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Updated: Sep 12, 2025
Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration
Published on: September 27, 2022
Extended ovarian transport for centralized tissue cryobanking: impact on primordial follicle integrity
Murat Erden1, Soner Celik1, Wengelawit Molla1
1Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut.
Objective:
To evaluate the impact of ovarian tissue transportation on human primordial follicle integrity before cortical tissue cryopreservation.
Design:
Translational research study.
Subjects:
Whole ovaries (n = 13) or ovarian cortex (n = 3) from 16 individuals (10 deceased organ donors and 6 undergoing clinical ovarian tissue cryopreservation) were transported to our center in appropriate media for cryopreservation. In the clinical group indications were as follows: planned (elective, n = 3), high-grade serous ovarian cancer (n = 1), endometriosis (n = 1), and Hodgkin lymphoma (n = 1).
Exposure:
Transport duration was calculated from cross-clamp time for organ donors, and from the time of oophorectomy or ovarian tissue excision for patients, until ovarian tissue cryopreservation.
Main Outcome Measures:
Antiactive caspase-3, γH2AX, and hypoxia-inducible factor 1-alpha immunostaining were performed to assess follicular and stromal cell apoptosis, oocyte deoxyribonucleic acid (DNA) damage, and tissue hypoxia. Follicle activation was assessed by primordial follicle growth initiation index, defined as the primary/primordial follicle ratio.
Results:
Primordial follicle antiactive caspase-3 (apoptotic death) and γH2AX (DNA damage) staining intensity significantly increased with transport duration (coefficient: 4.22, 95% CI: 0.26; 8.17 and coefficient: 0.73, 95% CI: 0.26; 1.45, respectively), although primordial follicle density and primordial follicle growth initiation index were not affected. Threshold analyses identified 20.58 and 3.15 hours as critical transport threshold times for apoptotic death and DNA damage. However, DNA damage, as semiquantified by γH2AX staining intensity, was minimal. In addition, stromal hypoxia-inducible factor 1-alpha and AC-3 expressions or stromal cell density did not correlate with transport duration, indicating that hypoxia or stromal damage was not an underlying mechanism of follicular damage.
Conclusion:
Extended ovarian tissue transportation does not affect primordial follicle integrity up until 21 hours. The significantly increased primordial follicle apoptosis after 21 hours of transport time does not seem to be due to increased tissue hypoxia, stromal damage, or increased growth activation. Although minimal, increased DNA damage may partially be responsible for transport duration-related apoptotic death of primordial follicles.
