Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro.

Endometriosis is a common gynecological disorder of unclear pathogenesis. We have established an in vitro model to investigate phenotypic similarities and differences between normal endometrial and endometriosis cells. Highly purified cultures of epithelial and stromal cells were isolated from normal endometrium and endometriosis implants. Morphological features as well as immunocytochemical markers confirm these isolates as epithelial and stromal cells. Potential hormone responsiveness was established by the documentation of estrogen receptor mRNA in epithelial and stromal cells isolated from both tissue types. Expression of this receptor protein was verified in stromal cells by competitive radioligand binding, revealing comparable receptor numbers and dissociation constants. CA-125 is selectively secreted in similar concentrations by epithelial cells isolated from both tissue types. PRL secretion is selectively exhibited by progestin-stimulated stromal cells from both tissue types. Our findings demonstrate that highly purified epithelial and stromal cells cultured from normal endometrial and endometriosis tissues express the same phenotypic and functional markers as their in vivo counterparts. These cultures provide useful models to identify endometriosis-specific cell products that contribute to the pathogenesis of this disorder.

Suppression of circulating delta 4-androstenedione and dehydroepiandrosterone sulfate during oral glucose tolerance test in normal females.

Extending a series of previous investigations on the regulatory interaction of insulin and androgens, this study tests the hypothesis that the physiological insulinemia after oral glucose suppresses circulating dehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S), and androstenedione (delta 4A) delta 4 in normal women. Accordingly, seven normal weight, ovulatory women were randomized to receive first either a 75 g glucose dose or a sham control for diurnal rhythm consisting of distilled water at 1700 h. After this insulin stimulus, DHEA-S suppressed below sham control at 90 and 120 min (P less than 0.05) whereas delta 4A suppress at 60, 90, and 120 min (P less than 0.05). Furthermore, as serum insulin increased after glucose, DHEA-S (r2 = 0.351, P less than 0.05) and delta 4A (r2 = 0.314, P less than 0.05) decreased in an inverse linear relationship with insulin. There was no significant suppression below sham at any point in time for DHEA, testosterone, or cortisol. Thus, the endogenous serum insulin response after oral glucose in normal women is associated with suppression of serum DHEA-S and delta 4A with absence of testosterone and cortisol suppression.

Divergent correlations of circulating dehydroepiandrosterone sulfate and testosterone with insulin levels and insulin receptor binding.

We evaluated the insulin response to a standard oral glucose tolerance test (OGTT) and in vitro insulin binding to erythrocytes (RBC) in 26 women from 3 groups: Group NW, normal women (n = 11); Group DS, women (n = 9) with elevated serum DHEAS concentrations, greater than 400 micrograms/dl (greater than 10.84 mumol/L); and Group IR, women (n = 6) with elevated basal plasma insulin concentrations (IRI). There was a significant linear correlation between the area under the insulin response curve (IRI-AUC) and serum testosterone (T) (r = 0.78, p = 0.0001). Using stepwise multiple linear regression, IRI-AUC was characterized as a function of both serum T and DHEAS; positively with T and negatively with DHEAS. In vitro (n = 17), there was a positive correlation between RBC-insulin binding and serum DHEAS (r = 0.54, p = 0.029) and a negative correlation between RBC-binding and T (r = -0.57, p = 0.017). We conclude that DHEAS may enhance insulin binding and action and that DHEAS and T have divergent functional relationships with IRI. DHEAS and T may therefore exert opposing effects on insulin secretion and action.

Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.

Retrograde menstruation is postulated as the initiating event in the histogenesis of endometriosis; however, subsequent steps in the pathogenesis of this common disorder remain poorly characterized. The ip accumulation of activated leukocytes and the infiltration of endometriosis lesions by macrophages and T cells are cytological markers of the inflammatory nature of this syndrome. The apparent recruitment of these leukocytes prompted us to search for chemokine expression by endometriosis cells. We reported previously that pelvic fluid RANTES (regulated upon activation, normal T cell expressed and secreted) concentrations correlated with the stage of endometriosis. In the current study, RANTES messenger ribonucleic acid (mRNA) was identified in normal endometrium and endometriosis lesions, and techniques were developed to localize RANTES protein within these tissues. Using isolated endometrial and endometriosis cell cultures, we demonstrated that RANTES mRNA and protein can be induced by the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma in endometrial stromal, but not in epithelial or adenocarcinoma cells. Immunocytochemical studies confirmed the biochemical findings. Metabolic labeling experiments verified that nascent RANTES secreted by cytokine-stimulated endometriosis stromal cells was the mature, 8-kDa protein predicted by the mRNA encoding this chemokine. The results indicate that RANTES is a normal constituent of the eutopic endometrium. We propose that secretion of RANTES by ectopic endometriosis implants provides a mechanism for peritoneal leukocyte recruitment.

Interleukin-6 secretion in vitro is up-regulated in ectopic and eutopic endometrial stromal cells from women with endometriosis.

An in vitro model developed to compare human endometrial and endometriosis stromal cells was used to examine basal and stimulated expression of interleukin (IL-6). Stromal cells isolated from normal endometrium (NE) exhibited the lowest level of IL-6 secretion (84 pg/10(6) cells-48 h), whereas those cells isolated from endometriosis implants (EI) secreted the highest concentration of this inflammatory cytokine (46,284 pg/10(5) cells-48 h; P < 0.01). Eutopic endometrial stromal cells from women with endometriosis (EE) expressed an intermediate concentration of IL-6 (831 pg/10(6) cells-48 h). Stimulation of the various cultures with IL-1 beta dramatically augmented stromal cell production of IL-6. The mean concentrations of stimulated IL-6 secretion were 16,257, 37,800, and 264,290 pg/10(5) cells-48 h for NE, EE, and EI cells, respectively (P < 0.03). Exposure of the cell cultures to 10 nmol/L estradiol had little direct effect on IL-6 production. The results indicate that endometrial stromal cells isolated from tissues of women with and without endometriosis express IL-6 under basal and cytokine-stimulated conditions. Differential responsiveness among the three cell sources indicates that NE, EE, and EI cells have intrinsic quantitative differences in cytokine regulation.

Failure to demonstrate significant antisperm antibodies in peritoneal fluid of patients with endometriosis.

Endometriosis, even in mild cases, decreases monthly fecundity. Immunologic disorders have been suggested as the mechanism. In light of possible increases in serum autoimmune antibodies, increased peritoneal macrophages, and increased sperm phagocytosis associated with this disease, we postulated that peritoneal fluid antisperm antibodies would be increased and might be the cause of increased sperm phagocytosis and its associated infertility. Peritoneal fluid, from 18 patients with endometriosis and 10 infertile controls, was tested with the antisperm antibody immunobead test validated for peritoneal fluid. One of 18 patients with endometriosis and none of 10 controls had antisperm antibodies present. Therefore, increased sperm phagocytosis is unlikely a result of peritoneal antisperm antibodies in endometriosis patients.

Effect on corpus luteum function of luteal phase administration of a potent gonadotropin-releasing hormone analog (nafarelin).

Fourteen women with ovulatory menstrual cycles were treated with a superactive agonistic analog of gonadotropin-releasing hormone (6-D-[2-naphthyl]-alanyl)-GnRH (nafarelin). Eight of the women received a single subcutaneous injection of nafarelin during the luteal phase at a dosage of 2, 5, or 100 micrograms for determination of the dose-response and pharmacokinetic characteristics of the drug. All doses stimulated the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Maximal release was obtained with the 5-micrograms dose (mean +/- standard deviation: delta LH = 297 +/- 75 mIU/ml; delta FSH = 29 +/- 7 mIU/ml), and there was no greater release of gonadotropin with the 100-micrograms dose. To investigate the contraceptive potential of nafarelin as a luteolytic agent, six of the women were treated with 100 micrograms of analog by daily injection for 10 days, beginning either 2 to 3 days or 5 to 7 days after ovulation. Gonadotroph desensitization or down-regulation developed within 24 hours, but serum concentrations of LH and FSH did not fall below normal values during treatment. There were no significant changes in mean estradiol or progesterone concentrations. There also was no change in mean length of the luteal phase (13.7 +/- 2.1 days [control] versus 13.6 +/- 1.4 days). Thus, nafarelin, like other superactive analogs of GnRH, does not appear to be clinically useful as a luteolytic agent in contraceptive development.

Elevated serum progesterone levels on the day of human chorionic gonadotropin administration do not predict outcome in assisted reproduction cycles.

OBJECTIVE: To determine if the level of serum P drawn on the day of hCG administration predicts assisted reproductive technology (ART) outcome in patients undergoing stimulation with hMG under GnRH agonist (GnRH-a) suppression. DESIGN: Retrospective P assay of stored serum. PATIENTS: One hundred seventy-one patients (189 cycles) who had undergone GnRH-a suppression (leuprolide acetate or nafarelin) and stimulation with hMG for an ART procedure. MAIN OUTCOME MEASURES: Progesterone RIA of serum obtained on the day of hCG administration. Measurement of sequential serum LH values by RIA in those patients with the highest P levels. RESULTS: Pregnancy rates per oocyte retrieval were not correlated with the P level before hCG administration. There were 18 of 54 (33.3%) clinical pregnancies in those cycles with P < 0.9 ng/mL (conversion factor to SI unit, 3.180) and 42 of 135 (31.1%) clinical pregnancies in cycles with a P > or = 0.9 ng/mL. Significantly higher serum E2 levels and numbers of of follicles and oocytes obtained were observed in the high P cycles. There were no differences in the number of oocytes fertilized, the number of embryos transferred, or the implantation rate. However, a significantly higher percentage of mature oocytes were fertilized in the low P cycles (73%), as compared with the high P cycles (60%). CONCLUSIONS: Serum P levels before hCG administration do not predict the outcome of ART cycles in patients suppressed with GnRH-a before hMG stimulation. Lower fertilization rates observed in the high P cycles did not have an effect on clinical outcome.

Birth after intracytoplasmic sperm injection with use of testicular sperm from men with Kartagener/immotile cilia syndrome.

OBJECTIVE: To describe two cases of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with testicular sperm in men with immotile cilia syndromes. DESIGN: Case report. SETTING: A university-based male infertility clinic and assisted reproduction unit. PATIENT(S): Two couples with male factor infertility due to Kartagener/immotile cilia syndrome. INTERVENTION(S): IVF/ICSI with testicular sperm. MAIN OUTCOME MEASURE(S): Semen characteristics, sperm viability, fertilization rate, and pregnancy. RESULT(S): With testicular sperm, the two pronuclear fertilization rates were 63% and 60% in two cases. One case resulted in the birth of normal healthy girl. CONCLUSION(S): With testicular sperm, successful oocyte fertilization after ICSI in couples with male Kartagener/immotile cilia syndrome is possible despite the lack of sperm motility.

Elevated ovarian follicular fluid stem cell factor concentrations are associated with improved pregnancy rates in in-vitro fertilization cycles.

OBJECTIVE: To determine whether ovarian follicular fluid (FF) stem cell factor concentrations are associated with successful IVF pregnancies. DESIGN: Nested case-control design evaluation of stem cell factor levels from the FF of oocytes fertilized and transferred after controlled ovarian hyperstimulation. SETTING: University-based ART program. PATIENT(S): Infertile women undergoing IVF in a university-based ART program. INTERVENTION(S): Fifty-seven FF samples from a cohort of patients (n = 13) with tubal factor and unexplained infertility were stored at -80 degrees C and subsequently evaluated for stem cell factor concentration. Patients with endometriosis, polycystic ovary disease, and male factor infertility were excluded. Stem cell factor concentrations were measured using a commercially available ELISA kit according to the manufacturer's specifications. The groups were analyzed using a one-way analysis of variance, and significance was determined using the chi2 analysis of contingency table, the unpaired t-test, or the Mann-Whitney rank-sum test. MAIN OUTCOME MEASURE(S): FF stem cell factor concentration, pregnancy. RESULT(S): Stem cell factor concentrations were significantly higher in the FF of the patients who achieved successful pregnancies than in those who did not (641.7+/-75.2 pg/mL versus 475.5+/-50.58 pg/mL). CONCLUSION(S): Elevated FF stem cell factor concentrations are associated with an increased likelihood of IVF success. Therefore, stem cell factor may play a role in human follicular and oocyte development, and increasing infrafollicular stem cell factor concentrations may improve pregnancy rates after oocyte retrieval, fertilization, and ET.

Hyperglycemia and hyponatremia during operative hysteroscopy with 5% dextrose in water distention.

Operative hysteroscopy may result in profound hyperglycemia and hyponatremia when crystaloids containing glucose are used as a distention medium. Four patients undergoing operative hysteroscopy developed hyperglycemia in proportion to increasing operative time. None of five monitored patients undergoing diagnostic hysteroscopy developed clinically significant hyperglycemia.

Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis.

OBJECTIVE: To investigate the presence of interleukin-8 (IL-8), a macrophage-derived angiogenic factor, in peritoneal fluid (PF) of women with and without endometriosis. DESIGN: Case-control study. SETTING: University hospital. PATIENTS: Eighteen women with laparoscopic findings of mild to severe endometriosis, and nine women with no visual evidence of pelvic pathology. MAIN OUTCOME MEASURES: Peritoneal fluid IL-8 levels were determined using an ELISA. Interleukin-8 concentrations were compared among women with and without endometriosis. Correlation between PF IL-8 concentration and endometriosis stage was investigated. RESULTS: Interleukin-8 was detectable in the PF of a majority of women (67%). Interleukin-8 concentrations were higher in the PF of women with endometriosis than in matched normal controls. A significant correlation between PF IL-8 concentration and endometriosis stage was noted. CONCLUSIONS: We hypothesize that IL-8 is an important angiogenic factor that contributes to the pathogenesis of endometriosis by promoting the neovascularization of ectopic endometrial implants.

Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in patients with nonobstructive azoospermia.

OBJECTIVE: To evaluate intracytoplasmic sperm injection (ICSI) outcomes in a cohort of men with nonobstructive azoospermia who underwent prior fine-needle aspiration (FNA) "maps" to localize sperm and guide testis sperm extraction (TESE). DESIGN: Retrospective clinical study. SETTING: University-based infertility practice. PATIENT(S): A consecutive cohort of 19 infertile, azoospermic men. INTERVENTION(S): Couples underwent IVF-ET in which TESE procedures were informed and directed by prior FNA maps of the testis. MAIN OUTCOME MEASURE(S): Sperm retrieval and pregnancy rates. RESULT(S): In 21 IVF-ET and ICSI cycles, sufficient sperm for all oocytes were retrieved in 20 TESE attempts (95%). A mean of 3.1 biopsies per patient were required, with an average size of 72 mg. Mean operative time for the TESE procedure was 88 minutes. Overall, the two-pronuclear fertilization rate was 66%; ongoing clinical pregnancies were obtained in 10 of 21 initiated cycles (48%). CONCLUSION(S): In an effort to reduce IVF-ET cancellation rates in cases of nonobstructive azoospermia, diagnostic testis FNA can define those patients who are good candidates for TESE. It also directs sperm retrieval and minimizes tissue removal from nonobstructed testes.

Enhanced post-receptor insulin effects in women following dehydroepiandrosterone infusion.

OBJECTIVE: We hypothesized that intravenous dehydroepiandrosterone (DHEA) would decrease insulin resistance in normal and insulin-resistant women. METHODS: Five insulin-resistant women diagnosed as having polycystic ovaries (PCO) with elevated testosterone and normal dehydroepiandrosterone sulfate (DHEAS) with amenorrhea were recruited. Obese controls (OC) with normal menses and normal testosterone and DHEAS were recruited and matched to each PCO woman for age and weight. The PCO women had a mean testosterone of 3.2 +/- 0.4 nmol/L, fasting serum insulin level of 330 +/- 55 pmol/L, and DHEAS level of 3.4 +/- 1.3 mumol/L. An oral glucose tolerance test (OGTT) was performed at 8 AM after an overnight fast. A DHEA infusion (1 mg/hour for 17 hours) was begun at 6 PM and continued until the completion of the second OGTT performed the following morning at 8 AM. T-lymphocytes were drawn at 8 AM each morning. RESULTS: The DHEA infusion had no significant effect on any of the in vivo indices of insulin sensitivity, ie, basal and OGTT insulin, C-peptide, and ratios of insulin/glucose. In vitro, DHEA significantly increased insulin binding to T-lymphocytes of PCO women but caused no significant change in OC women. There was, however, marked enhancement of T-lymphocyte pyruvate dehydrogenase (PDH) activities in both groups of study subjects following DHEA. CONCLUSION: We conclude that a 17-hour infusion of DHEA enhanced T-lymphocyte insulin binding and PDH activity while producing no detectable improvements in in vivo indices of insulin sensitivity.

Practical aspects of pulsatile gonadotropin-releasing hormone administration.

Pulsatile administration of gonadotropin-releasing hormone represents a major advance in the treatment of anovulation in women who fail to ovulate with clomiphene citrate and is an alternative for many women who currently receive human menopausal gonadotropin. Four issues must be addressed before administering pulsatile gonadotropin-releasing hormone: (1) safety, (2) efficacy, (3) convenience, and (4) cost. Each of these issues will be affected by the three major decisions a physician makes with gonadotropin-releasing hormone therapy: (1) patient selection, (2) route of administration, and (3) dose of gonadotropin-releasing hormone. The ideal candidate for gonadotropin-releasing hormone therapy is a patient with an absence of endogenous pulsatile gonadotropin-releasing hormone, as seen in hypothalamic amenorrhea. Although women with polycystic ovarian disease can be treated with pulsatile gonadotropin-releasing hormone, a decreased ovulation rate should be expected. The route of administration, intravenous or subcutaneous, and the degree of monitoring can be tailored by the physician to fit each patient's needs. Pulsatile gonadotropin-releasing hormone therapy is a safe, effective, convenient, and economical alternative to human menopausal gonadotropin for ovulation induction in women resistant to clomiphene.

GnRH agonists.

The development of GnRH agonists has had a major impact on the practice of gynecology and reproductive endocrinology. The clinical usefulness of GnRH agonists will increase as modes of administration are improved and indications become better defined. GnRH agonists and, potentially, antagonists will provide a prompt, effective, and reversible method of suppressing ovarian function. GnRH agonists may soon become a treatment of choice for many of the noncontraceptive uses of oral contraceptives. Current indications for GnRH agonist administration are best divided into two groups: short-term (less than 6 months) and long-term (greater than 6 months) suppression. Short-term administration of GnRH agonist include most of the current usage of GnRH agonists. Short-term administration avoids most of the side effects of GnRH agonist and offers the most potential for development of GnRH antagonists. Short-term therapy has been shown to be particularly effective in the preoperative treatment of fibroids, suppression of ovarian function before ovulation induction, for short-term suppression of endometriosis, and for diagnostic purposes to determine whether a medical illness is related to ovarian function. Chronic administration of GnRH agonist has produced varying degrees of success. The treatment of precocious puberty is probably the perfect indication for GnRH agonist suppression. The disease is completely reversed with a remarkable absence of side effects. Long-term administration for metastatic breast or prostatic cancer has been shown to be as efficacious as other forms of gonadal suppression and the potential benefits of suppression outweigh the potential side effects of long-term suppression. The risk-benefit ratio must be carefully analyzed for the other indications for long-term suppression. Long-term suppression could be used for medical illnesses exacerbated by the menstrual cycle, painful symptoms related to endometriosis, contraception, and suppression of hyperandrogynism. Although initial studies show the agonist to be quite effective in treating all of these disorders, long-term suppression also may result in potential serious side effects related to hypoestrogenism including hot flashes and osteoporosis. Long-term administration of GnRH agonist may become feasible by lowering the dose and degree of suppression or by combining GnRH agonist with estrogen or progestin replacement, or both.

Prolactin-secreting pituitary adenomas.

Prolactin-secreting pituitary adenoma is a common cause of gynecologic problems that include oligomenorrhea, infertility, amenorrhea and galactorrhea. Diagnosis requires a combination of endocrine testing and radiologic evaluation. The diagnosis of macroadenomas is usually straightforward and these large tumors may be associated with mass effects such as severe headache, nerve palsies or visual changes. Microadenomas may be more subtle in presentation, and the diagnosis of hyperprolactinemia without radiologic evidence of a tumor frequently is problematic. The management of prolactin-secreting adenoma remains controversial, with no clear consensus or indication for surgical versus medical treatment. Surgical intervention is a realistic option for those patients who have access to an experienced neurosurgeon and who have tumor characteristics that offer a reasonable hope for cure. Many questions remain to be answered, including the cause, natural history of development and the optimum treatment for individual cases.

Does human chorionic gonadotropin have human thyrotropic activity in vivo?

Current evidence indicates that thyroid cells are sensitive to human chorionic gonadotropin (hCG) stimulation. In turn, thyroid hormones appear to influence ovarian and endometrial physiology and reproductive function. Our studies addressed the possible effect of endogenous and exogenous hCG on in vivo thyroid function in normal pregnancy and controlled ovarian hyperstimulation, respectively. Circulating concentrations of hCG in pregnant women during gestation were positively correlated with serum free thyroxine (r = 0.43, p = 0.02) and negatively correlated with thyrotropin levels in the same patients (r = 0.42, p = 0.02). By contrast, exogenous administration of hCG to effect follicular maturation in non-pregnant patients undergoing ovarian hyperstimulation resulted in lower circulating hCG concentrations than seen in pregnancy and failed to alter free thyroxine or thyrotropin levels (p > 0.22). Endogenous isoforms of hCG in early pregnancy appear to have thyrotropic activity in vivo. However, the results indicate that, under clinical conditions of controlled ovarian hyperstimulation for assisted reproduction, exogenous hCG does not affect the hypothalamic-pituitary-thyroid axis.

The mini-micro-epididymal sperm aspiration for sperm retrieval: a study of urological outcomes.

Epididymal sperm aspiration and in-vitro fertilization (IVF) with intracytoplasmic sperm injection is an established treatment for obstructive azoospermia. Sperm aspiration is performed with either an incision or percutaneously. To control costs, minimize morbidity and retain the advantages of both approaches, we developed a mini-incision technique for epididymal aspiration and here report sperm retrieval and procedure-related outcomes. Twenty-six consecutive patients with obstructive azoospermia underwent epididymal sperm retrieval through a 1 cm incision with local anaesthesia to provide spermatozoa for concurrent IVF cycles. The quality of retrieved spermatozoa, the quantity of spermatozoa cryopreserved as well as anaesthetic requirement, recovery time and patient satisfaction were evaluated. Fresh epididymal spermatozoa were retrieved in 25 of 26 (96%) patients. In one patient, testicular sperm extraction was necessary. Excess motile spermatozoa were cryopreserved in 24 of 26 (92%) patients; a mean total motile count of 4.8x10(6) motile spermatozoa were banked. The procedure was performed with 62% of patients receiving minimal i.v. sedation. Post-procedure recovery was rapid, with a median time to return to work of 2.0 days with a median of 2.0 pain pills taken. Procedure-related satisfaction was high. The mini-micro-epididymal sperm aspiration achieves the goals of reliable retrieval of abundant epididymal spermatozoa with a single, minimally morbid procedure. It appears to combine the advantages of the incision and percutaneous approaches.