No abstract

In vitro maturation of human oocytes at the germinal vesicle (GV) stage could offer an alternative in several cases of female infertility. It however rests on a better knowledge of the quality of human oocyte. Using fluorescence imaging of DNA and of the transcription sites, combined with electron microscopy, we show that human oocytes follow size-dependent changes in chromatin configuration, transcription sites distribution and nuclear ultrastructure that follow those observed in mouse GV oocytes. We thus analyzed in mouse GV oocytes the phosphorylation dependence of the transcriptional activity. We show by Western blot that, while active GV oocytes have approximately the same proportion of hypo- and hyperphosphorylated forms of the RNA polymerase II (RNAP II), the hyperphosphorylated form is almost absent from inactive oocytes. We also show that (1) RNAP II-dependent transcription is much less sensitive to various kinase inhibitors in mouse oocytes than in somatic cells or mouse one-cell embryos, although the phosphorylation equilibrium of RNAP II was largely shifted towards the hypo-phosphorylated form upon treatment with these inhibitors (2) RNAP I is completely insensitive to kinase inhibitors in GV oocytes.

No abstract

Reactive oxygen species (ROS) have a negative impact on sperm DNA, leading to the formation of oxidative products such as 8-oxo-7,8-dihydroxyguanosine. This compound causes fragmentation and, thus, has a mutagenic effect. Patient treatment with oral antioxidant vitamins is, therefore, standard practice for male infertility, in an attempt to decrease formation of ROS and improve fertility. In this study, the DNA fragmentation index and the degree of sperm decondensation were measured using the sperm chromatin structure assay before and after 90 days treatment with antioxidant vitamins associated with zinc and selenium. Antioxidant treatment led to a decrease in sperm DNA fragmentation (-19.1%, P < 0.0004), suggesting that at least part of the decay was linked to ROS. However, it also led to an unexpected negative effect: an increase in sperm decondensation with the same order of magnitude (+22.8%, P < 0.0009). The opening of interchain disulphide bridges in protamines may explain this aspect, as antioxidant vitamins, especially vitamin C, are able to open the cystin net, thus interfering with paternal gene activity during preimplantation development. This observation might explain the discrepancy observed concerning the role of these antioxidant treatments in improving male fertility.

More than 17,000 intrauterine insemination (lUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. The quantity and motility of spermatozoa in the final preparation used for insemination had a positive effect on the outcome, as classically observed in the past. It was found that advanced maternal age had a negative effect on the pregnancy rate and was associated with increased miscarriage rate. More interestingly, an exactly parallel effect was found for paternal age. The impact of increased age on necrospermia and sperm DNA structure is discussed as a probable direct cause of this paternal effect.

Embryos are conventionally selected for transfer based on the evaluation of the cleavage speed and extent of blastomere fragmentation. Here we examined whether the predictive value of these criteria, as indicators of the chance of embryo implantation, can be further potentiated by adding previously described criteria reflecting the regularity of pronuclear development. In a group of embryos selected for transfer in 380 fresh embryo transfer cycles according to the conventional criteria, the transfer of only those embryos that developed from zygotes judged normal at the pronuclear stage (pattern 0) gave significantly higher pregnancy (44.8%) and implantation (30.2%) rates compared with the pregnancy (22.1%; P < 0. 05) and implantation rates (11.2%; P < 0.001) for the transfers of only those embryos that developed from zygotes judged abnormal (non-pattern 0). The transfer of only one pattern 0 embryo was sufficient for the optimal chance of pregnancy (no differences in pregnancy rates after transfer of one, two or three pattern 0 embryos), whereas the transfer of two pattern 0 embryos mostly resulted in a twin pregnancy. The inclusion of the criteria based on pronuclear morphology can thus lead to the application of a single embryo transfer policy and optimize the selection of embryos for transfer and cryopreservation.

Germ cell apoptosis was evaluated in 11 men suffering from nonobstructive azoospermia and enrolled in a spermatid conception programme. In six of these patients, round spermatids (Sa stage) were the most advanced spermatogenic cells recovered from testicular biopsy samples. This condition is referred to as complete spermiogenesis failure. In the remaining five men, a few late elongated spermatids (Sd stage) were unexpectedly found in the testicular biopsy samples on the day of treatment. This condition is referred to as incomplete spermiogenesis failure. Germ cell apoptosis in both groups of patients was examined by analysing cell smears prepared from mechanically disintegrated testicular tissues using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), which detects apoptosis-specific DNA fragmentation, and annexin-V binding, detecting apoptosis-related translocation of plasma membrane phosphatidylserine to the membrane's outer surface. Both methods were combined, in double-fluorescence labelling preparations, with immunocytochemical detection of proacrosin, a specific germline marker. Patients with complete spermiogenesis failure had significantly higher frequencies of primary spermatocytes and round spermatids carrying the apoptosis-specific DNA damage in comparison with patients with incomplete spermiogenesis failure. Surprisingly, apoptosis-related phosphatidylserine externalization occurs rarely until the advanced stages of spermiogenesis. Since externalized phosphatidylserine is expected to be involved in the recognition of apoptotic cells by phagocytes, apoptotic spermatocytes and round spermatids may not be removed easily by phagocytosis. The high frequency of DNA damage in round spermatids from patients with complete spermiogenesis failure explains the low success rates of spermatid conception in these cases. The evaluation of apoptosis can help predict success rates of spermatid conception.

Purpose Investigate in what extent sperm transcriptome of infertile men is different from that of fertile individuals. Methods Semen samples were collected for determination of sperm parameters as well as for RNA isolation. Gene expression profile was investigated in spermatozoa of 8 infertile and 3 fertile men by microarray analysis using the Affymetrix Chip HG-U133 Plus 2.0. Result(s)We observed up to 33-fold reduction expression of genes involved in spermatogenesis and sperm motility. Furthermore, there is an important decrease in expression of genes involved in DNA repair as well as oxidative stress regulation. In this study, we also show a striking drop in expression of histone modification genes. Conclusion(s) We found that transcription profile in germ cells of men with idiopathic infertility is different from that of fertile individuals. Interestingly, about 15% of the regulated genes (Eddy Rev Reprod 4:23-30, 1999) play a role in spermatogenesis.