In Vitro Fertilization induces reproductive changes in male mouse offspring and has multigenerational effects [RNA-Seq]

In vitro fertilization (IVF) is a non-coital method of conception used to treat infertility. Although safe, IVF has been associated with adverse outcomes in the fetus, placenta, and adult life, but studies focusing on the male reproductive system are limited. Here, we used a mouse model to assess the morphological and molecular effects of IVF on male offspring. We evaluated three developmental stages: 18.5-day fetuses and 12- and 39-week adults. Regardless of age, we observed changes in the testicular-to-body weight ratio, serum testosterone levels, testicular morphology, transcriptome, and DNA methylation. Also, sperm showed changes in morphology and DNA methylation. To assess multigenerational phenotypes, we mated IVF and naturally conceived males with wild-type females. Offspring from IVF males exhibited a decreased fetal weight-to-placental weight ratio regardless of sex. At 12-weeks-of-age, offspring showed higher body weights, differences in glucose, triglycerides, insulin, total cholesterol, HDL and LDL/VLDL levels. Both sexes showed changes in gene expression in liver, testes and ovaries, and decreased global DNA methylation. Collectively, our findings demonstrate that male IVF offspring exhibit abnormal testicular and sperm morphology and molecular alterations and transmit defects multigenerationally. These experiments contribute to our understanding of the long-term impacts of IVF on adult offspring health. Gene expression analysis using Total RNA extracted from F1: E18.5 testis (n=10, Natural=4 and IVF=6), 12 weeks testis (n=10, Natural=5 and IVF=5) and 39 weeks testis (n=10, Natural=4 and IVF=6). F2: 12 weeks testis (n=12, Natural=5 and IVF=7), 12 weeks male livers (n=10, Natural=5 and IVF=5), 12 weeks ovaries (n=9, Natural=4 and IVF=5), 12 weeks female livers (n=10, Natural=4 and IVF=4), using either Illumina NovaSeq6000 or NextSeq1000.