Enhancement of the efficiency of oocyte vitrification through regulation of histone deacetylase 6 expression.

OBJECTIVE: Successful oocyte vitrification (OV) is critical for cryopreservation of the oocytes from female patients with infertility, polycystic ovaries, and gynecologic cancers. Recent evidence suggests that relatively low levels of histone acetylation are critical for maintenance of the maturation capacity of cryopreserved oocytes. However, previous studies have only demonstrated a key role of histone deacetylases (HDAC) 1 and 2 in the cryopreservation of oocytes. METHODS: In this study, we investigated the role of HDAC6 in these settings. We found that mouse oocytes with low HDAC6 levels decreased survival rate, cleavage rate, and blastocyst rate after OV. Bioinformatics analyses were used to predict HDAC6-targeting microRNAs (miRNAs), while the functional binding of miRNAs to HDAC6 mRNA was evaluated by a dual luciferase reporter assay. RESULTS: Among all HDAC6-targeting miRNAs, we detected expression of miR-558, miR-527, and miR-762 in mouse oocytes. Specifically, we found that only miR-762 significantly inhibited protein translation of HDAC6 via binding to the 3'-UTR of the HDAC6 mRNA. Transfection of oocytes with HDAC6 or antisense of miR-762 significantly increased the survival rate, the cleavage rate, and blastocyst rate after OV. CONCLUSION: As a result, our data suggest that induction of HDAC6 levels by miR-762 suppression may improve the current protocol for OV.

Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer.

BACKGROUND: The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT. RESULTS: We investigated whether the in vitro development of reconstructed bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability, pregnancy rate up to day 90 of gestation, as well as percent of term births were higher in the homotype SCNT groups than in the heterotype groups. In addition, homotype and heterotype SCNT embryos showed different methylation patterns of histone 3-lysine 9 (H3K9) genome-wide and at pluripotency-related genes (Oct-4, Sox-2, Nanog). CONCLUSION: Both histone and DNA methylation show that homotype SCNT blastocysts have a more successful epigenetic asymmetry pattern than heterotype SCNT blastocysts, which indicates more complete nuclear reprogramming. This may result from variability in their epigenetic patterns and responses to nuclear reprogramming. This suggests that the compatibility of mtDNA haplotypes between donor cells and host oocytes can significantly affect the developmental competence of reconstructed embryos in SCNT, and may include an epigenetic mechanism.

ICSI treatment of severe male infertility can achieve prospective embryo quality compared with IVF of fertile donor sperm on sibling oocytes.

Azoospermia, cryptozoospermia and necrospermia can markedly decrease the ability of males to achieve pregnancy in fertile females. However, patients with these severe conditions still have the option to be treated by intracytoplasmic sperm injection (ICSI) to become biological fathers. This study analyzed the fertilization ability and the developmental viabilities of the derived embryos after ICSI treatment of the sperm from these patients compared with in vitro fertilization (IVF) treatment of the proven-fertile donor sperm on sibling oocytes as a control. On the day of oocyte retrieval, the number of sperm suitable for ICSI collected from two ejaculates or testicular sperm extraction was lower than the oocytes, and therefore, excess sibling oocytes were treated by IVF with donor sperm. From 72 couples (73 cycles), 1117 metaphase II oocytes were divided into 512 for ICSI and 605 for IVF. Compared with the control, husbands' sperm produced a lower fertilization rate in nonobstructive azoospermia (65.4% vs 83.2%; P< 0.001), crytozoospermia (68.8% vs 75.5%; P< 0.05) and necrospermia (65.0% vs 85.2%; P< 0.05). The zygotes derived in nonobstructive azoospermia had a lower cleavage rate (96.4% vs 99.4%; P< 0.05), but the rate of resultant good-quality embryos was not different. Analysis of the rates of cleaved and good-quality embryos in crytozoospermia and necrospermia did not exhibit a significant difference from the control. In conclusion, although the sperm from severe male infertility reduced the fertilization ability, the derived embryos had potential developmental viabilities that might be predictive for the expected clinical outcomes.

Endocrine gland-derived vascular endothelial growth factor concentrations in follicular fluid and serum may predict ovarian hyperstimulation syndrome in women undergoing controlled ovarian hyperstimulation.

OBJECTIVE: To assess the predictive value of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) concentrations in follicular fluid (FF) and serum for ovarian hyperstimulation syndrome (OHSS) in patients undergoing controlled ovarian hyperstimulation. DESIGN: Retrospective, case-control study. SETTING: University hospital, IVF center. PATIENT(S): Seventeen women with OHSS and 61 controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): FF and serum EG-VEGF and VEGF concentrations, IVF outcome. RESULT(S): FF and serum EG-VEGF concentrations showed a significant negative correlation with serum E(2) concentration on the day of hCG administration. FF, but not serum, VEGF concentrations also showed a significant negative correlation with serum E(2) concentrations on hCG day. The FF EG-VEGF, FF VEGF, and serum EG-VEFG concentrations were significantly lower in the OHSS group than in the non-OHSS group. There was no significant difference in serum VEGF concentrations. Among FF and serum EG-VEGF and VEGF concentrations, only FF EG-VEGF concentrations were significantly lower in patients with moderate OHSS than in those with mild OHSS. CONCLUSION(S): FF and serum EG-VEGF concentrations may predict OHSS occurrence. Furthermore, FF EG-VEGF concentrations were significantly correlated with OHSS severity; thus, EG-VEGF appears to be more valuable than VEGF for predicting OHSS.

Improved efficiency of bovine somatic cell nuclear transfer by optimizing operational procedures.

To improve bovine somatic cell nuclear transfer (SCNT) efficiency, we studied various aspects to optimize the experimental procedures. Firstly, donor cells were treated with pronase, which resulted in a higher fusion rate than that of cells without the pronase treatment (78.3 vs. 53.9%). Secondly, when fused embryos were activated either by chemical (ionomycin + cyclohemixide (CHX)) or electrical + CHX stimulation, the cleavage and blastocyst formation rates were comparable amongst these treatment groups (P>0.05); however, mortality following electrical + CHX activation was significantly higher than that observed with the chemical activation, regardless of the pronase treatment (P<0.05). Finally, we compared the culture conditions of the reconstructed embryos using ACM medium plus mouse embryonic fibroblasts (MEF) vs. B2 medium plus granulose cells (GC), and the results clearly demonstrated that the former culture conditions led to a higher blastocyst rate, 90-day pregnancy rate, and newborn rate, than that observed for culture in B2 medium plus GC (46.7 vs. 34.7%, 36.1 vs. 9.6% and 25.9 vs. 5.8% for the blastocyst, pregnancy and newborn rates, respectively). In summary, the efficiency of bovine SCNT can be greatly improved using optimized operational procedures, including treating the donor cells with pronase, activation of fused embryos by ionomycin + CHX and the culture of the reconstructed embryos in ACM + MEF media.