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Objective: To investigate whether polymorphisms in estrogen receptor alpha (ERalpha) or beta (ERbeta) genes are associated with a risk of miscarriage., Design: A retrospectively analyzed, prospectively obtained database of cases and controls., Setting: University hospital menopause unit., Patient(s): 177 women with at least one spontaneous abortion and 442 controls with at least one live birth and no history of miscarriage., Intervention(s): None., Main Outcome Measure(s): Genotype frequencies and odd ratios for abortion risk in cases and controls for four single nucleotide polymorphisms (SNPs) located in intron 1 (C>T and A>G), intron 4 (A>T), and exon 8 (T>C) for the ERalpha gene, and two SNPs located in intron 2 (C>T) and intron 8 (G>A) for the ERbeta gene., Result(s): A statistically significant association was found between spontaneous abortion and SNPs rs2234693 (C>T, defined by restriction enzyme PvuII) and rs9340799 (A>G, defined by restriction enzyme XbaI) in intron 1 of the ERalpha gene. The age-adjusted odds ratio for abortion risk was 1.29 for the TA haplotype (defined by PvuII-XbaI) with respect to the CG haplotype in women with at least one abortion, which increased to 1.9 in women with two or more abortions., Conclusion(s): The TA haplotype defined by PvuII and XbaI was associated with an increased risk of reproductive loss. No association was found for the ERbeta gene., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Postmenopausal women treated with an isopropanolic extract of Cimicifuga racemosa underwent a decrease in the urinary concentration of N-telopeptides, a marker of bone resorption, and an increase in alkaline phosphatase, a marker of bone formation, at the third month of therapy. Serum from treated women did not modify the activity of alkaline phosphatase or the expression of three genes, runt-related transcription factor-2 (Runx-2), alkaline phosphatase, and osteocalcin, when added to the MC3T3-E1 osteoblastic cell line.

Objective: To examine the proliferative effect of of raloxifene on human umbilical-vein endothelial cells (HUVECs), and to investigate whether there is an associated increased expression of some key regulators of the cell cycle., Design: Cell culture for different incubation times., Setting: University research laboratory., Patient(s): Sources of HUVECs., Intervention(s): Measurement of cell proliferation, of protein levels of cyclin A, cyclin B1, cyclin D1, cyclin-dependent protein kinase (CDK) 2, CDK4, and p27(Kip1), and of messenger RNA expression of cyclin A, cyclin B1, and p27(Kip1)., Main Outcome Measure(s): Cell proliferation was measured by the 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the bromo-2'-deoxyuridine assay, and the sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate assay. Changes in protein expression were measured by immunoblotting, and modifications in gene expression were measured by quantitative real-time polymerase chain reaction., Result(s): Both 1 nM and 10 nM of either E(2) or raloxifene achieved a similar increase in cell proliferation. The pure antiestrogen ICI 182780 only blocked the E(2)-induced proliferative effect. Western blot experiments detected an increase in the expression of only cyclin A and B1, and a decreasing trend for p27(Kip1). Enhancements in gene expression were observed in response to E(2) and raloxifene for cyclin A and B1. No significant changes were found for p27(Kip1). The ICI 182780 effectively abrogated the increased gene expression associated with E(2) for cyclin B1, but not for cyclin A. In contrast, ICI 182780 was ineffective in the case of raloxifene., Conclusion(s): Raloxifene increased the proliferation of HUVECs in association with enhanced gene expression of cyclins A and B1.

We investigated the effect of a therapeutic dose of raloxifene on the plasma levels of myeloperoxidase and F2alpha-isoprostanes, two markers of oxidative stress recently described as reliable indicators of coronary heart disease. Contrary to changes described in the literature for estrogens (E), raloxifene did not modify the levels of either myeloproxidase or F2alpha-isoprostanes after 3 or 6 months of treatment.

Objective: To examine the effects of raloxifene on prostacyclin production by human umbilical vein endothelial cells (HUVEC) and to shed light on the molecular details of that action., Design: Cell culture for 4, 8, 16, 24, and 48 hours., Setting: University research laboratory., Patient(s): Source of HUVEC., Intervention(s): Measurement of prostacyclin production and of protein levels and mRNA expression of cyclooxygenase (COX)-1 and -2., Main Outcome Measure(s): Prostacyclin production was measured by enzyme immunoassay, the mRNA expression of COX-1 was measured by quantitative real time-polymerase chain reaction, and the protein levels of COX-1 and -2 were measured by immunoblotting., Result(s): Raloxifene significantly increased prostacyclin release in a time- and dose-dependent manner, being higher than control after 24 hours. Raloxifene, at 0.1-10 nM, increased the mRNA expression of COX-1 and the protein content of both COX-1 as well as COX-2. All of these effects were independent of the classical pathway for estrogen receptor (ER) activation because the treatment of cells with the ER antagonist ICI 182780 did not eliminate any of the effects. Although treatment with either the selective COX-1 inhibitor SC-560 or the selective COX-2 inhibitor NS-398 significantly diminished prostacyclin release (20% +/- 5% and 24% +/- 7%, respectively), co-treatment with raloxifene and either SC-560 or NS-398 was followed by a smaller increase than that achieved by raloxifene alone. The nonselective COX inhibitor indomethacin, however, reduced prostacyclin production to 37% +/- 11% of control values., Conclusion(s): Raloxifene increased HUVEC prostacyclin release through a mechanism possibly distinct from the classical ER pathway and involving enhanced COX-1 and COX-2 expression and activity.

Objective: To determine the effects of menopause and three different formulations of E2 plus medroxyprogesterone acetate on serum concentrations of basal and growth hormone-releasing hormone (GHRH)-stimulated growth hormone (GH), insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein (IGFBP)-1, IGFBP-3, insulin, and C peptide., Design: Prospective, controlled trial., Setting: Menopausal outpatient clinic at an academic tertiary care hospital., Patient(s): Nineteen postmenopausal women with different menopausal ages. Seventeen premenopausal women were included as controls., Intervention(s): Oral estrogen (E2 valerate, 2 mg/d) or transdermal estrogen (50-microg or 100-microg E2 patch) was administered for 8 weeks. Medroxyprogesterone acetate (5 mg/d) was administered during weeks 3, 4, 7, and 8 of each protocol. Blood samples were collected before treatment and after the completion of each protocol from postmenopausal women, and on cycle days 6-8 from premenopausal women., Main Outcome Measure(s): Levels of GH, IGF-1, IGFBP-1, IGFBP-3, insulin, and C peptide., Result(s): Basal GH levels were negatively correlated with age in premenopausal women but not in postmenopausal women. The area under the GHRH-induced GH curve decreased in older postmenopausal women after the oral estrogen protocol. Levels of IGF-1 diminished after the oral E2 protocol in postmenopausal women., Conclusion(s): The administration of oral, but not transdermal, E2 plus medroxyprogesterone acetate at the usual clinical doses used in postmenopausal women decreased IGF-1 levels and the response of GH to GHRH in older women. No substantial changes were detected in IGFBP-1, IGFBP-3, insulin, or C peptide levels.

Objective: To compare compliance, symptom control, bleeding patterns, endometrial response, and lipid changes in postmenopausal women treated with transdermal E2 and a regimen of either intermittent or continuous dosing of progestin., Design: Randomized, prospective study., Setting: Menopausal Outpatient Clinic at an academic tertiary care hospital., Patient(s): One hundred women who had reached menopause naturally and had been amenorrheic for at least 1 year. Fifty women were randomly assigned to receive each regimen., Intervention(s): All patients received 50-microg E2 patches and medroxyprogesterone acetate, either 5 mg twice per week or 2.5 mg daily. The bleeding pattern was registered in diary cards. Endometrial status was assessed by vaginal ultrasound and endometrial biopsy. Lipid levels were measured by ELISA., Main Outcome Measure(s): The number of patients who dropped out of the study and the number of days each patient reported spotting or bleeding were recorded. Endometrial thickness was measured and histologic examination of the endometrial tissue was performed. Plasma levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were measured., Result(s): Fourteen women dropped out of the intermittent dosing group and 13 dropped out of the continuous dosing group. Irregular spotting or bleeding occurred at a similar rate in both groups. Biopsies performed at the end of the study showed adequate endometrial stabilization in both groups. No significant changes in lipid levels were detected with either regimen., Conclusion(s): Both regimens were similarly effective. The high rate of atrophic endometria suggests the possibility of further reduction of the progestin dose.

Objective: To evaluate the heparin-glutathione test (HEGLUT) for the selection of viable sperm for intracytoplasmic sperm injection (ICSI)., Design: A prospective study., Setting: Department of Pediatrics, Obstetrics and Gynecology, University of Valencia and Instituto Valenciano de Infertilidad., Patient(s): Semen samples from healthy donors and patients with infertility., Intervention(s): Sperm samples were kept in culture for different periods in Ham's F-10 medium supplemented or not supplemented with heparin, reduced glutathione (GSH), or a heparin-GSH mixture. Control and heparin-GSH-treated spermatozoa were injected into hamster oocytes. The HEGLUT and ICSI were performed., Main Outcome Measure(s): Sperm nuclear decondensation, progressive and nonprogressive motility, and male pronucleus formation., Result(s): The maximum proportion of sperm nuclear decondensation (28.7%+/-2.1% versus 2.6%+/-0.5% in the control group) was reached after 60 minutes of incubation in the presence of a heparin-GSH mixture. Differences in the percentages of progressive and nonprogressive motility among treatments and times of incubation, although statistically significant, were biologically negligible. No statistically significant differences were observed in the rate of sperm head decondensation (8.2% [4/49] versus 11.1% [6/54]) and male pronucleus formation (18.4% [9/49] versus 22.2% [12/541) after the injection of control and treated spermatozoa into hamster oocytes., Conclusion(s): The HEGLUT may offer an alternative to the hypo-osmotic swelling test for the selection of viable sperm for ICSI.

Objective: To investigate the changes induced by age in the function and secretory pattern of the human ovary. Immunoreactive alpha-inhibin, E2, and P secretion in vivo and in vitro have been compared in two different populations., Design: Prospective study. Women undergoing IVF-ET were divided into two groups according to age: group 1 (32.0 +/- 0.7 years; mean +/- SEM) and group 2 (40.3 +/- 0.3 years)., Setting: In vitro fertilization program at the Instituto Valenciano de Infertilidad., Patients: A total of 33 infertile women with regular menses, undergoing IVF-ET., Interventions: Follicle aspiration performed by transvaginal ultrasound. Four follicles per patient were aspirated in individual plastic tubes. Granulosa-luteal cells isolated with Percoll columns and cultured in vitro up to 4 days in the presence of hCG., Main Outcome Measures: In vitro fertilization parameters, serum levels of E2, immunoreactive alpha-inhibin, and P, as well as the secretion of immunoreactive alpha-inhibin and P by the cultured granulosa-luteal cells., Results: Serum immunoreactive alpha-inhibin levels the day of ovum pick-up were significantly lower in group 2 compared with group 1. Incubation of cells for 96 hours showed a significantly higher ability to accumulate immunoreactive alpha-inhibin in group 1 than 2. Human chorionic gonadotropin stimulated immunoreactive alpha-inhibin production after 96 hours. Cells from younger women displayed a significantly higher ability to secrete P than cells from older women. Human chorionic gonadotropin was able to significantly stimulate P production in group 1., Conclusions: These results confirm previous observations showing a reduced production of immunoreactive alpha-inhibin and steroids of ovaries from older women and suggest that a reduced cellular function, rather than a decrease in the follicular population, is the main mechanism by which these changes are produced.

Objective: To determine the effect of FSH/LH in vivo and epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in vitro on human oocyte maturation., Design: Oocyte-cumulus complexes were harvested from three different groups of patients: [1] unstimulated ovaries from women undergoing surgery; [2] multifollicular development achieved with a combination of FSH and LH in the absence of an ovulatory dose of hCG; and [3] oocyte-cumulus complexes retrieved after appropriate ovarian stimulation with FSH/LH and hCG for IVF purposes., Setting, Patients: In vitro fertilization program and patients undergoing surgery for benign disorders at the Instituto Valenciano de Infertilidad, Valencia, Spain., Interventions: Oocyte-cumulus complexes from unstimulated ovaries collected at surgery by follicular puncture and washing. Oocyte-cumulus complexes from stimulated cycles obtained by ultrasound-guided transvaginal aspiration. Oocyte-cumulus complexes cultured in vitro in the absence or presence of different concentrations of EGF and IGF-I., Main Outcome Measure: Germinal vesicle breakdown and metaphase-II stage after 24 and 48 hours., Results: Comparison of the spontaneous resumption of meiosis and metaphase II oocytes among groups showed significant differences between unstimulated and stimulated ovaries after 24 and 48 hours in culture. Administration of hCG accelerated the percentage of maturation by 24 hours. Further incubation of unstimulated oocyte-cumulus complexes with EGF and IGF-I significantly increased the percentage of metaphase-II oocytes after 24 and 48 hours in culture., Conclusions: Epidermal growth factor and IGF-I are able to augment spontaneous maturaion in immature human oocytes. Because spontaneous maturation is mainly observed when follicles have been exposed to pharmacological doses of hMG, it is suggested that increasing FSH levels within the follicle is coincident with the generation of a positive signal necessary to complete oocyte maturation in humans. This signal may be linked to the dynamics of growth factors within the follicle itself.

Objective: To analyze different biopsy methods, embryo stages, and cellular masses that can be removed for preimplantation diagnosis of genetic diseases to find optimal biopsy conditions compatible with the subsequent development of the conceptus, the acquisition of intact viable blastomeres, and the reliability of the genetic analysis., Data Identification: The most important published studies have been identified through a computerized bibliographical search (MEDLINE; Dialog, Palo Alto, CA)., Study Selection: Studies reporting different embryo biopsy methods practiced at different stages have been selected., Results: The analysis carried out in the current review shows, at the present time, the following: [1] the displacement and push methods may be more suitable than the stitch and pull and aspiration (puncturing the zona pellucida) approaches at cleavages stages; [2] the aspiration and stitch and pull procedures may assure higher success rates than the herniation procedure at the blastocyst stage; [3] the mechanical division method and the use of acid Tyrode's solution would not be advisable before the eight-cell stage; [4] human embryos at the two-cell and blastocyst stages may not be suitable for preimplantation diagnosis because of an excessive reduction of cellular mass at the two-cell stage and a low or zero pregnancy rate after transfer at the blastocyst stage; and [5] biopsy of a quarter of the embryonic cellular mass on day 2 after insemination may increase biochemical pregnancies if the cleavage rate is not preserved., Conclusions: At the present time, biopsy of a quarter of the embryo on day 3 after insemination may be the most feasible approach for preimplantation diagnosis.

Objective: To assess any reduction in viability and development in vitro after biopsy of a quarter of the cells of human embryos on day 2 after insemination., Design: A prospective study in which normally fertilized surplus embryos of good morphology with two to eight cells approximately 48 hours after insemination were randomly allocated to a control or biopsied group, respectively., Setting: In vitro fertilization (IVF) unit and laboratories of the Hammersmith Hospital, Institute of Obstetrics and Gynaecology, London University., Patients, Participants: One hundred twenty-nine embryos from 28 infertile IVF patients., Interventions: Follicular aspiration by ultrasound-guided transvaginal puncture and embryo biopsy by micromanipulative procedures., Main Outcome Measure(s): Pyruvate uptake and cell number at the blastocyst stage., Results: Embryo biopsy did not have an adverse effect on either the proportion developing to the blastocyst stage (50% [32 of 64] and 47.7% [31 of 65] for the control and biopsied groups, respectively) or embryo viability, measured indirectly through pyruvate uptake. However, the proportion of embryos that reached the morula stage after day 4 (retarded embryos) was significantly higher (44%, 11 of 25 versus 8.7%, 2 of 23) in the biopsied group. The total number of cells (29.6 +/- 3.1 versus 62.4 +/- 4.7), numbers of inner cell mass (7.7 +/- 2.2 versus 24.5 +/- 1.4) and trophectoderm (24.0 +/- 5.2 versus 45.0 +/- 6.4) cells, and the inner cell mass:trophectoderm ratio (34.7 +/- 7.9 versus 59.5 +/- 11.7) were strikingly reduced at the blastocyst stage in the biopsied group. This reduction was greater in embryos that reached the morula stage after day 4., Conclusions: More investigation is needed to assess whether the detrimental effects observed were because of the biopsy method used in this study or to a high sensitivity of human embryos at early stages to manipulation in vitro.

Objective: To investigate possible causes of in vitro fertilization (IVF) failure., Design: A retrospective cytogenetic study of human oocytes divided into four groups or, alternatively, into two groups according to fertilization rates and whether the patients became pregnant or not. Two additional groups included oocytes in which there was no or only partial fertilization., Setting: Primary treatment of infertility in an institutional practice., Patients, Participants: Two hundred fifty-three inseminated-unfertilized oocytes from 87 women entering the IVF program because of tubal, unknown, and male infertility. Immunological infertility was excluded., Interventions: Ultrasound-guided transvaginal follicular aspiration., Main Outcome Measure(s): Planned after data collection., Results: The rate of chromosome anomalies did not show any significant difference among the four groups established according to the fertilization rate and between pregnant and nonpregnant patients. Independently, our data identified male factor as responsible for 41%, chromosome anomalies 19.3%, oocyte immaturity 11.8%, and unknown etiology 41% of fertilization failures (based on analysis of 161 oocytes)., Conclusions: Fertilization rate and pregnancy outcome after IVF are not related to the incidence of oocyte chromosome anomalies.

Objective: To determine possible effects on chromosomal status of human oocytes in vitro., Design: A prospective cytogenetic study of human oocytes fixed at 4, 20 (noninseminated oocytes), or approximately 52 hours after pick-up (inseminated-unfertilized oocytes)., Setting: Primary treatment of infertility in an institutional practice., Patients, Participants: Eighty-eight consecutive in vitro fertilization (IVF) patients (262 inseminated-unfertilized oocytes) and 47 IVF patients with greater than 8 oocytes retrieved (95 noninseminated oocytes)., Interventions: Ultrasound-guided transvaginal follicular aspiration., Main Outcome Measure(s): Planned before data collection began., Results: The incidence of chromosome anomalies did not differ between groups. However, a negative correlation between oocytes apparently without chromosomes and oocytes with fragmented chromosomes was found., Conclusions: Incubation of oocyte in vitro does not affect the chromosome complement. The mechanisms leading to oocytes apparently without chromosomes and with fragmented chromosomes are self-excluding.

Several reports have shown decreased fertilization and implantation rates in high-responder patients to gonadotropins. In this cytogenetic study of unfertilized human oocytes, we have investigated possible reasons for this clinical evidence. Three groups of patients were established according to the number of oocytes retrieved: group 1 (n = 21), 1 to 5 oocytes; group 2 (n = 32), 6 to 10 oocytes; and group 3 (n = 35), greater than or equal to 11 oocytes. Patients in group 3 had lower estradiol levels per follicle developed, lower follicular volume, higher incidence of diploid oocytes, and higher rate of oocytes with an additional set of prematurely condensed sperm chromosomes of the presynthetic gap than groups 1 and 2. These results suggest that women with greater than or equal to 11 oocytes retrieved have a relative higher cytoplasmic immaturity that could explain the lower fertilization and implantation rates found in other studies.