Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.
Ovarian cortical tissue was obtained during gynaecological operations by biopsy or after oophorectomy from 20 women aged 25-42 years. It was placed in organ culture, either fresh or following thawing after cryopreservation, for 1-4 months. The tissue was cut in slices 0.1-0.3 mm in diameter and transferred to 12 mm inserts in 24-well culture plates. These slices were cultured for 4-21 days in either alpha minimum essential medium (alpha-MEM) or Earle's balanced salt solution with added pyruvate. Both media were supplemented with 10% human serum, insulin, gonadotrophins and antibiotics. Half of the inserts were precoated with extracellular matrix (Matrigel). Histological samples revealed that there were viable, non-atretic, primordial, primary and secondary follicles in all the cultures. Mitoses were seen in the granulosa cells of the secondary follicles. Although the proportion of atretic follicles increased during culture, non-atretic follicles were still present after 21 days. After 4-11 days the proportion of viable follicles was significantly higher when cultured in Earle's solution supplemented with pyruvate, than when cultured in MEM (77 versus 38%, P < 0.001). In cultures with extracellular matrix the proportion of viable follicles was significantly higher after 10-15 days than it was without matrix (85 versus 19%, P < 0.001). Culture after thawing frozen ovarian tissue did not affect the density or the proportion of the viable follicles. Two-thirds of follicles in cryopreserved tissue were viable after 10-15 days in culture. The results indicate that it is possible to culture human primary and primordial follicles in vitro, and follicles in cryopreserved tissue are viable.
Hovatta
O
O
Infertility Clinic, The Family Federation of Finland, Helsinki.
Silye
R
R
Abir
R
R
Krausz
T
T
Winston
R M
RM
D000328:Adult; D001706:Biopsy; D015925:Cryopreservation; D046508:Culture Techniques; D005109:Extracellular Matrix / Q000502:physiology*; D005260:Female; D005496:Follicular Atresia / Q000502:physiology; D005615:Freezing; D006801:Humans; D006080:Ovarian Follicle / Q000166:cytology / Q000502:physiology*; D010052:Ovariectomy; D010053:Ovary / Q000166:cytology* / Q000473:pathology; D013997:Time Factors