Isolation of theca cells from human antral follicle membranes using ANPEP-based magnetic cell sorting.

RESEARCH QUESTION: Can ANPEP-based magnetic activated cell sorting (MACS) be used to isolate and functionally enrich human steroidogenic theca interna cells from small antral follicle membranes?

METHODS

This experimental study used ovarian tissue from eight women (mean age 33.7 years) undergoing fertility preservation. Membranes from small antral follicles (2-9 mm) were enzymatically digested and cultured for 48 h. A mixed cell sample was obtained before MACS separation into ANPEP-positive and ANPEP-negative cell fractions, followed by an additional 48-h culture. Cell purity and viability were assessed by flow cytometry (FACS) using ANPEP and FSHR antibodies. Gene expression (ANPEP, CYP17A1, LHCGR, FSHR, CYP19A1) was analyzed by RT-qPCR. Steroid secretion (pregnenolone, progesterone, 17-OH-pregnenolone, 17-OH-progesterone, DHEA, androstenedione) was quantified using LC-MS/MS.

RESULTS

Before cell sorting, 29.6-82.9% of viable cells expressed ANPEP. After MACS, ~ 68% of cells in the ANPEP-positive fraction expressed ANPEP, compared with ~ 3% in the negative fraction (P < 0.05). CYP17A1 expression was higher in pre-MACS and ANPEP-positive populations than in the negative fraction. ANPEP-positive cells secreted androgen precursors (17-OH-pregnenolone, 17-OH-progesterone, DHEA, androstenedione), whereas the negative fraction produced relatively more progesterone, consistent with theca cell enrichment.

CONCLUSIONS

ANPEP-based MACS enables enrichment of viable, steroidogenic human theca interna cells. The ANPEP-positive fraction exhibits molecular and functional characteristics consistent with theca lineage and provides a useful model for studying ovarian steroidogenesis.