Loss of Centromere Cohesion in Aneuploid Human Oocytes Correlates with Decreased Kinetochore Localization of the Sac Proteins Bub1 and Bubr1.

In human eggs, aneuploidy increases with age and can result in infertility and genetic diseases. Studies in mouse oocytes suggest that reduced centromere cohesion and spindle assembly checkpoint (SAC) activity could be at the origin of chromosome missegregation. Little is known about these two features in humans. Here, we show that in human eggs, inter-kinetochore distances of bivalent chromosomes strongly increase with age. This results in the formation of univalent chromosomes during metaphase I (MI) and of single chromatids in metaphase II (MII). We also investigated SAC activity by checking the localization of BUB1 and BUBR1. We found that they localize at the kinetochore with a similar temporal timing than in mitotic cells and in a MPS1-dependent manner, suggesting that the SAC signalling pathway is active in human oocytes. Moreover, our data also suggest that this checkpoint is inactivated when centromere cohesion is lost in MI and consequently cannot inhibit premature sister chromatid separation. Finally, we show that the kinetochore localization of BUB1 and BUBR1 decreases with the age of the oocyte donors. This could contribute to oocyte aneuploidy.

Female aging alters expression of human cumulus cells genes that are essential for oocyte quality.

Impact of female aging is an important issue in human reproduction. There was a need for an extensive analysis of age impact on transcriptome profile of cumulus cells (CCs) to link oocyte quality and developmental potential with patient's age. CCs from patients of three age groups were analyzed individually using microarrays. RT-qPCR validation was performed on independent CC cohorts. We focused here on pathways affected by aging in CCs that may explain the decline of oocyte quality with age. In CCs collected from patients >37 years, angiogenic genes including ANGPTL4, LEPR, TGFBR3, and FGF2 were significantly overexpressed compared to patients of the two younger groups. In contrast genes implicated in TGF-ß signaling pathway such as AMH, TGFB1, inhibin, and activin receptor were underexpressed. CCs from patients whose ages are between 31 and 36 years showed an overexpression of genes related to insulin signaling pathway such as IGFBP3, PIK3R1, and IGFBP5. A bioinformatic analysis was performed to identify the microRNAs that are potential regulators of the differentially expressed genes of the study. It revealed that the pathways impacted by age were potential targets of specific miRNAs previously identified in our CCs small RNAs sequencing.

Comparative gene expression profiling in human cumulus cells according to ovarian gonadotropin treatments.

In in vitro fertilization cycles, both HP-hMG and rFSH gonadotropin treatments are widely used to control human follicle development. The objectives of this study are (i) to characterize and compare gene expression profiles in cumulus cells (CCs) of periovulatory follicles obtained from patients stimulated with HP-hMG or rFSH in a GnRH antagonist cycle and (ii) to examine their relationship with in vitro embryo development, using Human Genome U133 Plus 2.0 microarrays. Genes that were upregulated in HP-hMG-treated CCs are involved in lipid metabolism (GM2A) and cell-to-cell interactions (GJA5). Conversely, genes upregulated in rFSH-treated CCs are implicated in cell assembly and organization (COL1A1 and COL3A1). Interestingly, some genes specific to each gonadotropin treatment (NPY1R and GM2A for HP-hMG; GREM1 and OSBPL6 for rFSH) were associated with day 3 embryo quality and blastocyst grade at day 5, while others (STC2 and PTX3) were related to in vitro embryo quality in both gonadotropin treatments. These genes may prove valuable as biomarkers of in vitro embryo quality.

Laparoscopic-assisted colopexy and sterilization in male dogs: short-term results and physiologic consequences.

OBJECTIVE: To describe laparoscopic-assisted colopexy and sterilization, and evaluate outcome and effects in healthy male dogs. STUDY DESIGN: Experimental study. ANIMALS: Male Beagle dogs (n=7). METHODS: A laparoscopic-assisted, extracorporeally sutured colopexy, and sterilization by ligation and section of the testicular vessels and ductus deferens were evaluated 11 weeks after surgery. Ex vivo tensile tests were performed on the colopexy sites and loss of testicular function was assessed by monitoring serum testosterone, and by ultrasonographic and histologic examinations of the testes. Systemic inflammation and potential iatrogenic colonic functional disorders were investigated by monitoring serum C-reactive protein (CRP) in the perioperative period and from a sulfapyridine (SP) kinetic profile obtained before and 10 weeks after surgery. RESULTS: No intraoperative complications were recorded and clinical outcome was considered fair in all dogs. A mean tensile force of 42 N was required to disrupt colopexies. No relevant postoperative increase in CRP concentrations or changes in SP kinetics were observed. Testicular function was lost. CONCLUSIONS: Laparoscopic-assisted colopexy achieved adhesion of the colon to the abdominal wall and testicular endocrine function and spermatogenesis were eliminated by laparoscopic castration.

Oocyte recovery post human follicular fluid centrifugation in modified natural cycle and achieving embryo.

This case reports a successful live birth by intracytoplasmic sperm injection (ICSI) following human follicular fluid (HFF) centrifugation for oocyte retrieval in the modified natural cycle of a poor responder patient. A 37-year-old patient presenting with a severe ovarian defect underwent a modified natural cycle with HFF centrifugation prior to ICSI. As there was only one oocyte under direct binocular observation, HFF was centrifuged and a second oocyte was collected. ICSI was performed on both oocytes. Embryo quality and outcome were not compromised by HFF centrifugation. A live birth was achieved in April 2008. In a modified natural cycle, HFF centrifugation avoided loss of oocytes, optimized the IVF treatment, and achieved the development of two embryos.