Altered DNA methylation in estrogen-responsive repetitive sequences of spermatozoa of infertile men with shortened anogenital distance

Clin Epigenetics. 2022 Dec 26;14(1):185. doi: 10.1186/s13148-022-01409-1.


Background: It has been suggested that antenatal exposure to environmental endocrine disruptors is responsible for adverse trends in male reproductive health, including male infertility, impaired semen quality, cryptorchidism and testicular cancer, a condition known as testicular dysgenesis syndrome. Anogenital distance (AGD) is an anthropomorphic measure of antenatal exposure to endocrine disruptors, with higher exposure levels leading to shortened AGD. We hypothesized that exposure to endocrine disruptors could lead to changes in DNA methylation during early embryonic development, which could then persist in the sperm of infertile men with shortened AGD.

Results: Using fluorescence activated cell sorting based on staining with either YO-PRO-1 (YOPRO) or chromomycin-3 (CMA3), we isolated four sperm fractions from eleven infertile men with short AGD and ten healthy semen donors. We examined DNA methylation in these sorted spermatozoa using reduced representation bisulfite sequencing. We found that fractions of spermatozoa from infertile men stained with CMA3 or YOPRO were more likely to contain transposable elements harboring an estrogen receptor response element (ERE). Abnormal sperm (as judged by high CMA3 or YOPRO staining) from infertile men shows substantial hypomethylation in estrogenic Alu sequences. Conversely, normal sperm fractions (as judged by low CMA3 or YO-PRO-1 staining) of either healthy donors or infertile patients were more likely to contain hypermethylated Alu sequences with ERE.

Conclusions: Shortened AGD, as related to previous exposure to endocrine disruptors, and male infertility are accompanied by increased presence of hormonal response elements in the differentially methylated regulatory sequences of the genome of sperm fractions characterized by chromatin decondensation and apoptosis.

Keywords: Anogenital distance; Apoptosis; Epigenome; Estrogen response element; Histone; Infertility; Methylation; Protamine; Spermatozoa; Testicular dysgenesis syndrome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Methylation
  • Endocrine Disruptors* / metabolism
  • Estrogens / metabolism
  • Female
  • Humans
  • Infertility, Male* / chemically induced
  • Infertility, Male* / genetics
  • Male
  • Pregnancy
  • Repetitive Sequences, Nucleic Acid
  • Semen
  • Semen Analysis
  • Spermatozoa / metabolism
  • Testicular Neoplasms* / genetics


  • Endocrine Disruptors
  • Estrogens