Isolation of theca cells from human antral follicle membranes using ANPEP-based magnetic cell sorting
4 affiliations
- 1Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen 2100, Denmark
- 2Drug Toxicology, Metabolism and Analysis Group, Department of Pharmacy, University of Copenhagen, Copenhagen 2100, Denmark
- 3The Fertility Clinic, Department of Gynaecology, Fertility and Obstetrics, University Hospital of Copenhagen, Rigshospitalet, Copenhagen 2100, Denmark
- 4Department of Clinical Medicine, University of Copenhagen, Copenhagen 2200, Denmark
Abstract
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.
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s10815-026-03983-2.
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BibTeX
@article{jensen2026isolation,
title = {{Isolation of theca cells from human antral follicle membranes using ANPEP-based magnetic cell sorting}},
author = {Lea Bejstrup Jensen and Malene Louise Johannsen and Jane Alrø Bøtkjær and Anna Ronnefeld Veng and Kirsten Tryde Macklon and Anette Tønnes Pedersen and Bjarne Styrishave and Stine Gry Kristensen},
journal = {Journal of Assisted Reproduction and Genetics},
year = {2026},
month = jul,
doi = {10.1007/s10815-026-03983-2},
url = {https://doi.org/10.1007/s10815-026-03983-2},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Isolation of theca cells from human antral follicle membranes using ANPEP-based magnetic cell sorting
AU - Lea Bejstrup Jensen
AU - Malene Louise Johannsen
AU - Jane Alrø Bøtkjær
AU - Anna Ronnefeld Veng
AU - Kirsten Tryde Macklon
AU - Anette Tønnes Pedersen
AU - Bjarne Styrishave
AU - Stine Gry Kristensen
PY - 2026
DA - 2026/07/23
JO - Journal of Assisted Reproduction and Genetics
DO - 10.1007/s10815-026-03983-2
UR - https://doi.org/10.1007/s10815-026-03983-2
AB - 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. Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s10815-026-03983-2.
SN - 1058-0468
SN - 1573-7330
ER -