OBJECTIVE

To determine the genetic and epigenetic stability of human spermatogonial stem cells (SSCs) during long-term culture.

DESIGN

Experimental basic science study.

SETTING

Reproductive biology laboratory.

PATIENT(S): Cryopreserved human testicular tissue from two prostate cancer patients with normal spermatogenesis.

INTERVENTION(S): None.

MAIN OUTCOME MEASURE(S): Testicular cells before and 50 days after culturing were subjected to ITGA6 magnetic-activated cell sorting to enrich for SSCs. Individual spermatogonia were analyzed for aneuploidies with the use of single-cell 24-chromosome screening. Furthermore, the DNA methylation statuses of the paternally imprinted genes H19, H19-DMR (differentially methylated region), and MEG3 and the maternally imprinted genes KCNQ1OT1 and PEG3 were identified by means of bisulfite sequencing.

RESULTS(S): Aneuploidy screening showed euploidy with no chromosomal abnormalities in all cultured and most noncultured spermatogonia from both patients. The methylation assays demonstrated demethylation of the paternally imprinted genes H19, H19-DMR, and MEG3 of 11%-28%, 43%-68%, and 18%-26%, respectively, and increased methylation of the maternally imprinted genes PEG 3 and KCNQ1OT of 13%-50% and 30%-38%, respectively, during culture.

CONCLUSION(S): In the current culture system for human SSCs propagation, genomic stability is preserved, which is important for future clinical use. Whether the observed changes in methylation status have consequences on functionality of SSCs or health of offspring derived from transplanted SSCs requires further investigation.