species (CT-1) and against two MUC1 ectodomain epitopes, HMFG-1 and HMFG-2, we demonstrate that MUC1 expression in the luminal epithelium is maintained throughout the menstrual cycle. The staining observed with CT-1 correlates with that seen with HMFG-2, but not HMFG-1. HMFG-1 reactivity was high in all regions except basal glands in the mid proliferative endometrium and fell to very low levels throughout the tissue in the mid secretory phase. In all cases, HMFG-1 reactivity could be restored by predigestion with keratanase or neuraminidase which removes keratan sulphates and sialic acids, respectively. These observations suggest that regionally restricted glycosylation generates an altered external structure of MUC1. These alterations appear to decrease accessibility to the MUC1 protein core region and are maximal in luminal epithelium at the receptive phase. Due to their large highly extended structures, MUC1 ectodomains are very likely to be among the first cell surface components encountered during human blastocyst attachment to the luminal epithelium. Thus, MUC1 either must be locally removed during the attachment process or functions actually to promote the initial steps in embryo adhesion to the apical surface of the human uterine epithelium.

Endometrial leukocytes are altered numerically and functionally in women with implantation defects.

PROBLEM: A significant cohort of women with autoimmune thyroid disease (ATD) also suffer from reduced fertility. The finding that neither exogenous hormones nor donor eggs correct the infertility suggests that the problem involves an inherent endometrial defect. METHOD OF STUDY: Endometrial leukocyte populations in women with ATD were quantitated by immunohistochemistry. A semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to examine the expression of transforming growth factor (TGF)-beta 1, interleukin (IL)-4, IL-10, and interferon (IFN)-gamma in the endometrial samples. RESULTS: A significant increase in the endometrial T-cell population was observed in women with ATD compared to controls. The relative abundance of IL-4 and IL-10 was decreased in women with ATD compared to controls, whereas IFN-gamma was increased. No difference was noted in the abundance of TGF-beta 1. The source of cytokine production for IL-4, IL-10, and IFN-gamma was the endometrial leukocytes. CONCLUSIONS: Both the leukocyte numbers and cytokine expression profile were altered significantly in a well-defined group of women with implantation defects.

Differential expression of G1 cyclins during human placentogenesis.

Cyclins are proteins that support the progression of cell-cycle stages in proliferating cells. The purpose of this study was to determine which of the cyclin genes is involved in the regulation of normal human trophoblast proliferation. The presence and cellular localization of four G1 cyclins D1, D2, D3 and E, were determined by immunohistochemistry. This analysis indicated that cyclins E and D3 are the predominant cyclins in villous trophoblast. D2 was present only within the villous core, in fetal macrophages. Positive immunoreactivity for cyclin D1 was strongest in second and third trimester placentae, in the cells lining the intravillous vessels with additional reactivity in extravillous cytotrophoblasts. Because cyclin E protein was present in a greater percentage of cells than those that are dividing, Western blot analysis was performed to validate the fidelity of the immunohistochemistry data. The results of the Western analysis revealed that two forms of cyclin E protein of the appropriate size were present. Data collected from this study suggest that within the trophoblast lineage, cyclins D3 and E are important cell cycle regulatory proteins, and further, that cyclin E may function in trophoblast terminal differentiation as well.

Decidual leukocyte populations in ectopic pregnancies.

OBJECTIVE: To determine whether the recruitment of decidual leukocytes during pregnancy is the same in intrauterine pregnancies (IUPs) versus ectopic pregnancies (EPs). DESIGN: Intrauterine decidual samples from both EPs and IUPs were obtained for the evaluation of leukocyte populations. SETTING: In vitro experiment. PATIENT(S): Women with EPs and women with IUPs. MAIN OUTCOME MEASURE(S): Immunohistochemical identification of decidual leukocyte populations. RESULT(S): We have analyzed the decidual leukocyte populations from three women with EPs by immunohistochemical analysis. The data demonstrate a leukocyte infiltration similar to that found in decidua from normal pregnancies. CONCLUSIONS(S): These data support the hypothesis that decidual leukocyte recruitment and/or increases during pregnancy is primarily hormonally regulated.

Endometrial Th2 cytokine expression throughout the menstrual cycle and early pregnancy.

Embryonic implantation and maintenance of pregnancy is influenced by the maternal immunological response. Type 2 T-helper (Th2) cells secrete interleukins 4, 5, 6 and 10 and are associated with suppression of cell-mediated immunity. Temporal changes in the expression of these cytokines were detectable by immunohistochemistry throughout the menstrual cycle. In pregnancy, 10-fold or greater increases in interleukin 6 and 10 secretion were detectable by enzyme-linked immunoassay compared with the non-pregnant state. The pattern of expression of Th2 cytokines suggests that progesterone may influence endometrial cytokine secretion. During pregnancy, Th2 expression paralleled that of vimentin, a stromal cell marker, suggesting that these cells may be a principal source of Th2 cytokines may be a mechanism of suppressing cell-mediated immunity in the endometrium, which may in turn enhance embryonic implantation and maintenance of pregnancy.

Adhesion formation from release of dermoid contents in the peritoneal cavity and effect of copious lavage: a prospective, randomized, blinded, controlled study in a rabbit model.

OBJECTIVES: To determine, in a rabbit model, whether peritoneal exposure to dermoid cyst material produces inflammation and adhesions above control levels and whether saline lavage reduces the degree of peritoneal reaction. DESIGN: A prospective, randomized, blinded, controlled study of adhesion formation. Thirty New Zealand white female rabbits were assigned randomly to five experimental groups: [1] laparoscopy with intraperitoneal injection of human dermoid material, [2] laparoscopy with intraperitoneal injection of human dermoid material and subsequent lavage to clear all visible dermoid material, [3] laparoscopy with saline lavage, [4] laparoscopy with intraperitoneal injection of human follicular fluid (antigenic control), and [5] laparoscopy alone. MAIN OUTCOME MEASURES: Six weeks after initial laparoscopy, inflammation and adhesions were scored in several categories via visual assessment (range 0 to 15) and histologic microscopic evaluation (range 0 to 24). Data were evaluated using Kruskal-Wallis and Mann-Whitney U nonparametric tests. RESULTS: For groups 1, 2, 3, 4, and 5, respectively, mean +/- SEM total inflammation-adhesion scores were 13.85 +/- 0.55, 2.90 +/- 1.15, 0 +/- 0, 1.50 +/- 1.00, and 0 +/- 0 for clinical evaluation and 16.83 +/- 1.22, 7.33 +/- 1.76, 0 +/- 0,0 +/- 0, and 0 +/- 0 for histologic evaluation. Using nonparametric tests, significant differences were found between groups in clinical and histologic scores. CONCLUSIONS: Dermoid material produces a significant peritonitis. Results of the clinical evaluation demonstrate that saline lavage brings inflammation and adhesion formation close to control levels. However, results of the histologic evaluation suggest that the decrement in inflammation as a result of lavage may be less dramatic than that found by clinical evaluation.

Vascular permeability factor and vascular endothelial growth factor in ovarian hyperstimulation syndrome: a preliminary report.

OBJECTIVE: To determine whether serum levels of vascular permeability factor (VPF) are elevated in patients with ovarian hyperstimulation syndrome (OHSS) and to determine if luteinizing granulosa cells may be a source of VPF. DESIGN: Prospective observational study. SETTING: University IVF and GIFT program. PATIENTS: Eight consecutive IVF and GIFT patients at high risk for OHSS. MAIN OUTCOME MEASURES: Vascular permeability factor concentration in serum and follicular fluid. RESULTS: Serum VPF was significantly higher (15.2 +/- 4.0 pM; mean +/- SEM) on day +14 in the group who developed severe OHSS compared with those who did not. Follicular fluid VPF (171.5 +/- 18.5 pM) was approximately 100-fold greater than serum (1.7 +/- 1.3 pM) or peritoneal fluid (2.5 +/- 1.3 pM) 36 hours after hCG administration. CONCLUSION: Vascular permeability factor is elevated in patients with severe OHSS and the ovary may be a source of VPF secretion.

Comparison of transdermal versus oral estradiol on endometrial receptivity.

OBJECTIVE: To compare the effects of oral micronized E2 with transdermal E2 on endometrial receptivity in women undergoing oocyte donation. DESIGN: Prospective, randomized, crossover trial. Serum E2 and P concentrations were measured on cycle days 14 and 22 (luteal day +8). Endometrial biopsies were obtained on day 22 and read in a blinded fashion for histology and beta-3-integrin expression. SETTING: University-based donor oocyte program. PATIENTS: Twenty-seven patients presenting for donor oocytes. MAIN OUTCOME MEASURES: Endometrial histology and beta-3-integrin expression. RESULTS: The endometrial glandular histology in women given oral micronized E2 was delayed by a mean of 1.6 days in comparison to that of women given transdermal E2. Seventy percent of women given oral E2 displayed a lag > or = 4 days whereas 29.6% given transdermal E2 displayed a similar lag. Serum E2 levels were 1,194 +/- 108.8 pg/mL (mean +/- SEM; conversion factor to SI unit, 3.671) in women on oral micronized E2 and 117.4 +/- 14.0 pg/mL in those on transdermal E2. CONCLUSION: The supraphysiologic serum E2 levels associated with oral micronized E2 may have a deleterious impact on endometrial receptivity. The development of more physiologic hormone replacement protocols may enhance endometrial receptivity and lead to improved clinical pregnancy rates.

Infertility evaluation in fertile women: a model for assessing the efficacy of infertility testing.

A standard infertility evaluation consists of a semen analysis, hysterosalpingogram, post-coital test, endometrial biopsy and laparoscopy. Although these tests are well grounded in clinical experience, information on their ability to discriminate between fertile and infertile couples is limited. In this study, we performed standard infertility tests plus two others--sperm antibodies and cervical culture for Mycoplasma hominis and Ureaplasma urealyticum--on fertile and infertile couples. Women in the fertile group were selected from those who had delivered a child within the previous 2 years and who were scheduled for a laparoscopic tubal ligation. Women in the infertile group were selected from those presenting for an infertility evaluation (mean duration of infertility 4.2 years), and they were matched by age (+/- 3 years) and race with fertile subjects. Subjects were recruited from both private and clinic patients. A total of 64 couples (32 matched pairs) completed the evaluation. At least one 'abnormal' infertility test was found in 69% of fertile and 84% of infertile couples. With the exception of tubal damage and endometriosis, which as expected were more common in infertile couples, no significant differences between groups for remaining infertility factors could be demonstrated. Despite the small size of the current study, these results confirm the feasibility and importance of comparisons of the prevalence of infertility factors in fertile and infertile couples.

Endometriosis impairs the efficacy of gamete intrafallopian transfer: results of a case-control study.

OBJECTIVE: To determine whether pelvic endometriosis impairs the efficacy of GIFT. DESIGN: Matched follow-up study. SETTING: University-based assisted reproduction program. PARTICIPANTS: Patients undergoing GIFT between 1987 and 1991. Cases had a primary diagnosis of endometriosis. Controls had no endometriosis and were matched with cases according to age, number of mature eggs transferred, and sperm grade. INTERVENTION: Gamete intrafallopian transfer was performed in all patients in an identical manner independent of their underlying diagnosis. MAIN OUTCOME MEASURES: Pregnancy and delivery rates. RESULTS: Of 114 laparoscopic egg retrievals performed in the endometriosis group, there were 37 pregnancies (32.5%) and 27 deliveries (23.7%). Of the 214 retrievals in the control group, there were 101 pregnancies (47.2%) and 76 deliveries (35.5%). Mantel-Haenszel estimates of relative risk indicated that endometriosis significantly impaired pregnancy and delivery rates. There was no statistically significant difference in pregnancy rates according to severity of disease among endometriosis cases. There was no statistically significant difference in pregnancy rates according to severity of disease among endometriosis cases. CONCLUSIONS: Our finding that GIFT pregnancy rates were lower in women with a primary diagnosis of endometriosis than in matched controls suggests that endometriosis is associated with reduced efficacy of GIFT.

Sperm recovery and survival: two tests that predict in vitro fertilization outcome.

OBJECTIVES: To determine if human sperm recovery during swim-up and sperm survival after 24 hours, as obtained from a screening semen specimen, are predictive of subsequent IVF and clinical pregnancy rates (PRs) and to determine if these techniques can identify men with normal semen analysis parameters and poor IVF success. DESIGN: Historical prospective study. SETTING: All semen evaluations and IVF cycles were performed at the University of Pittsburgh, Magee-Womens Hospital, Pittsburgh, Pennsylvania. PATIENTS, PARTICIPANTS: Couples undergoing IVF at Magee-Womens Hospital from August 1988 through June 1993. INTERVENTIONS: A screening semen analysis and swim-up procedure were performed on all couples undergoing IVF. The number of spermatozoa recovered after swim-up and the percentage of motile spermatozoa present after a 24-hour incubation were recorded. MAIN OUTCOME MEASURES: Fertilization and PRs were compared according to the parameters obtained from routine semen analysis, the number of spermatozoa obtained with swim-up, and the percentage of motile spermatozoa at 24 hours. RESULTS: Using chi2 or Fisher's exact test, fertilization rates were significantly different according to the number of spermatozoa recovered after swim-up (< or = 2.0 and > 2.0 x 10(6) spermatozoa recovered, 48.3% versus 71.4%) as were PRs (16.9% versus 29.8%). Similarly, the percentage of motile spermatozoa present at 24 hours (< or = 20% and > 20%) discriminated between fertilization rates (45.9% versus 65.8%) and PRs (16.4% versus 36.5%). Among a subset of men with normal semen analyses and total motile sperm counts > or = 40 x 10(6), the results from swim-up and survival discriminated between men with high and low fertilization and PRs. Receiver operating characteristic analysis revealed that swim-up results better discriminated between pregnant and nonpregnant IVF patients than sperm motility, but that the percentage of motile spermatozoa present at 24 hours was no better in this regard than sperm motility. CONCLUSIONS: The number of spermatozoa recovered after swim-up and the percentage of spermatozoa that maintain their motility after 24 hours were both helpful in assessing IVF and PRs and may be helpful in altering physicians to a subset of men having normal semen analysis parameters yet poor IVF success.

Aromatase cytochrome P450 in rat ovarian granulosa cells before and after luteinization: adenosine 3',5'-monophosphate-dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5' genomic DNA.

The complete nucleotide sequence for rat ovarian aromatase cytochrome P450 (P450arom) has been derived from four cDNA clones isolated from three rat granulosa/luteal cell lambda gt11 cDNA expression libraries. The composite P450arom cDNA extends 1597 basepairs, encodes a protein of 508 amino acids (calculated mol wt = 58,263), and hybridizes to three mRNA transcripts (3.3, 2.6, and 1.9 kilobases in size) in rat ovarian tissues. A 5' genomic fragment was isolated from a rat genomic library and shown to contain exon I and 538 basepairs of 5' flanking sequences, including putative promoter elements. Further, we document that P450arom mRNA and estradiol (E) biosynthesis are regulated by cAMP-dependent mechanisms in granulosa cells of preovulatory (PO) follicles, but are maintained by cAMP-independent mechanisms after LH/hCG-induced luteinization. The transition of the PO granulosa cell to the luteal cell (PO + hCG) phenotype requires 5 h of exposure to hCG in vivo. Once the luteal cell phenotype is programed, P450arom mRNA and E biosynthesis are maintained in the luteinized cells for up to 10 days in a constitutive manner in the absence of hormones or agents that increase intracellular cAMP. Furthermore, when PO + hCG (7 h) follicles were isolated and incubated for 1-3 h with reversible inhibitors of transcription (actinomycin-D) or translation (cycloheximide) before harvesting the granulosa cells, neither morphological nor functional luteinization of granulosa cells in culture was impaired. Thus, rapid cellular and molecular events occur in granulosa cells within 5-7 h after an ovulatory LH/hCG surge that alter the hormonal regulation of the aromatase gene.

Regulation of aromatase mRNA and estradiol biosynthesis in rat ovarian granulosa and luteal cells by prolactin.

Regulation by PRL of aromatase (P450arom) mRNA and protein and estradiol (E) biosynthesis was examined in granulosa cells during early stages of luteinization in vitro and in vivo. PRL caused a dose-dependent (10-1000 ng/ml) decrease in P450arom mRNA and E biosynthesis (greater than 99%) in luteinized rat granulosa cells in vitro, even when the cells were cultured in the presence of insulin and hydrocortisone (hormones known to synergize with PRL to induce proteins in mammary tissue) or in the presence of forskolin (a nonhormonal stimulator of cAMP). PRL also prevented the marked increases in aromatase mRNA and E biosynthesis stimulated by FSH and forskolin in nonluteinized preovulatory granulosa cells in culture. These effects of PRL on granulosa cells in culture were specific for aromatase and were not observed for other proteins, such as cholesterol side-chain cleavage cytochrome P450 (P450scc) and alpha 2-macroglobulin. PRL also decreased P450arom mRNA and protein during the early stages of luteinization in vivo. PRL administered to rats beginning day 1 postovulation to mimic hormone release during pseudopregnancy reduced the progressive increase in P450arom mRNA occurring in corpora lutea on days 3-4 in ovulated rats not treated with PRL. CB 154, a dopamine agonist that inhibits pituitary release of PRL, caused P450arom mRNA and protein to decrease 50% if given to pregnant rats on days 8-10 of gestation, but increased P450arom mRNA and protein if given to pregnant rats on days 10-12 of gestation. These diverse effects of PRL in pregnancy suggest that placental factors may modify the response of luteal cells to PRL during gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450scc) in rat ovarian granulosa cells and corpora lutea. cDNA and deduced amino acid sequence of rat P-450scc.

We report the isolating and sequencing of three cDNA clones encoding rat P-450scc, the nucleotide and protein sequences of which are highly homologous to those of bovine and human P-450scc, especially in the putative heme and steroid binding domains. We document that different molecular mechanisms regulate P-450scc in granulosa cells of preovulatory (PO) follicles prior to and after luteinization. Luteinizing hormone/human chorionic gonadotropin (LH/hCG) and cAMP are obligatory to induce P-450scc mRNA in PO granulosa cells in vivo and in vitro. Once P-450scc mRNA is induced as a consequence of the LH/hCG surge it is constitutively maintained by luteinized cells in vivo (0-4 days) and in vitro (0-9 days) in the absence of gonadotropins, is susceptible to modulation by prolactin and is no longer regulated by cAMP. Exposure to elevated concentrations of hCG in vivo for 5-7 h was required for PO granulosa cells to undergo a functional transition establishing the stable luteal cell phenotype. Transient exposure of PO + hCG (7 h) follicles in vitro to the RNA synthesis inhibitor actinomycin D (1 microgram/ml) or the protein synthesis inhibitor cycloheximide (10 micrograms/ml), for 1-5 h prior to culturing the granulosa cells failed to disrupt the induction of P-450scc mRNA, progesterone biosynthesis, and appearance of the luteal cell morphology. Inhibitors of protein kinase A (Rp-cAMPS; 1-500 microM and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8); 1-200 microM) added directly to the luteinized cell cultures also failed to alter P-450scc mRNA in these cells, although the cells contain in vivo amounts of mRNA for RII beta, RI alpha, and C alpha, the primary subunits of protein kinase A found in the rat ovary. These data suggest that expression of the P-450scc gene in rat ovarian follicular cells is regulated in a sequential manner by cAMP-dependent and cAMP-independent mechanisms associated with granulosa cells and luteal cells, respectively.