Paternal developmental exposure to endometriosis causes negative transgenerational impacts on male fertility

Endometriosis is a gynecological disease causing pain and infertility in about 15 percent of women worldwide. Little is known regarding male fertility following prenatal exposure to endometriosis. Our hypothesis is that developmental exposure of male embryos and fetuses to endometriosis reduces spermatozoa quality and fertility. We used a well-established surgical rodent model of endometriosis (Endo rat) with a surgical control (Sham surgery). Four weeks post-surgery, founder F0 Endo and Sham rats were bred with proven fertile males to produce three subsequent generations. The phenotype of the male progeny (F1, F2, and F3 generations) was evaluated from gestational days (GD) seven to 70. At GD 70, males were randomly divided into two groups. One-half of the males from each generation were euthanized at postnatal day (PND) 70 to collect cauda epididymal spermatozoa and testes around the time of sexual maturity. The remaining half of each generation were bred with WT female rats to produce pups. Following gestation, whelping, nursing and uterine involution, males were euthanized at PND 200 to collect spermatozoa testis. Spermatozoa were isolated using a swim out method and fixed in 10 percent formaldehyde. Epifluorescence microscopy and image-based flow cytometry (IBFC) with biomarkers of histone H3 methylation (H3K9me3; retention is associated with reduced sperm quality in rodents) and sperm surface glycosylation (lectin Lens culinaris agglutinin/LCA has high affinity for defective sperm surface) were used to assess spermatozoa quality. Data analysis indicated elevated H3K9me3 and LCA labeling between Endo and Sham rat spermatozoa as well as between generations. For the first time, embryonic and fetal exposure to maternal endometriosis in founder rats influences sperm quality in male offspring of the paternal lineage. The multiplex IBFC analysis may provide future clinical applications to enhance human sperm analyses.