Significance: Sex ratio is an important indicator of reproductive health, and its skewing reflects disturbed embryonic development. This study focused on sex skewing, which has recently identified in human in vitro fertilization (IVF) babies. We reported herein that the skewed sex ratio in mouse IVF offspring was due to the impaired imprinted X chromosome inactivation via suppressing the ring finger protein 12 ( Rnf12 )/X-inactive specific transcript ( Xist ) pathway; the sex skewing can be corrected by overexpressing Rnf12 or by supplementation of retinoic acid in embryo culture medium. Hence, our study not only identified a major epigenetic error responsible for sex skewing in IVF offspring, but also implicated a potential strategy for preventing sex skewing and IVF-associated complications by targeting erroneous epigenetic modifications induced by IVF.

Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, in vivo and in vitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

Decidualization is a progesterone-dependent cellular differentiation process that is essential for establishing pregnancy. Robust activation of glycolysis and lactate synthesis during decidualization is remarkable, but their developmental functions remain largely unknown. Herein, we identify that endometrial lactate production plays a critical role in establishing local histone lactylation, a newly identified histone modification, and is important for ensuring normal decidualization. Enhanced endometrial glycolysis is the hallmark metabolic change and is tightly coupled with H4K12la during decidualization. Inhibition of histone lactylation impaired decidualization, in either physiological conception or in vivo and in vitro induced decidualization models. Mechanistic study based on CUT&Tag and ATAC-seq revealed that a transcriptional factor hypoxia-inducible factor 1 α (Hif1α) is the critical regulatory target of H4K12la, and in turn forms a H4K12la-Hif1α-glycolysis feedback loop to drive decidualization. Moreover, we demonstrate that the loop is directly activated by progesterone during decidualization. Together, our study not only advance the current knowledge of the role of lactate in regulating uterine function, but also establish a novel functional link among the major endocrine factor, endometrial metabolic change and epigenetic modification during endometrial remodeling. These findings present valuable clues to develop clinical intervention strategies to improve pregnancy outcomes following both natural conception and assisted reproduction.