Assisted reproduction treatment and epigenetic inheritance

Background: The subject of epigenetic risk of assisted reproduction treatment (ART), initiated by reports on an increase of children with the Beckwith–Wiedemann imprinting disorder, is very topical. Hence, there is a growing literature, including mouse studies. Methods: In order to gain information on transgenerational epigenetic inheritance and epigenetic effects induced by ART, literature databases were searched for papers on this topic using relevant keywords. Results: At the level of genomic imprinting involving CpG methylation, ART-induced epigenetic defects are convincingly observed in mice, especially for placenta, and seem more frequent than in humans. Data generally provide a warning as to the use of ovulation induction and in vitro culture. In human sperm from compromised spermatogenesis, sequence-specific DNA hypomethylation is observed repeatedly. Transmittance of sperm and oocyte DNA methylation defects is possible but, as deduced from the limited data available, largely prevented by selection of gametes for ART and/or non-viability of the resulting embryos. Some evidence indicates that subfertility itself is a risk factor for imprinting diseases. As in mouse, physiological effects from ART are observed in humans. In the human, indications for a broader target for changes in CpG methylation than imprinted DNA sequences alone have been found. In the mouse, a broader range of CpG sequences has not yet been studied. Also, a multigeneration study of systematic ART on epigenetic parameters is lacking. Conclusions: The field of epigenetic inheritance within the lifespan of an individual and between generations (via mitosis and meiosis, respectively) is growing, driven by the expansion of chromatin research. ART can induce epigenetic variation that might be transmitted to the next generation

Analysis of H19 methylation in control and abnormal human embryos, sperm and oocytes

International audienceART is suspected to generate increased imprinting errors in the lineage. Following an ICSI (Intra Cytoplamic Sperm Injection) procedure, a certain number of embryos fail to develop normally and imprinting disorders may be associated to the developmental failure. To evaluate this hypothesis, we analysed the methylation profile of H19DMR, a paternally imprinting control region, in high graded blastocysts, in embryos showing developmental anomalies, in the matching sperm and in oocytes of the concerned couples when they were available. Significant hypomethylation of the paternal allele was observed in half the embryos, independently of the stage at which they were arrested (morula, compacted morula, pre blastocyst or BC graded blastocysts). Conversely, some embryos showed significant methylation on the maternal allele, whereas few others showed both, hypomethylation of the paternal allele and abnormal methylation of the maternal allele. The matching sperm at the origin of the embryos exhibited normal methylated H19 patterns. Thus, hypomethylation of the paternal allele in the embryos does not appear inherited from the sperm but likely reflects instability of the imprint during the demethylating process which occurred in the early embryo. Analysis of a few oocytes suggests that the defect in erasure of the paternal imprint in the maternal germ line may be responsible for the residual methylation of the maternal allele in some embryos. None of these imprinting alterations could be related to a particular stage of developmental arrest; compared to high grade blastocysts, embryos with developmental failure are more likely to have abnormal imprinting at H19 (p<0.05)

Differential expression of parental alleles of BRCA1 in human preimplantation embryos

Gene expression from both parental genomes is required for completion of embryogenesis. Differential methylation of each parental genome has been observed in mouse and human preimplantation embryos. It is possible that these differences in methylation affect the level of gene transcripts from each parental genome in early developing embryos. The aim of this study was to investigate if there is a parent-specific pattern of BRCA1 expression in human embryos and to examine if this affects embryo development when the embryo carries a BRCA1 or BRCA2 pathogenic mutation. Differential parental expression of ACTB, SNRPN, H19 and BRCA1 was semi-quantitatively analysed by minisequencing in 95 human preimplantation embryos obtained from 15 couples undergoing preimplantation genetic diagnosis. BRCA1 was shown to be differentially expressed favouring the paternal transcript in early developing embryos. Methylation-specific PCR showed a variable methylation profile of BRCA1 promoter region at different stages of embryonic development. Embryos carrying paternally inherited BRCA1 or 2 pathogenic variants were shown to develop more slowly compared with the embryos with maternally inherited BRCA1 or 2 pathogenic mutations. This study suggests that differential demethylation of the parental genomes can influence the early development of preimplantation embryos. Expression of maternal and paternal genes is required for the completion of embryogenesis

DPY19L2, its mutation in about half globozoospermia

International audienceIntroduction: Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection is a promising assessment in reproductive practice particularly for patients with non obstructive azoospermia (NOA). There was evidence that impaired spermatogenesis could be related to an imbalance in the intratesticular oestradiol/testosterone ratio. We carried out a prospective observational study in order to evaluate the putative variation of the expression of genes implicated in the estrogen synthesis (aromatase) and mediation of estrogen action (estrogen receptors and GRP30 for the respective initiation of genomic and non-genomic pathways) in human testicular biopsies and to understand the mechanisms involved in different testicular disorders in relation to NOA such as hypospermatogenesis (Hsg), germ cell arrest (GCA) and Sertoli Cell Only (SCO) syndrome. Material and Methods: Histological evaluation, sperm recovery and ARN extraction followed by the measurement of relative mRNA level of cyp19, Esr1, Esr2 and gpr30 using real time polymerase chain reaction were realized in testicular bilateral fragments (n = 98) providing from 49 azoospermic patients. Taking into account the existence of potential discordant patterns in bilateral biopsies, the high prevalence of mixed patterns in a same testes and the fact that histological evaluation was always performed in a testicular biopsy different from this studied, we have reported the expression of specific genes considered as cells markers (Prm1 for round spermatids, H1t for pachytene spermatocytes and vimentin for mature Sertoli cells) for the selection of pure and homogeneous NOA forms. Then the expression of genes encoding for aromatase, estrogen receptors and GPR 30 has been evaluated in obstructive azoospermia group (0A) used as control and NOA groups (Hsg,GCA and SCO). Results are expressed as means + S.E.M. Statistical analysis was performed using ANOVA (Graphpad Instat 3, GraphPad Software, San Diego, CA, USA) and means are compared using Tukey-Kramer multiple comparisons test. Statistical significance was accepted at p < 0.05. Results: We have at first described specific patterns of pure forms of Hsg, GCA, SCO and OA with the helpful of cell markers. A pure form of SCO could be defined as a relative expression of vimentin transcript higher than 2 associated with an absence of Prm 1 or H1t transcripts. The level of Prm1 transcripts and the ratio Prm1 mRNA/H1t mRNA are significantly correlated with the number of spermatozoa recovered by TESE. A reduced expression of GRP30 is observed in all groups but seems more elevated in GCA group. Levels of the two isoforms ERalpha and ERbeta transcripts are significantly increased in OA and Hsg groups. But only the ERalpha level is strongly correlated with that of Prm1 and sperm recovery. Aromatase expression doest not differ significantly in the four groups studied. However we have found an intensive expression of aromatase and ERalpha in the SCO group associated with Leydig cell hyperplasia. Conclusions: Studying the putative variation of transcripts implicated in the estrogen synthesis and mediation of estrogen action in testicular biopsies could represent a helpful for the understanding of mechanisms involved in the pathogenesis of NOA forms and bring new insights about the role of estrogens during spermatogenesis. GRP30 expression seems to be restricted to testicular cells implicated in the first steps of spermatogenesis. The relative important expression of the two isoforms ERalpha and ERbeta in post-meiotic cells suggests their role during spermiogenesis. But an enhanced expression of ERalpha in Leydig cell hyperplasia and a tight correlation between ERalpha and Prm1 expression could argue the case for a differential role of the two ER isoforms during spermatogenesis