The Study Of Mechanisms Underlying The Skewed Sex Ratio At Birth After In Vitro Fertilization

In vitro fertilization (IVF), one of the most effective and successful assisted reproductive technologies, is widely used to treat human infertility and improve intensity of genetic selection from genetically superior individuals in domestic animals. Although the great majority of IVF-conceived offspring are in good health, epidemiologic analyses in humans and laboratory studies in animals show that IVF is associated with various short- or long-term consequences, such as miscarriage, preterm birth, birth defects, skewed sex ratio, as well as higher disease risks in later life, such as metabolic diseases and cancer. IVF-induced epigenetic errors are suspicious of key spoilers of these health complications. Unfortunately, the nature of these epigenetic errors and their roles have never been determined, impeding the prevention of any related complications.Skewed sex ratio is an issue of great concern among the IVF-associated complications. Since 1991, this phenomena has been confirmed repeatedly in bovine, porcine and human IVF embryos. However, the underlying mechanisms remain unclear. Sex ratio is an important indicator of reproductive health, its skewing has a potential effect on long-term social and behavior consequences. IVF-associated sex skewing reflects potential gender-biased embryonic developmental defects, which are reminiscent of the disrupted gender-specific epigenetic events. Thus, we hypothesized that the IVF process may disturb gender-specific epigenetic events, thereby leading to the skewed sex ratio. Here, we generated mouse embryos under standardized IVF conditions (IVF group) and used in vivo fertilized embryos as the control (IVO group). We found that sex ratio is skewed in mouse IVF offspring, and this is due to female-biased developmental defects during periimplantation stage. Functional experiments demonstrated that the sex skewing is caused by impaired imprinted X-chromosome inactivation (iXCI) via reduced Rnf12/Xist expression. Accordingly, correcting this epigenetic error by overexpression of Rnfl2 to compensate iXCI via Xist upregulation or by incubation of the IVF embryos with an epigenetic modulator retinoic acid redeemed the skewed sex ratio. Thus, our data show that iXCI is one of the major epigenetic barriers for the developmental competence of female embryos during preimplantation stage and targeting erroneous epigenetic modifications may provide a potential approach for preventing IVF-associated complications.Moreover, comparative microRNA profiling between IVO and IVF mouse embryos at E3.5, E7.5, and E10.5 showed that the dysregulated microRNAs were mainly associated with the regulation of genes involved in carcinogenesis, genetic information processing, glucose metabolism, cytoskeleton organization, and neurogenesis. Further analysis showed that only miR-199a-5p was consistently downregulated in IVF embryos when compared with their IVO counterparts from E3.5 to El0.5. Functional experiments demonstrated that IVF-induced downregulation of miR-199a-5p plays an important role in the higher glycolytic rate, the lower developmental potential, and the lower survival post-implantation development in IVF embryos. Therefore, preventing the downregulation of miR-199a-5p may become an effective strategy for improving the development of IVF peri-implantation embryos.