Sex-specific epigenetic profile of inner cell mass of mice conceived in vivo or by IVF.

The preimplantation stage of development is exquisitely sensitive to environmental stresses, and changes occurring during this developmental phase may have long-term health effects. Animal studies indicate that IVF offspring display metabolic alterations, including hypertension, glucose intolerance and cardiac hypertrophy, often in a sexual dimorphic fashion. The detailed nature of epigenetic changes following in-vitro culture is, however, unknown. This study was performed to evaluate the epigenetic (using whole-genome bisulfite sequencing (WGBS) and assay for transposase-accessible chromatin using sequencing (ATAC-seq)) and transcriptomic changes (using RNA-seq) occurring in the inner cell mass (ICM) of male or female mouse embryos generated in vivo or by IVF. We found that the ICM of IVF embryos, compared to the in-vivo ICM, differed in 3% of differentially methylated regions (DMRs), of which 0.1% were located on CpG islands. ATAC-seq revealed that 293 regions were more accessible and 101 were less accessible in IVF embryos, while RNA-seq revealed that 21 genes were differentially regulated in IVF embryos. Functional enrichment analysis revealed that stress signalling (STAT and NF-kB signalling), developmental processes and cardiac hypertrophy signalling showed consistent changes in WGBS and ATAC-seq platforms. In contrast, male and female embryos showed minimal changes. Male ICM had an increased number of significantly hyper-methylated DMRs, while only 27 regions showed different chromatin accessibility and only one gene was differentially expressed. In summary, this study provides the first comprehensive analysis of DNA methylation, chromatin accessibility and RNA expression changes induced by IVF in male and female ICMs. This dataset can be of value to all researchers interested in the developmental origin of health and disease (DOHaD) hypothesis and might lead to a better understanding of how early embryonic manipulation may affect adult health.

Ovulation induction is associated with altered growth but with preservation of normal metabolic function in murine offspring.

OBJECTIVE: To study the effects of ovulation induction on mouse postnatal health, with a focus on growth pattern and glucose tolerance. To study the effect of ovulation induction on DNA methylation, we took advantage of the agouti viable yellow (Avy) mouse. DESIGN: Animal study. SETTING: University Setting. ANIMALS: Agouti viable yellow (Avy) mice on a C57BL/6 background. INTERVENTION(S): Avy female mice were either allowed to mate spontaneously (control group, C) or after superovulation with 5 IU of PMSG and hCG (ovulation induction group, OI). MAIN OUTCOME MEASURE(S): Birth parameters and postnatal growth of the offspring were followed up to 29 weeks of age. Body composition analysis was performed by EchoMRI; fasting insulin, intraperitoneal glucose tolerance tests, and glucose-stimulated insulin secretion by beta cells were assessed to study glucose metabolism. RESULT(S): Mice born to superovulated dams had lower survival rates, shorter anogenital distances, and shorter crown-rump lengths. Female mice generated by OI weighed less at birth, whereas male mice generated by OI had lower weight gain and had reduced lean mass. Glucose parameters, including islet functions, did not differ between the groups. No difference in agouti coat color was noted between the groups. CONCLUSION(S): Ovulation induction resulted in mice having increased morphometric differences at birth and male mice showing reduced weight gain but no difference in glucose tolerance or agouti coat color.

Effect of culture conditions and method of conception on mouse live birth rate.

OBJECTIVE: To understand in a mouse model whether there are differences in the decidua and live birth rate after transfer of blastocysts generated by in vitro fertilization (IVF) or by superovulation with spontaneous mating into unstimulated recipients. DESIGN: Animal experiment. SETTING: University-affiliated tertiary hospital. ANIMAL(S): Mice. INTERVENTION(S): IVF embryos were generated and cultured in either Whitten medium (WM, suboptimal conditions) and 20% O2 or KSOM medium with amino acids (KAA, optimal conditions) and 5% O2. The control blastocysts from superovulated mice were flushed out of the uterus 3.5 days (E3.5) after mating (FB group). The resulting blastocysts were transferred to nonsuperovulated CF1 recipients mated to vasectomized males. To understand whether anomalies of decidua were present, the expression of genes involved in decidual development and inflammation was analyzed at E7.5 and E18.5. Similarly, immunostaining was used to evaluate whether the pathways involved in activation of mTORC1 (p-S6) and Cox2 signaling (Cox 2 staining) were altered. MAIN OUTCOME MEASURE(S): Live birth rate, gene expression, and immunostaining of decidua. RESULT(S): Implantation rates at E7.5 were similar, but in vivo embryos (FB groups) were predicted to result in live births 3.3 times higher (2.2-5.1) and 6.6 times higher (4.7-9.3) compared with optimal and suboptimal cultures, respectively. Expression of genes involved in decidual development and inflammation or localization and intensity of staining for p-S6 (mTOR pathway), or inflammation (Cox 2 pathway) were not different among the groups. CONCLUSION(S): The predicted live birth rate was decreased in mouse embryos generated by IVF compared with embryos generated by mating, whereas the implantation rate was not different. Suboptimal culture conditions resulted in lower birth rate. We did not find evidence of abnormalities in decidualization that could explain these findings. These data indicate that blastocysts cultured in stressful conditions are less competent, suggesting that decreasing the number of embryonic manipulations may result in higher live birth rates.