Impact of metformin therapy on ovarian stimulation and outcome in 'coasted' patients with polycystic ovary syndrome undergoing in-vitro fertilization.

This study was designed to determine if metformin therapy improves ovarian stimulation and IVF outcomes in coasted patients with clomiphene-resistant polycystic ovarian syndrome (PCOS). A retrospective data analysis was performed on women with clomiphene citrate-resistant PCOS treated with or without metformin, who underwent 72 cycles of IVF-embryo transfer with intracytoplasmic sperm injection (ICSI). In 59 cycles, patients were coasted to allow oestradiol concentrations to drop before human chorionic gonadotrophin administration. In patients undergoing coasting, the outcome of IVF with ICSI was compared in those who were treated and untreated. In patients treated with metformin, follicular fluid concentrations of testosterone and insulin were significantly lower (60.5 +/- 5 versus 79.1 +/- 6 ng/dl; P < 0.05 and 18 +/- 2.5 versus 22 +/- 2.4 micro IU/ml; P < 0.05 respectively), and the mean number of oocytes retrieved (22.3 +/- 2.4 versus 19.7 +/- 1.6) did not differ. The metformin-treated group showed an increase in the mean number of mature oocytes, oocytes fertilized and cleaving embryos (4-cell or greater by 72 h). However, in the group of patients undergoing coasting, maximum oestradiol concentrations and number of days of coasting were all lower in the metformin-treated group with increased clinical pregnancy rates (71 versus 30%, P < 0.05). Therefore, metformin use appears beneficial in improving IVF outcomes in clomiphene citrate-resistant PCOS patients.

Metformin treatment of patients with polycystic ovary syndrome undergoing in vitro fertilization improves outcomes and is associated with modulation of the insulin-like growth factors.

OBJECTIVE: To determine if metformin therapy improves in vitro fertilization (IVF) outcomes in patients with clomiphene-resistant polycystic ovarian syndrome (PCOS). DESIGN: Retrospective data analysis of selective groups of patients. SETTING: A private IVF unit. PATIENT(S): Forty-six women with clomiphene citrate-resistant PCOS underwent 60 cycles of IVF embryo transfer with intracytoplasmic sperm injection. INTERVENTION(S): In half of the cycles, patients received metformin (1000 to 1500 mg) daily, starting the cycle prior to gonadotropin treatment. MAIN OUTCOME MEASURE(S): Total number of follicles; serum estradiol (E2) on the day of hCG administration and the cycle's E2 maximum; total number of oocytes, mature oocytes, embryos, fertilization, and pregnancy rates; and follicular fluid levels of insulin-like growth factors (IGF-I, IGF-II) and IGF-binding proteins (IGFBP-1, IGFBP-3). RESULT(S): In patients treated with metformin, the total number of follicles on the day of hCG treatment was decreased (23 +/- 1.2 vs. 33 +/- 2.6) with no change in follicles > or = 14 mm in diameter (21 +/- 1.2 vs. 25 +/- 1.7). Metformin treatment did not affect the mean number of oocytes retrieved (22 +/- 1.9 vs. 20.3 +/- 1.5). However, the mean number of mature oocytes (18.4 +/- 1.5 vs. 13 +/- 1.5) and embryos cleaved (12.5 +/- 1.5 vs. 5.9 +/- 0.9) were increased after metformin treatment. Fertilization rates (64% vs. 43%) and clinical pregnancy rates (70% vs.30%) were also increased. Metformin led to modulation of preovulatory of follicular fluid IGF levels with increases of IGF-I (140 +/- 8 vs. 109 +/- 7ng/mL) and decreased of IGFBP-1 (133 +/- 8 vs.153 +/- 9ng/mL). CONCLUSION(S): Metformin use appears to improve IVF outcomes in patients with clomiphene citrate-resistant PCOS.

Cauterization of hydrosalpinges before in vitro fertilization is an effective surgical treatment associated with improved pregnancy rates.

OBJECTIVE: Our purpose was to determine whether proximal tubal cauterization is an effective method of reversing the decreased pregnancy rates seen in patients undergoing in vitro fertilization-embryo transfer with hydrosalpinges present. STUDY DESIGN: We studied a group of 94 patients with tubal factor infertility. Sixty patients had hydrosalpinges documented by either hysterosalpingography or laparoscopy, or both. Forty-five had surgical treatment of hydrosalpinges by salpingectomy or by proximal tubal cauterization. In vitro fertilization-embryo transfer was performed within 3 months after surgery. Pregnancy and implantation rates were compared. RESULTS: Patients with hydrosalpinx had significantly decreased clinical pregnancy and implantation rates per cycle (14% and 8%, respectively) compared with those of patients undergoing proximal tubal cauterization before the in vitro fertilization cycle (73% and 36%, respectively). These pregnancy and implantation rates are comparable with those found in patients with tubal factor infertility without hydrosalpinges (53% and 22%, respectively), as well as in salpingectomy-treated patients (46% and 24%, respectively). CONCLUSIONS: Proximal tubal cauterization is effective in reversing the adverse effects of hydrosalpinges.

Cadherin function is required for axon outgrowth in retinal ganglion cells in vivo.

The cell-cell adhesion molecule N-cadherin strongly promotes neurite outgrowth in cultured retinal neurons. To test whether cadherins regulate process outgrowth in retinal neurons in vivo, we have blocked cadherin function in single cells by expression of a dominant negative N-cadherin mutant. We report that when cadherin function is inhibited, axon and dendrite outgrowth are severely impaired, particularly in retinal ganglion cells. Laminar migration and cell type specification, by contrast, appear unaffected. Further, expression of the catenin-binding domain of N-cadherin, which blocks cadherin-mediated adhesion in early embryos, does not affect axon outgrowth, suggesting that outgrowth and adhesion are mediated by distinct regions of the cytoplasmic domain. These findings indicate that cadherins play an essential role in the initiation and extension of axons from retinal ganglion cells in vivo.

Application of the cavitron ultrasonic surgical aspirator (CUSA) for gynecological laparoscopic surgery using the rabbit as an animal model.

OBJECTIVE: To study the potential application of the cavitron ultrasonic surgical aspirator (CUSA) in gynecological laparoscopic surgery using a rabbit animal model. DESIGN: Twenty-six rabbits were prospectively randomized into two groups. Laparoscopically directed standard injuries were made on the randomly assigned horn and sidewall in all animals with the CUSA. Contralateral injuries were made with a contact neodymium-yttrium aluminum garnet (Nd:YAG) laser in group 1 and with bipolar cautery in group 2. Adhesion and inflammation scores were assessed for two animals in each group at 24, 48, and 72 hours, and seven animals in each group at 14 days. SETTING: University animal research facility. MAIN OUTCOME MEASURES: Adhesion and inflammation scores were compared between animals in the CUSA versus Nd:YAG study and the CUSA versus bipolar cautery at 14 days. RESULTS: No significant difference in uterine or sidewall adhesion scores was noted between the CUSA versus Nd:YAG or the CUSA versus bipolar cautery. Bipolar cautery produced significantly less inflammation on the uterine horn compared with the CUSA (3.0 +/- 0.2 versus 5.3 +/- 0.7, P = 0.0001), but no difference in sidewall inflammation was noted between the CUSA compared with bipolar cautery. No difference in inflammation was observed between the CUSA and the Nd:YAG laser. CONCLUSIONS: The bipolar cautery appears to be preferable to the CUSA for coagulation of uterine lesions, although dissection of the uterus is not possible with bipolar cautery. The CUSA and the Nd:YAG appear to be comparable for uterine horn dissection. Because the CUSA causes similar adhesion formation and tissue inflammation at the sidewall when compared with the Nd:YAG laser and bipolar cautery and may be less likely to damage blood vessels, ureters, or other collagen-rich tissues, the CUSA may represent a promising new surgical tool for laparoscopically directed peritoneal dissection.

Effects of clomiphene citrate and leuprolide acetate on luteal-phase hyperprolactinemia during ovarian stimulation with menopausal gonadotropins.

Hyperprolactinemia, a known modulator of reproductive function, occurs commonly in women undergoing ovarian stimulation with human menopausal gonadotropins (hMG). Clomiphene citrate (CC) and gonadotropin releasing hormone analogues (GnRHa), when administered during the luteal phase, attenuate the hyperprolactinemic response to hMG. We asked whether follicular-phase administration of CC and GnRHa, as employed clinically in women undergoing ovarian stimulation for in vitro fertilization or gamete intrafallopian transfer, would alter the incidence and severity of hMG-induced luteal-phase hyperprolactinemia. Seventy-five percent of all patients had at least one luteal prolactin level greater than 25 ng/ml, and 40% had mean luteal-phase prolactin levels greater than 25 ng/ml. The incidence of hyperprolactinemia was similar in pregnant and nonpregnant cycles. The incidence of hyperprolactinemia was similar for both the GnRH agonist-treated group and those given clomiphene citrate. The increase in mean luteal prolactin levels over the follicular-phase baseline level was significantly greater in the CC-treated group (P = 0.03). This was due to the significant suppression of follicular-phase baseline prolactin levels in patients receiving CC. We conclude that neither CC nor GnRHa administration in the follicular phase prevents luteal-phase hyperprolactinemia in women undergoing ovarian stimulation with hMG.

Serum progesterone levels predict success of in vitro fertilization/embryo transfer in patients stimulated with leuprolide acetate and human menopausal gonadotropins.

Serum progesterone (P4) levels greater than 2.86 nmol/L (0.9 ng/mL) on the day of hCG administration are reportedly associated with decreased pregnancy rates in in vitro fertilization/embryo transfer (IVF/ET) cycles. To further assess this phenomenon we measured serial serum P4, LH, and estradiol levels in 115 consecutive patients undergoing stimulation for IVF/ET with midluteal leuprolide acetate and human menopausal gonadotropins. IVF/ET cycle outcome was retrospectively correlated with P4 levels on the day of hCG administration. Two critical breakpoints were identified, 1.27 nmol/L (0.4 ng/mL) and 286 nmol/L (0.9 ng/mL). Clinical pregnancies occurred in 9 of 18 patients in group I (P4, less than 1.27 nmol/L) compared to 11 of 81 patients in group II (1.27 less than P4 less than 2.86 nmol/L; P = 0.001) and 0 of 14 patients in group III (P4, less than or equal to 2.86 nmol/L) (P = 0.001). Eleven patients in group III had cryopreservation of embryos during that cycle. Six subsequently underwent frozen embryo transfer, and clinical pregnancies occurred in 2, both of whom have delivered. These findings demonstrate that even modest increases in serum P4 levels (greater than 1.27 nmol/L) are associated with reduced pregnancy rates in IVF/ET cycles. In addition, it appears that the mechanism may not exclusively involve poor oocyte quality.

Nucleic acid binding characteristics of group A/B hnRNP proteins.

hnRNP proteins are primarily defined by their specific sedimentational, reconstitutional, and extraction properties and are presumed to be RNA binding. However, it is not clear whether all these proteins have RNA binding capabilities. Recently, using two monoclonal antibodies, fA12 and AC88, we reported that the abundance of a subclass of the highly basic A/B hnRNP proteins was specifically down regulated during terminal differentiation of human and murine cells in vitro. In this report we have examined the nucleic acid binding characteristics of this subclass and other members of the A/B hnRNP proteins in vitro. All members of class A/B hnRNP proteins appear to have similar but not identical nucleic acid binding characteristics. However, the subclass of proteins recognized by AC88 and fA12 exhibit stronger binding affinities and are shown to be highly selective in their binding to RNA vs DNA in vitro. These proteins also preferentially bind poly(U) RNA, suggesting that in vivo they may bind effectively to uridine rich motifs critical in pre-mRNA processing.

Macrophage-conditioned media enhance endometrial stromal cell proliferation in vitro.

It has been hypothesized that the activated peritoneal macrophages of endometriosis, rather than being a response to ectopic endometrium, may contribute to its pathogenesis. This study attempts to define the effect of macrophage-derived growth factors on endometrial stromal cell growth in vitro, as well as the interaction between estrogen and these growth factors. Mouse endometrial stromal cells were prepared and cultured in either a serum-containing, insulin-free medium or a serum-free, insulin-containing medium, and the effects of adding 10% macrophage-conditioned medium on [3H]thymidine incorporation were assessed. Results indicate that macrophage-conditioned medium will increase incorporation in the presence of insulin but not in insulin-free media. Serum can substitute for macrophage-conditioned medium, but the two together show no additive or synergistic effect. The effect of estrogen on this system was determined, and results indicate that estrogen will perform the function of insulin, with an optimal concentration of 10(-10) to 10(-12) mol/L. Thus macrophage-conditioned medium appears to function as a competence growth factor, and estrogen (in appropriate concentrations) functions as a progression growth factor.

c-myc protooncogene polypeptide expression in endometriosis.

The c-myc protooncogene and its polypeptide product are important regulators of cell proliferation and differentiation, and ovarian steroids are believed to stimulate growth of various uterine cell types through altered expression of the c-myc gene. To determine whether c-myc expression may also be involved in the growth and development of endometriosis, we assessed c-myc expression in eutopic and ectopic endometrial tissue obtained from women undergoing surgery for endometriosis. Immunocytochemistry using a monoclonal antibody to the c-myc protein demonstrated positive staining of glandular and stromal cell nuclei, and cytoplasmic staining of glandular but not stromal cells in both eutopic and ectopic endometrium. These findings suggest that c-myc expression may be an important regulator of cell proliferation in endometriotic tissue.

Immediately preovulatory levels of messenger ribonucleic acid for inhibin alpha-subunit are diminished in granulosa cells from successful in-vitro fertilization-embryo transfer.

Inhibin, a gonadal glycoprotein which suppresses pituitary gonadotrophin secretion, preferentially follicle stimulating hormone, has been extensively characterized. It consists of two covalently bound subunits, the alpha- and beta-subunits, encoded by separate genes. In this study, the expression of messenger ribonucleic acid (mRNA) for the inhibin alpha-subunit was studied by Northern blot analysis in granulosa cells of women undergoing in-vitro fertilization/embryo transfer (IVF/ET). Three patient groups were studied: women who failed to become pregnant (n = 11), women who became pregnant but experienced early spontaneous abortion (n = 3) and women who conceived normal ongoing pregnancies (n = 4). Granulosa cells were obtained at the time of follicle aspiration. Levels of alpha-subunit mRNA were 40% lower in patients establishing normal pregnancies than in those who failed to become pregnant or who spontaneously aborted. Thus, a relative diminution of immediately preovulatory levels of mRNA for inhibin alpha-subunit is a marker of success in clinical IVF/ET cycles. This marker of IVF/ET success can be related to previously established markers of success (increased follicular fluid oestradiol and decreased follicular fluid cyclic adenosine monophosphate) by known physiological mechanisms.

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice.

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.

Effects of tamoxifen on corpus luteum function and luteal phase length in cynomolgus monkeys.

Previous data in nonhuman primates have demonstrated that tamoxifen prolongs the luteal phase without altering reproductive hormone levels. A small study in humans found no effect on menstrual cycle length, but an increase in luteal ovarian steroid levels. In view of these conflicting results, we studied the effect of tamoxifen on corpus luteum (CL) function in monkeys (n = 20). Blood was obtained daily beginning cycle day 8, and sera assayed for estradiol (E2), progesterone (P), luteinizing hormone, and follicle-stimulating hormone. Four days after the midcycle E2 peak, laparoscopy confirmed CL formation, and the animals were administered (1) lactose (n = 7), (2) tamoxifen, 0.5 mg.kg-1.d-1 (n = 6), or (3) tamoxifen, 3.0 mg.kg-1.d-1 (n = 7) for 12 consecutive days. Serum collection continued until cycle day 50 or menses, whichever came first. Results indicate a biphasic response among tamoxifen-treated animals, with 7 of 13 developing prolonged luteal phases. There was, however, no significant difference in luteal phase length among the three groups, although when the two groups given tamoxifen were combined, the difference in luteal phase length versus controls approached significance. No differences were found among peak P levels, mean luteal phase P levels, or mean luteal phase gonadotropin levels. No variables were found to correlate significantly with luteal phase length. These results suggest that luteal phase administration of the antiestrogen tamoxifen does not alter pituitary gonadotropin secretion or CL function. However, tamoxifen does prolong luteal phase length in a subset of monkeys, perhaps via a direct effect on the endometrium.

Identification of a nucleotide exchange-promoting activity for p21ras.

The biological activity of proteins encoded by the ras family of oncogenes is dependent on whether they are bound to GTP or GDP: the type of nucleotide bound is dependent on the rate of GTP hydrolysis (promoted by the GTPase-activating protein, GAP) and the rate of nucleotide exchange with cytosolic pools. A protein that stimulates the rate of exchange of guanine nucleotide on p21ras has been identified and characterized in cytoplasmic extracts of human placenta. The exchange-promoting protein runs on a gel filtration column with an apparent relative molecular weight of about 60,000. It is sensitive to heat and to trypsin. The exchange-promoting protein acts reversibly and does not cause degradation of p21ras. It is inactive towards the alpha subunit of a heterotrimeric GTP-binding protein (Go alpha) but acts on a large number of different mutant ras proteins, including transforming and effector mutants that are insensitive to the action of GAP. This protein, which we have termed REP (ras exchange-promoting), has the characteristics expected of a physiological activator of p21ras in cellular growth-signal-transduction pathways.

Premature luteinizing hormone surges in menopausal gonadotropin-stimulated cycles in monkeys: lack of initiation by progesterone.

The occurrence of spontaneous luteinizing hormone (LH) surges in women receiving human menopausal gonadotropins (hMG) for in vitro fertilization-embryo transfer is a significant clinical problem. One hypothetical mechanism is that premature progesterone (P) secretion occurring in the high estradiol (E2) milieu produced by hMG triggers the spontaneous LH surge. To investigate this possibility, 11 rhesus and cynomolgus monkeys were stimulated with hMG. At maximal ovarian stimulation, monkeys were injected with 15 micrograms/kg P (n = 3), 30 micrograms/kg P (n = 3), or 1,000 IU human chorionic gonadotropin (hCG) (n = 5; controls). Blood for E2, P, and LH was drawn twice daily in the periovulatory period and daily before and after this period. Laparoscopy was performed after P or hCG injection. In the 6 monkeys receiving P, no LH surges were detected. Further, postinjection P profiles and laparoscopy showed no evidence of ovulation. Controls demonstrated laparoscopic and hormonal evidence of ovulation. These findings suggest that P does not trigger LH surges in hMG-stimulated cycles.

Effects of gonadotropin-releasing hormone agonist and clomiphene citrate on estrogen/progesterone-induced hyperprolactinemia in monkeys.

The effects of follicular phase clomiphene citrate (CC) and two regimens of leuprolide acetate on estrogen-progesterone-induced hyperprolactinemia in nonhuman primates were studied. All groups received estradiol (E2) benzoate (12.5 micrograms intramuscularly on cycle days 2 to 33) and progesterone (P) (silastic implant for cycle days 14 to 33). A gonadotropin-releasing hormone agonist (GnRH-a) (Lupron 0.5 mg daily, TAP Pharmaceuticals, Chicago, IL) was administered from cycle day 2 to 14 in group II and from day 20 of the previous cycle until cycle day 14 in group III. Oral CC was given on cycle days 3 through 7 in group IV. No significant differences of mean E2 and P concentrations were noted between groups. Neither GnRH-a nor CC had an overall effect on E2/P-induced hyperprolactinemia. However, for the 5-day interval at the onset of the P treatment there was a significant increase in prolactin (PRL) secretion for group II (130.4 +/- 30.6) versus group I (53.9 +/- 3.3), group III (64.4 +/- 11.1), and group IV (68.8 +/- 14.3). This suggests that leuprolide may exert a delayed stimulatory effect on PRL secretion, or that complete suppression of the putative paracrine regulation of PRL stimulation may require more than 13 days of GnRH-a administration.

Steroid hormone regulation of the Achlya ambisexualis 85-kilodalton heat shock protein, a component of the Achlya steroid receptor complex.

The steroid hormone antheridiol regulates sexual development in the fungus Achlya ambisexualis. Analyses of in vivo-labeled proteins from hormone-treated cells revealed that one of the characteristic antheridiol-induced proteins appeared to be very similar to the Achyla 85-kilodalton (kDa) heat shock protein. Analysis of in vitro translation products of RNA isolated from control, heat-shocked, or hormone-treated cells demonstrated an increased accumulation of mRNA encoding a similar 85-kDa protein in both the heat-shocked and hormone-treated cells. Northern (RNA) blot analyses with a Drosophila melanogaster hsp83 probe indicated that a mRNA species of approximately 2.8 kilobases was substantially enriched in both heat-shocked and hormone-treated cells. The monoclonal antibody AC88, which recognizes the non-hormone-binding component of the Achyla steroid receptor, cross-reacted with Achlya hsp85 in cytosols from heat-shocked cells. This monoclonal antibody also recognized both the hormone-induced and heat shock-induced 85-kDa in vitro translation products. Taken together, these data suggest that similar or identical 85-kDa proteins are independently regulated by the steroid hormone antheridiol and by heat shock and that this protein is part of the Achyla steroid receptor complex. Our results demonstrate that the association of hsp90 family proteins with steroid receptors observed in mammals and birds extends also to the eucaryotic microbes and suggest that this association may have evolved early in steroid-responsive systems.

Cocaine disrupts estrous cyclicity and alters the reproductive neuroendocrine axis in the rat.

Although a common drug of abuse, cocaine's effects on cyclic reproductive functions and the neuroendocrine systems regulating these functions have not been studied. Here, we report the effects of cocaine on (1) estrous cyclicity and ovulation rates and (2) the stimulated in vitro release of hypothalamic GnRH and aminergic neurotransmitters directly involved in regulating or modulating GnRH release. Within 7 days of treatment with 10 mg kg-1 day-1 of cocaine HCl subcutaneously, rats demonstrated significant estrous cycle irregularity including repetitive days of estrus and prolonged periods of diestrus. After 6 weeks of treatment, cocaine-treated rats exhibited a 44.3% decrease in ovulation rates. For the in vitro studies, bilaterally ovariectomized rats were injected with cocaine (10 mg kg-1 day-1) or with saline for 2 weeks. Each rat received estradiol benzoate (50 mg kg-1 day-1 s.c.) for 2 days before sacrifice. Hypothalamic slices were prepared, placed in 0.1 ml microchambers and perfused with modified Krebs buffer (pH 7.4) using a programmable perfusion system. Basal release of norepinephrine (NE) and serotonin (5HT) was significantly increased in the cocaine-treated group versus controls. Ten-minute pulses of 10(-7)M progesterone (P4) increase NE and 5HT, but not dopamine (DA), release in the saline-treated group. In contrast, pulses of P4 increased NE, but not 5HT or DA, in the cocaine-treated rats. Ten-minute pulses of 0.1 microM NE increased GnRH release in both saline- and cocaine-treated rats. However, the response to pulsed NE was significantly attenuated in the cocaine-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)