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Objective: To determine matrix metalloproteinase 2 (MMP-2) levels in sera and peritoneal fluids of women with or without endometriosis-associated infertility and to determine whether MMP-2 had correlation with 17β-E2 and P in the sera and peritoneal fluids of the patients with endometriosis.Design: A case–control study.Setting: A women''s hospital in China.Patient(s): Forty patients with endometriosis-associated infertility undergoing laparoscopy and 18 women undergoing tubal ligation.Intervention(s): Sera and peritoneal fluids were collected from women with or without endometriosis.Main outcome measure(s): Matrix metalloproteinase 2, 17β-E2, and P concentration.Result(s): Compared with the control women, the patients with endometriosis had higher MMP-2 levels in their sera and peritoneal fluids. Serum and peritoneal fluid MMP-2 levels were higher in the proliferative phase than in the secretory phase. The level of MMP-2 in the patients was correlated positively with 17β-E2 in serum and peritoneal fluid and negatively with P.Conclusion(s): The results demonstrated a correlation between MMP-2 concentrations and steroid hormones in sera and peritoneal fluids of the patients with endometriosis-associated infertility. Estradiol might up-regulate MMP-2, resulting in the formation of endometriosis. Progesterone might down-regulate MMP-2 to inhibit the formation and development of endometriosis. [Copyright &y& Elsevier]

Y chromosome microdeletions were detected in 33.3% (6 out of 18) of idiopathic azoospermic patients with high serum follicle-stimulating hormone (FSH) levels in the present study. The results suggest that it may be necessary to detect microdeletions in patients with azoospermia, especially for those with high serum FSH levels, before assisted reproductive technology (ART) is provided to them. [Copyright &y& Elsevier]

Objective: To investigate the effects of cryoprotectants on the expression of AQP7 in oocytes. Design: Experimental animal study. Setting: University-based research laboratory. Animal(s): Adult female C57BL/6J mice. Intervention(s): In metaphase II (MII) oocytes obtained from adult female C57BL/6J mice and from donations by fertile women, the mouse oocytes were treated with human tubal fluid medium containing 8% ethylene glycol (EG), 9.5% dimethylsulfoxide (DMSO), and 0.5 M sucrose, respectively; 293T cells transfected with GFP-hAQP7 expression vector were treated with the same solutions. Main Outcome Measure(s): AQP7 expression in oocytes examined by reverse-transcriptase-nested polymerase chain reaction and immunofluorescence, changes in the volume of mouse oocytes treated with different solutions calculated to determine their permeability to water, and survival rates of vitrified oocytes. Result(s): AQP7 is expressed in human and mouse oocytes. Cryoprotectants, including EG, DMSO, and sucrose, up-regulated AQP7 expression in mouse oocytes and 293T cells transfected with GFP-hAQP7 expression vector. Compared with other cryoprotectants, DMSO stimulated higher expression of AQP7, and this was associated with faster cell volume recovery and lower survival rates of vitrified oocytes. Conclusion(s): DMSO up-regulates AQP7 expression in mouse oocytes more than EG. This may facilitate water diffusion and reduce the time for oocytes to reach osmotic balance with the cryoprotectant solution during cryopreservation. [ABSTRACT FROM AUTHOR]

Objective: To investigate whether the embryotoxic effect of peritoneal fluid (PF) from infertile women with mild endometriosis on mouse oocytes and embryos is associated with changes in embryonic epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and their receptors. Design: Experimental animal study. Setting: University-based research laboratory. Animal(s): Adult ICR mice. Intervention(s): Peritoneal fluid was obtained from fertile women with no endometriosis (PF-NE) and infertile women with mild endometriosis (PF-E). In vitro fertilization was performed, and mouse two-cell stage embryos were cultured in human tubal fluid medium with or without PF. Main Outcome Measure(s): Rates of fertilization, cleavage, and blastulation. The embryonic EGF and IGF-I levels in culture medium were analyzed by enzyme-linked immunosorbent assay, and EGF receptor, IGF-I receptor, and phosphorylated extracellular signal-regulated protein kinases (p-ERK) expression was determined by immunofluorescence and confocal microscopy. Result(s): When oocytes and embryos were cultured in media with PF-E, the fertilization capability of oocytes and the development potential of embryos were decreased. The levels of embryonic EGF, IGF-I, and their receptors were increased. However, p-ERK of the postreceptor signal transduction pathway was down-regulated. Conclusion(s): Endometriotic PF may attenuate oocyte and embryo development by impairing embryonic growth factor/receptor/signal transduction, resulting in endometriotic infertility. [Copyright &y& Elsevier]

Objective: To investigate the possible roles of cyclin B1/cyclin-dependent kinase (cdc2) and Polo-like kinase 1 (Plk1) in the pathogenesis of endometriosis.Design: A case-control study.Setting: University hospital.Patient(s): Patients with or without endometriosis were diagnosed by pathological examination or laparoscopy. The patients with the following criteria within the past 6 months were excluded: endocrine or inflammatory diseases, pregnancy or lactation, hormonal therapy, and neoplasm in the uterine cavity.Intervention(s): Eutopic and ectopic endometria were obtained at the time of surgery. Blood was collected on the same day as surgery.Main Outcome Measure(s): The mRNA/protein expression and localization of cyclin B1, cdc2, and Plk1 in endometrium, and serum levels of E(2) and P.Result(s): The expression levels of cyclin B1 and Plk1, but not cdc2, in ectopic endometria were significantly higher than in eutopic endometria. The immunohistochemical staining of cyclin B1 and Plk1 was detected in the nuclei of ectopic and eutopic endometrial cells. Furthermore, ectopic endometrial expression levels of cyclin B1 or Plk1 were positively correlated with serum E(2) levels.Conclusion(s): Cyclin B1 and Plk1 may play important roles in the pathogenesis of endometriosis by mediating ectopic endometrial cell proliferation under regulation of ovarian hormones. [ABSTRACT FROM AUTHOR]

Objective: To assess the effects of autologous mature follicular fluid (FF) on the in vitro maturation (IVM) outcome of human cumulus-removed oocytes from routine stimulated ovaries, particularly on the IVM co-culture outcome with autologous cumulus cells. Design: Prospective comparison study. Setting: A provincial reproductive medicine and research center. Patient(s): A total of 196 germinal vesicle (GV) and 192 metaphase I (MI) stage oocytes were recruited from 151 infertile women by ovarian stimulation for routine intracytoplasmic sperm injection (ICSI) program. Intervention(s): Two novel IVM systems assisted with either autologous mature FF alone or both autologous mature FF and autologous cumulus cells were developed to mature in vitro MI and GV stage oocytes, respectively. A standard IVM system was used as the control. Main Outcome Measure(s): Cumulative IVM rate of oocytes and the subsequent fertilization, cleavage, and preimplantation embryonic development. Result(s): By adding autologous mature FF to the medium of the IVM system, the nuclear maturation of human cumulus-removed oocytes was significantly promoted in both speed and rate, but not the corresponding fertilization, cleavage, and preimplantation embryonic development. Conclusion(s): Autologous mature FF favors the IVM of human cumulus-removed oocytes, including the IVM co-culture with autologous cumulus cells, mainly in nuclear, but not cytoplasmic, maturation. [Copyright &y& Elsevier]

Objective: To investigate c-fos and matrix metalloproteinase-9 (MMP-9) expression in the endometrium from women with or without endometriosis throughout the menstrual cycle, and to explore the correlation of c-fos expression with MMP-9 expression and 17β-E2 levels in serum. Design: Molecular studies in human tissue. Setting: A women''s hospital in China. Patient(s): Fifty-five premenopausal women (25 with endometriosis and 30 without endometriosis) undergoing laparoscopic surgery or hysterectomy. Intervention(s): Eutopic and ectopic endometrium tissue were obtained at the time of surgery. Peripheral sera were also collected on the same day. Main Outcome Measure(s): Immunohistochemical localization of c-fos in the endometrium, c-fos and MMP-9 protein levels in the endometrium, and 17β-E2 levels in the serum. Result(s): c-fos protein was predominantly located in the nuclei of glandular epithelial cells and stromal cells. c-fos and MMP-9 protein levels in paired eutopic and ectopic endometria from women with endometriosis were significantly higher than those in the endometrium from women without endometriosis. No significant difference in c-fos or MMP-9 protein levels was observed between paired eutopic and ectopic endometria. c-fos protein levels in endometrium positively correlated with endometrial MMP-9 levels and serum 17β-E2 levels. Conclusion(s): Expression of c-fos in the human endometrium may be regulated by 17β-E2, and c-fos may be involved in development of endometriosis by promoting MMP-9 gene expression and subsequently the invasive potential of endometrial explants. [Copyright &y& Elsevier]

Objective: To investigate the risks of chromosome mutation after ART for couples with comparable genetic backgrounds. Design: Prospective clinical observational study. Setting: In vitro fertilization center at a tertiary-care, university-affiliated teaching hospital. Patient(s): Ninety-seven male children whose fathers have normal spermatogenesis were recruited, including 19 babies conceived through IVF, 18 babies conceived through intracytoplasmic sperm injection (ICSI), and 60 naturally conceived babies, as well as the babies'' fathers. Intervention(s): Collection of peripheral and umbilical cord blood samples. Main Outcome Measure(s): The Yq genetic status of the babies and fathers according to 13 Y-specific markers covering four azoospermia factor (AZF) subregions, the karyotype, and the neonatal examination. Result(s): We found that all children had a normal 46, XY karyotype, but de novo Y-chromosome microdeletions were identified in 1 (5.3%) of 19 IVF offspring and in 3 (16.7%) of 18 ICSI offspring. The incidence of de novo Y-chromosome microdeletion in male children conceived through ICSI or IVF was statistically significantly higher than that in those conceived naturally (10.8% vs. 0). In four babies with microdeletion, one was complicated, with hypospadias. Conclusion(s): Our results, for the first time, indicate that risks of gene mutation may increase in the ART offspring, even though their fathers have normal spermatogenesis and genetic background. Hence, intense attention should be placed on genetic safety in the ART children, and the benefits and risks of adopting ART should be balanced gingerly. [Copyright &y& Elsevier]

Objective: To explore the importance of follicle-stimulating hormone receptor (FSHR) in granulosa cells in the ovarian response to gonadotropin stimulation. Design: Prospective study. Setting: A women’s hospital in China. Patient(s): One hundred infertile women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (rFSH). Intervention(s): These women were divided into three groups: poor, moderate, and high responders, according to the number of follicles with diameter ≥14 mm. The FSHR expression at both mRNA and protein levels was determined by either reverse transcription–polymerase chain reaction or Western blot in granulosa cells. E2 concentrations in serum and FSH levels in serum/follicular fluid (FF) were measured by electrochemiluminescence immunoassay. Main Outcome Measure(s): Relative expression of mRNA and protein of FSHR in granulosa cells, serum E2, FSH level in serum and FF, and the number of mature follicles. Result(s): The expression of FSHR, at both the mRNA and protein levels, was significantly different among the three groups, with the lowest expression in the poor responders. The level of FSHR protein was positively correlated with the peak level of serum E2 and the number of mature oocytes. FSH levels in FF and the dosage of rFSH used were significantly different among the three groups, with the highest values in the poor responders. Conclusion(s): Different levels of FSHR expression in granulosa cells result in different ovarian response, and lower expression of FSHR may account for poor ovarian response to gonadotropin stimulation, which suggests the critical role of FSHR in the ovarian response to gonadotropin stimulation. [Copyright &y& Elsevier]

Objective: To establish a mouse model for endometriosis and to evaluate roles of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in the formation of disease.Design: Experimental laboratory study.Setting: A women's hospital in China.Patient(s) and Animal(s): Ten women with endometriosis and 10 control women, as well as ICR mice.Intervention(s): Endometrial fragments were transplanted in the peritoneal cavities of mice at minilaparotomy. Transplants were observed and then removed for the assessment of morphology and immunohistochemical staining of VEGF and MMP-2.Main Outcome Measure(s): Observation of transplants, expression of VEGF and MMP-2.Result(s): On days 1 and 2, glandular and stromal cells were viable at the margins of transplants. On day 3, the transplants were surrounded by mesothelial cells, and the endometrial glands and stromal cells were clearly viable at the interface. The scores of VEGF and MMP-2 of viable glandular cells of transplants were increased compared with the ones before transplantation. The scores of VEGF and MMP-2 of transplants from women with endometriosis were higher than those of control women.Conclusion(s): Endometrial transplants from the patients with endometriosis express more VEGF and MMP-2 than endometrium in control women, suggesting that VEGF and MMP-2 may expedite the formation of endometriosis in its early stage. [ABSTRACT FROM AUTHOR]

The growth differential factor-9 (GDF-9) gene, an oocyte-specific factor, was screened in 106 Chinese women with diminished ovarian reserve (DOR), and three single-nucleotide polymorphisms, c.G169A, c.C447T and c.G546A, were detected. We found GDF-9 c.G546A, but not c.G169A or c.C447T, to be correlated with the poor ovarian stimulation and in vitro fertilization outcomes in women with DOR. [ABSTRACT FROM AUTHOR]

Objective: To evaluate cardiovascular and metabolic features of offspring conceived by in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI).Design: Literature review and meta-analysis.Setting: Not applicable.Patient(s): Offspring from IVF-ICSI versus natural conception.Intervention(s): None.Main Outcome Measure(s): Systolic and diastolic blood pressure (SBP and DBP), cardiovascular function, body mass index (BMI), and lipid and glucose profiles.Result(s): We included 19 studies that had recruited 2,112 IVF-ICSI and 4,096 naturally conceived offspring, ranging from childhood to early adulthood. The blood pressure levels of IVF-ICSI offspring were statistically significantly higher than those of naturally conceived offspring (weighted mean differences and confidence intervals: 1.88 mm Hg [95% CI, 0.27, 3.49] for SBP and 1.51 mm Hg [95% CI, 0.33, 2.70] for DBP). In addition, cardiac diastolic function was suboptimal and vessel thickness was higher among IVF-ICSI offspring. Compared with the metabolism of naturally conceived offspring, IVF-ICSI offspring displayed comparable BMI, lower low-density lipoprotein cholesterol levels, and higher fasting insulin levels.Conclusion(s): Children conceived by IVF-ICSI manifested a minor yet statistically significant increase in blood pressure without the clustering of increased BMI or impaired lipid metabolism by early adulthood. Our findings indicate a risk of cardiovascular disease among IVF-ICSI offspring, which calls for longer-term follow-ups and further investigation. [ABSTRACT FROM AUTHOR]

Objective To examine epithelial Na + channel (ENaC) expression in endometrium of overweight/obese women with polycystic ovary syndrome (PCOS) during the window of implantation, and to explore the mechanism linking leptin-mediated reduction of γ-ENaC to low endometrial receptivity. Design Controlled, prospective, clinical, experimental study. Setting University-based infertility center. Patient(s) Blood and endometrium samples were collected from 12 control women and 12 overweight/obese PCOS patients. Pregnancy outcomes were obtained from 245 women with male-factor infertility (533 cycles) and 57 infertile women with PCOS (120 cycles) who underwent intrauterine insemination. Intervention(s) Human endometrial biopsies. Main Outcome Measure(s) Expression of ENaC mRNA and protein in endometrium. Result(s) The expression of γ-ENaC decreased in the secretory phase endometrium of PCOS patients who showed increased serum leptin levels. In cultured endometrial cells (Ishikawa cells), leptin dose-dependently down-regulated the expression of γ-ENaC and reduced the JAr spheroid attachment rate, which could be blocked by knockdown of STAT3, a signal in the pathway of leptin receptor activation. The overweight/obese PCOS patients with increased serum leptin levels showed a significantly increased biochemical pregnancy rate, suggesting that high leptin might attenuate endometrial receptivity and increase very early pregnancy loss. Conclusion(s) High serum leptin may reduce endometrial receptivity by activating the STAT3 signal pathway and down-regulating γ-ENaC expression in the endometrium. These results provide valuable new insights into the molecular mechanisms linking abnormal ENaC gene expression to early pregnancy loss in overweight/obese PCOS patients. [ABSTRACT FROM AUTHOR]

Objective: To assess the expression patterns of SAM68 in the testes of azoospermic patients with normal and abnormal spermatogenesis. Design: Retrospective study and in vitro study. Setting: University hospital. Patient(s): Testicular biopsies of azoospermic men with normal spermatogenesis (OAZ; n = 20), with maturation arrest at the spermatocyte stage (MA; n = 20), and with Sertoli cell–only syndrome (SCOS; n = 10). Intervention(s): No interventions with patients. Knockdown of Sam68 was performed in the GC-2spd(ts) cell line. Main Outcome Measure(s): SAM68 expression was analyzed using quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry analysis in tissues. Moreover, Sam68 was knocked down in GC-2spd(ts) cells. Cell viability was measured using the MTT assay, and the apoptosis rate was detected using flow cytometry with the Annexin V–FITC kit. Result(s): Using qRT-PCR, the expression level of testicular SAM68 mRNA in MA and SCOS patients was statistically reduced compared with in OAZ patients. In addition, using qRT-PCR, Western blot, and immunohistochemistry analyses, mRNA and protein expressions of SAM68 were absent or barely detectable in testicular tissues in 45% (9 of 20) of patients with MA and in all patients with SCOS. Furthermore, decreased expression of Sam68 suppressed germ cell proliferation and induced apoptosis in transfected GC-2spd(ts) cells. Conclusion(s): Deficient SAM68 expression was observed in the human testis with MA at the spermatocyte stage and SCOS. These results may offer new perspectives on the molecular basis of abnormal spermatogenesis. [ABSTRACT FROM AUTHOR]

Objective: To evaluate whether the genomic imprinting status of assistant reproductive technology (ART)–conceived offspring is stable. Design: Prospective clinical observational study. Setting: In vitro fertilization (IVF) center, university-affiliated teaching hospital. Patient(s): Sixty ART-conceived babies (30 IVF and 30 intracytoplasmic sperm injection [ICSI]) and 60 naturally conceived babies. Intervention(s): Collection of umbilical cord blood and peripheral blood samples. Main Outcome Measure(s): Expression profile was examined by microarray and real-time reverse-transcription polymerase chain reaction (PCR), allele-specific expression was studied by direct sequencing after PCR, and DNA methylation status was investigated by sodium bisulfite sequencing. Result(s): Hierarchic clustering demonstrated no obvious clustering between the ART- and naturally conceived offspring, suggesting similar genomic imprinting expression between the two groups. Three differentially expressed genes were identified in ART-conceived offspring, with PEG10 and L3MBTL up-regulated and PHLDA2 down-regulated. Allele-specific expression of the differentially expressed imprinted genes was maintained in the majority of the ART- and naturally conceived offspring. However, in one ICSI case, monoallelic expression of L3MBTL was disrupted and all CpGs were completely unmethylated. These were not inherited from the parents. Conclusion(s): The global profile of imprinting is stable in children conceived through ART. However, imprinting of a few specific imprinted genes may be vulnerable in a fraction of ART-conceived children. [Copyright &y& Elsevier]