| Background: Controlled ovarian stimulation(COS)is an important step in assisted reproduction clinics for obtaining multiple mature oocytes,a process that results in aberrant DNA methylation of oocytes,particularly loss of imprinted gene imprints.Because COS promotes the simultaneous development of multiple follicles,maternal serum estradiol(β-estradiol,E2)levels are elevated to more than ten times physiological levels.Studies have shown that the increased risk of low birth weight in offspring due to assisted reproductive technology(ART)is closely related to elevated serum E2 levels.Our team previously found a negative correlation between birth weight and E2 levels in IVF infants in frozen transfer cycles.Therefore,we believe that high estrogen levels in COS affecting oocytes may be an important factor contributing to low birth weight in IVF.Based on the literature reports and the previous research results of our group,we speculate that hyperestrogenism may lead to abnormal placental development and function by affecting the DNA methylation of maternal imprinted genes related to placental development,which ultimately leads to low birth weight in IVF.As the mechanism of COS hyperestrogenism causing low birth weight in IVF is still unclear,it is an urgent scientific issue to investigate the mechanism and provide new ideas for clinical prevention and treatment of low birth weight in ART offspring.Objective: Through a combination of in vivo animal models and in vitro cellular experiments,this study aims to clarify the effects of COS hyperestrogenism on the DNA methylation level of oocytes,to reveal the mechanism of its occurrence,and to explore the role of the altered DNA methylation level of the key maternal-origin imprinted genes in it,so as to provide a new idea for the intervention and treatment of the COS that leads to the adverse pregnancy outcome in IVF.Methods: Part 1In vivo and in vitro experiments were used to analyse the effects of COS and high estrogen environment on DNA methylation and hydroxymethylation levels in oocytes.Firstly,COS mouse model was constructed and E2 concentration in serum was detected by radioimmunoassay.Secondly,oocytes at GV and MII stages and prokaryotic embryos were obtained from female mice in the COS and natural estrus groups,and the effects of COS on the methylation and hydroxymethylation levels of DNA in oocytes at GV and MII stages and in the prokaryoticDNA of fertilised eggs were analysed by immunofluorescence.Finally,oocytes were cultured in vitro and 1 nM and 100 nM estrogen were added to simulate physiological levels and the high estrogen environment of COS,respectively,and the effects of the high estrogen environment on the DNA methylation level and hydroxymethylation level of oocytes were analysed by immunofluorescence staining.Part II In order to investigate whether hyperestrogenic-induced DNA hypomethylation in oocytes persisted during embryonic and placental development,we firstly subjected MII stage oocytes to lone female activation,and observed the immunofluorescence intensities of 5mC and 5hmC in lone female embryos at different stages by immunofluorescence.Subsequently,solitary embryos that had developed to the blastocyst stage were transferred into naturally oestrous-surrogate female rats,and placental tissues from E10.5 solitary females were collected to detect overall 5mC and 5hmC levels by Enzyme-Linked Immunosorbent Assay(ELISA).Finally,in order to investigate which methylation sites in oocytes are altered by the hyperestrogenic environment of COS,we sequenced the placental tissues of E10.5 orphan females by Agilent DNA capture methylation sequencing,and selected genes enriched for differentially methylated CpGs(DMCs)based on the sequencing results and sequenced them after bisulfite treatment(bisulfite sequencing polymerase chain reaction(BSP).Through this part of the experiment,we hope to reveal the long time-course effect of DNA hypomethylation in oocytes caused by high estrogen and identify the key differentially methylated genes.Part III In order to reveal the possible molecular mechanisms of aberrant DNA demethylation in oocytes caused by high estrogen in COS,we first collected oocytes at stage MII from natural estrus and COS groups,orphan embryos and placental tissues from E10.5 orphan females,and detected ERα and expression levels of related DNA demethylases.In order to further verify that the process of DNA demethylation in oocytes is affected by the estrogen signalling pathway,we cultured stage MII oocytes by simulating the high estrogen environment of COS in vitro,and added the estrogen receptor alpha(ERα,ESR1)inhibitor,tamoxifen(TAM),which was detected by qRT-PCR after 24 h.The expression levels of ERα and related DNA demethylation enzymes were measured by real-time fluorescence quantitative PCR(qRT-PCR).The expression levels of ERα and related DNA demethylases were detected by qRT-PCR after 24 h.Finally,in order to elucidate the molecular mechanism of DNA demethylation in oocytes caused by COS hyperestrogenism,we chose parthenogenetic embryonic stem cells(pESCs)instead of oocytes for the mechanistic study.pESCs were isolated and characterised in vitro,and were treated for 24 h with 1 nM E2 and 100 nM E2,respectively.The pESCs were isolated and identified in vitro and treated with 1 nM E2 and 100 nM E2 in culture medium for 24 h.The levels of 5mC and 5hmC were detected by immunofluorescence and ELISA,and ERα,TET2 and Mest genes were detected by Chromatin immunoprecipitation(Ch IP),Co-immunoprecipitation(Co-IP)and BSP.The binding relationship between ERα,TET2 and Mest gene was detected by chromatin immunoprecipitation(Ch IP),Co-immunoprecipitation(Co-IP)and BSP.Results: 1.COS hyperestrogenism leads to aberrant DNA demethylation in oocytesAfter successfully constructing the COS mouse model,we used natural estrus mice as the Control group.Compared with the Control group,the 5mC levels of both GV and MII stage oocytes in the COS group were significantly lower(P < 0.001),and the 5hmC levels were significantly higher(P < 0.001).Compared with the Control group,the 5mC levels of female prokaryotes in prokaryotic stage embryos of the COS group were both significantly lower(P < 0.01)and higher(P < 0.01),and there was no significant difference between the 5mC levels of male prokaryotes and 5hmC levels of prokaryotic stage embryos of the two groups(P > 0.05).The 5mC and 5hmC levels were detected after culturing GV and MII stage oocytes in an in vitro simulated COS hyperestrogenic environment(100 nM E2).Compared with the physiological level(1 nM E2)group,the 5mC levels of GV and MII stage oocytes in the hyperestrogenic group were significantly(P < 0.001)lower,and the 5hmC levels were significantly(P < 0.001)higher.The results of this part of the experiment suggest that COS hyperestrogen can lead to DNA demethylation process in oocytes.2.COS hyperestrogenic state leads to abnormal DNA demethylation process and loss of methylation of maternal imprinted gene Mest in placental tissues of E10.5 orphansThe Control and COS groups were cultured in vitro to mature to the blastocyst stage,and it was found that compared with the Control group,the 5mC levels of the orphan embryos in the COS group were significantly lower(P < 0.05)and the 5hmC levels were significantly higher(P < 0.05)at all times.Using ELISA to measure the 5mC and 5hmC levels in placental tissue of E10.5 females,it was found that the 5mC level of placental tissue DNA of E10.5 females in the COS group was significantly lower(P < 0.05)and the 5hmC level was significantly higher(P < 0.05)compared with that of the Control group.The results of Agilent DNA capture methylation sequencing of placental tissues from the two groups of orphan females were analysed,and DNA methylated CpG sites with differential methylation levels greater than 10% and statistically significant differences(P < 0.05)were selected for KEGG pathway analysis,and it was found that these pathways were mainly involved in the endocrine system and the immune system.In addition,among the enriched pathways with significant differences(Top10),the estrogen signalling pathway had the highest enrichment score.Therefore,we performed a Wayne analysis of the downstream target genes of ERα,genes related to placental developmental functions,and maternally imprinted genes,and screened out the mesoderm-specific transcript homologue(Mest)of maternally imprinted genes for subsequent study.By BSP experiment,it was found that the COS process could lead to the loss of DNA methylation in the promoter region of Mest in the placental tissues of E10.5 orphan females.By qRT-PCR and Western Blot experiments,it was found that the mRNA and protein expression levels of MEST were elevated in the placenta tissues of E10.5 orphan females in the COS group(P < 0.01).The results of this part of the experiment suggest that oocyte DNA hypomethylation caused by COS hyperestrogenism persists during embryonic and placental development,and may lead to abnormal placental function and fetal development through DNA demethylation of the maternally imprinted gene Mest.3.The high estrogenic state of COS leads to significant DNA demethylation in the Mest promoter region of pESCs through ERα-TET2Compared with the Control group,the mRNA expression levels of Dnmt1,Dnmt3 a and Dnmt3 b in stage MII oocytes of the COS group were not significantly different(P > 0.05),nor were the mRNA expression levels of Tet1 and Tet3(P > 0.05),while the mRNA expression levels of Tet2 and ERα were significantly higher(P < 0.05).The mRNA and protein expression levels of TET2 were significantly higher(P < 0.05)in the tissues of lone female embryos and placentas of lone females on day E10.5 in the COS group compared with the Control group.Culture of stage MII oocytes by in vitro addition of E2 revealed that 5mC levels were significantly lower(P < 0.01)and 5hmC levels were significantly higher(P < 0.01)in the 100 nM group compared with the 1 nM group,while 5mC levels were higher(P < 0.01)and 5hmC levels were lower(P < 0.01)in the 100 nM + TAM group than in the 100 nM group,which indicated that TAM was able to partially inhibit the effect of high estrogen on DNA demethylation in stage MII oocytes.After pESCs were cultured and identified in vitro,1 nM and 100 nM E2 estrogen were added,respectively,and it was verified by Ch IP and Co-IP experiments that TET2 and Mest genes were direct transcriptional targets of ERα,and that the TET2 protein was a coactivator of ERα.The results of the BSP experiments showed that silencing of Tet2 and Esr1 significantly elevated the level of DNA methylation at the CpG site of the Mest promoter region.methylation level was significantly increased.The results of this part of the experiment suggest that the high estrogenic state of COS can lead to the DNA demethylation process in the Mest promoter region in pESCs.In this process,the activation of ERα by estrogen promotes the expression of TET2 on the one hand,and assists the binding of TET2 to the Mest promoter region to exert DNA demethylation on the other hand.Conclusion: The experimental results of the present study demonstrated for the first time that the estrogen signalling pathway plays a role in the aberrant DNA demethylation of genomic imprinted genes in oocytes caused by COS,and revealed for the first time the molecular mechanism by which ERα-TET2 regulates the process of DNA demethylation in the promoter region of Mest by substituting oocytes with pESCs,and preliminarily elucidated the possible mechanism of action of the high estrogenic environment of COS by affecting oocytes to cause low birth weight in the offspring.In this study,we found that the activation of ERα by high estrogen in COS promotes the expression of TET2 on the one hand,and on the other hand,assists TET2 to bind to the Mest promoter region to play the role of demethylation,leading to the abnormal development and function of the placenta.The findings of this study provide new insights into the disruption of genomic imprinting during oogenesis due to COS,and offer new ideas for the clinical application of COS and the prevention and treatment of adverse pregnancy outcomes in IVF. |
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