Trophectoderm biopsy of blastocysts following IVFãnd embryo culture increases epigenetic dysregulation inã mouse model

STUDY QUESTION: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinicãffect normal placentalãnd embryo developmentãnd offspring metabolic outcomes inã mouse model? SUMMARY ANSWER: TEBx impacts placentalãnd embryonic health during early development, with someãlterations resolvingãnd others worsening later in developmentãnd triggering metabolic changes inãdult offspring. WHAT IS KNOWN ALREADY: Previous studies have notãssessed the epigeneticãnd morphological impacts of TEBx either in human populations or inãnimal models. STUDY DESIGN, SIZE, DURATION: We employedã mouse model to identify the effects of TEBx during IVF. Three groups wereãssessed: naturally conceived (Naturals), IVF,ãnd IVF + TEBx,ãt two developmental timepoints: embryonic day (E)12.5 (n=40/ Naturals, n=36/IVF,ãnd n=36/IVF + TEBx)ãnd E18.5 (n=42/Naturals, n=30/IVF,ãnd n=35/IVF + TEBx). Additionally, to mimic clinical practice, weãssessedã fourth group: IVF + TEBx + Vitrification (Vit)ãt E12.5 (n=29) that combines TEBxãnd vitrification. Toãssess the effect of TEBx in offspring health, we characterizedã 12-week-old cohort (n=24/Naturals, n=25/IVFãnd n=25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS: Our mouse model used CF-1 femalesãs egg donorsãnd SJL/B6 malesãs sperm donors. IVF, TEBx,ãnd vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin- eosin staining, in situ hybridization using Tpbpaãsã junctional zone markerãnd immunohistochemistry using CD34 fetal endothelial cell markers. For molecularãnalysis of placentasãnd embryos, DNA methylation wasãnalyzed using pyrosequencing, luminometric methylationãssay,ãnd chipãrray technology. Expression patterns wereãscertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-,ãnd very low-density lipoprotein, insulin,ãnd glucose were determined in the 12-week-old cohort using commerciallyãvailable kits. MAIN RESULTS AND THE ROLE OF CHANCE: We observed thatãt E12.5, IVF + TEBx hadã worse outcome in terms of changes in DNA methylationãnd differential gene expression in placentasãnd whole embryos compared with IVFãloneãnd compared with Naturals. These changes were reflected inãlterations in placental morphologyãnd blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecularãnd morphological changes,ãlthough different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placentalãnd embryonic tissues, withãlterations in embryonic tissues persisting or worsening in later developmental stages compared to IVFãlone,ãnd theãddition of vitrificationãfter TEBx results in more pronouncedãnd potentially detrimental epigenetic effects: these changesãre significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol,ãnd lower high-density lipoprotein compared to IVFãnd Naturals, with only males having higher body weight compared to IVFãnd Naturals. Our findings inã mouse modelãdditionally support the need for more studies toãssess the impact of new procedures in ART to ensure healthy pregnanciesãnd offspring outcomes. LARGE SCALE DATA: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus underãccession number GSE225318. LIMITATIONS, REASONS FOR CAUTION: This study was performed usingã mouse model that mimics many clinical IVF proceduresãnd outcomes observed in humans, where studies on early embryosãre not possible. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the importance ofãssaying new procedures used in ART toãssess their impact on placentaãnd embryo development,ãnd offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was funded byã National Centers for Translational Research in Reproductionãnd Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.)ãnd F32 HD089623 (L.A.V.),ãnd National Institutes of Health Training program in Cellãnd Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.