Genome wide abnormal DNA methylome of human blastocyst in assisted reproductive technology

Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development. However, it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development. Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology （ART） practices. Our data reveal that the global methylation levels of high-quality blastocysts are similar （0.30 ± 0.02, mean ± SD）, while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts. The proportion of blastocysts with a methylation level falling within the range of 0.30± 0.02 in different grades correlates with the live birth rate for that grade. Moreover, abnormal methylated regions are associated with the failure of embryonic development. Furthermore, we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts. Our data indicate that global abnormal methylome reprogramming often occurs in human embryos, and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.

Fargeted elimination of mutant mitochondria DNA in MELAS-iPSCs by mitoTALENs

Mitochondrial diseases are maternally inherited hetero- geneous disorders that are primarily caused by mitochondrial DNA （mtDNA） mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruc- tion of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells （MiPSCs） that harbored a high proportion of m.3243A〉G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes （MELAS）. We engineered mitochondrial-targeted transcription activator-like effector nucleases （mitoTALENs） and successfully eliminated the m.3243A〉G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Further- more, we successfully achieved reduction in the human m.3243A〉G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

Chlamydia trachomatis infection in the genital tractis associated with inflammation and hypospermia inthe infertile male of China

Chlamydia trachomatis (CT) infection is the most prevalent sexually transmitted bacterial disease worldwide. However, unlikethat in female infertility, the role of CT infection in male infertility remains controversial. The objective of this retrospective studywas to explore the impacts of CT infection in the genital tract on sperm quality, sperm acrosin activity, antisperm antibody levels,and inflammation in a large cohort of infertile males in China. A total of 7154 semen samples were collected from infertile malesubjects, 416 of whom were CT positive (CT+ group) and 6738 of whom were CT negative (CT− group), in our hospital betweenJanuary 2016 and December 2018. Routine semen parameters (semen volume, pH, sperm concentration, viability, motility,morphology, etc.), granulocyte elastase levels, antisperm antibody levels, and sperm acrosin activity were compared between theCT+ and CT− groups. Our results showed that CT infection was significantly correlated with an abnormally low semen volume, aswell as an increased white blood cell count and granulocyte elastase level (all P < 0.05) in the semen of infertile males;otherroutine semen parameters were not negatively impacted. The antisperm antibody level and sperm acrosin activity were not affectedby CT infection. These findings suggested that CT infection might contribute to inflammation and hypospermia but does not impairsperm viability, motility morphology, and acrosin activity or generate antisperm antibodies in the infertile males of China.