Isolation of pre-antral follicles from human ovarian medulla tissue

BACKGROUND Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the medulla is discarded. The present study describes the development of a gentle method for isolating pre-an...

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Bibliographic Details
Published in:Human reproduction (Oxford) 2011-01, Vol.26 (1), p.157-166
Main Authors: Kristensen, Stine Gry, Rasmussen, Annette, Byskov, Anne Grete, Andersen, Claus Yding
Format: Article
Language:English
Subjects:
Adolescent
Adult
Age Factors
Biological and medical sciences
Child
Child, Preschool
Cryopreservation
Female
Fertility
Gynecology. Andrology. Obstetrics
Humans
Medical sciences
Neutral Red
Ovarian Follicle - anatomy & histology
Ovary - anatomy & histology
Tissue Preservation - methods
Young Adult
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Summary:BACKGROUND Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the medulla is discarded. The present study describes the development of a gentle method for isolating pre-antral follicles from human ovarian medulla and evaluating its follicular content. METHODS Medulla was collected from 40 girls/women aged 3–35 years undergoing cryopreservation of the ovarian cortex. Follicle density was assessed for all patients and pre-antral follicles were isolated from 22 patients. On the basis of the neutral red (NR) staining of follicles and enzymatic digestion with a mixture of Collagenase IV and Liberase Thermolysin Medium, viable pre-antral follicles were isolated. RESULTS NR accumulated in follicles resulting in a distinct red staining within the medulla. Follicle density of the medulla varied from 0 to 9824 follicles/gram of medulla and was significantly higher (P< 0.001) in the 3–9-year age group when compared with older groups (10–35 years). Enzymatic digestion combined with follicle identification by NR yielded a high output of isolated and viable pre-antral follicles from medulla, of which, 3607 follicles were collected and classified. The percentage of primordial and growing follicles decreased and increased, respectively, with age (P< 0.0001 and
ISSN:0268-1161
1460-2350
DOI:10.1093/humrep/deq318