Jian-Zhong Sheng

Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics‐based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2‐DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, α‐SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.

ORIGINAL ARTICLE: High Expression of l‐Selectin Ligand in Secretory Endometrium is Associated with Better Endometrial Receptivity and Facilitates Embryo Implantation in Human Being

Problemu2002 l‐selectin ligand has displayed mediating adhesion at the maternal–fetal interface. Therefore, we investigated the impact of l‐selectin ligand on establishing pregnancy in women undergoing in vitro fertilization and embryo transfer (IVF‐ET).

General imprinting status is stable in assisted reproduction–conceived offspring

OBJECTIVEnTo evaluate whether the genomic imprinting status of assistant reproductive technology (ART)-conceived offspring is stable.nnnDESIGNnProspective clinical observational study.nnnSETTINGnInxa0vitro fertilization (IVF) center, university-affiliated teaching hospital.nnnPATIENT(S)nSixty ART-conceived babies (30 IVF and 30 intracytoplasmic sperm injection [ICSI]) and 60 naturally conceived babies.nnnINTERVENTION(S)nCollection of umbilical cord blood and peripheral blood samples.nnnMAIN OUTCOME MEASURE(S)nExpression 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.nnnRESULT(S)nHierarchic 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.nnnCONCLUSION(S)nThe 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.

Adriamycin induces H2AX phosphorylation in human spermatozoa

AIMnTo investigate whether adriamycin induces DNA damage and the formation of gammaH2AX (the phosphorylated form of histone H2AX) foci in mature spermatozoa.nnnMETHODSnHuman spermatozoa were treated with adriamycin at different concentrations. gammaH2AX was analyzed by immunofluorescent staining and flow cytometry and double-strand breaks (DSB) were detected by the comet assay.nnnRESULTSnThe neutral comet assay revealed that the treatment with adriamycin at 2 microg/mL for different times (0.5, 2, 8 and 24 h), or for 8 h at different concentrations (0.4, 2 and 10 microg/mL), induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of gH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP1 with gammaH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, abolished the co-appearance of these two proteins with gammaH2AX.nnnCONCLUSIONnHuman mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/repair proteins as somatic cells.

Reduced Expression and Function of Aquaporin- 3 in Mouse Metaphase-II Oocytes Induced by Controlled Ovarian Hyperstimulation were Associated with Subsequent Low Fertilization Rate

Background/Aims: Aquaporin-3 (AQP3), one isoform of water channel family, has been found to be expressed in mouse oocytes. The present study aimed to investigate whether functional AQP3 was expressed in oocytes induced by controlled ovarian hyperstimulation (COH), and whether altered oocyte AQP3 expression was associated with changes in fertilization rate. Methods: Sixty ICR female mice were divided into two groups: COH and control. AQP3 mRNA expression of mouse metaphase II (MII) oocytes was quantified by real-time RT-PCR. The water permeability of oocytes was assessed with cell swelling test. The fertilization profiles of oocytes were generated via in vitro fertilization. Results: AQP3 mRNA was expressed in both natural and COH-induced mouse oocytes. COH significantly reduced AQP3 mRNA expression. The volume of oocytes was significantly increased after exposure to hypotonic medium and pretreatment with HgCl2 attenuated hypotonic medium-induced increase in oocyte volume and water permeability coefficient (Pf). Furthermore, the expression of AQP3, Pf and the fertilization rate were significantly lower in COH oocytes than those in control. Conclusion: AQP3 might play an important role in controlling oocyte quality and a low in vitro fertilization rate of COH mice might, in part, result from reduced AQP3 expression and water permeability in mouse oocytes.

Cardiovascular and metabolic profiles of offspring conceived by assisted reproductive technologies: a systematic review and meta-analysis

OBJECTIVEnTo evaluate cardiovascular and metabolic features of offspring conceived by inxa0vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI).nnnDESIGNnLiterature review and meta-analysis.nnnSETTINGnNot applicable.nnnPATIENT(S)nOffspring from IVF-ICSI versus natural conception.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nSystolic and diastolic blood pressure (SBP and DBP), cardiovascular function, body mass index (BMI), and lipid and glucose profiles.nnnRESULT(S)nWe 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: 1.88xa0mm Hg [95% CI, 0.27, 3.49] for SBP and 1.51xa0mm 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.nnnCONCLUSION(S)nChildren 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.

G546A polymorphism of growth differentiation factor-9 contributes to the poor outcome of ovarian stimulation in women with diminished ovarian reserve

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.

Leptin down-regulates γ-ENaC expression: a novel mechanism involved in low endometrial receptivity

OBJECTIVEnTo 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.nnnDESIGNnControlled, prospective, clinical, experimental study.nnnSETTINGnUniversity-based infertility center.nnnPATIENT(S)nBlood 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.nnnINTERVENTION(S)nHuman endometrial biopsies.nnnMAIN OUTCOME MEASURE(S)nExpression of ENaC mRNA and protein in endometrium.nnnRESULT(S)nThe 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.nnnCONCLUSION(S)nHigh 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.

Cryoprotectants up-regulate expression of mouse oocyte AQP7, which facilitates water diffusion during cryopreservation

OBJECTIVEnTo investigate the effects of cryoprotectants on the expression of AQP7 in oocytes.nnnDESIGNnExperimental animal study.nnnSETTINGnUniversity-based research laboratory.nnnANIMAL(S)nAdult female C57BL/6J mice.nnnINTERVENTION(S)nIn 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.nnnMAIN OUTCOME MEASURE(S)nAQP7 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.nnnRESULT(S)nAQP7 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.nnnCONCLUSION(S)nDMSO 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.