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.