Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue.
OBJECTIVE
To develop an efficient isolation technique for human primordial follicles.


METHODS
Prospective, experimental study of ovarian biopsies collected from healthy women undergoing elective cesarean section. Ovarian blocks either were fixed for histology and follicle counting or partially disaggregated with type 1A collagenase before or after cryopreservation. After partial disaggregation, follicles were isolated by microdissection.


METHODS
Leeds General Infirmary.


METHODS
Follicle viability was assessed with live-dead stains using 5-(and 6-) carboxyfluorescein diacetate, succinimidyl ester and propidium iodide, respectively, and using electron microscopy. The numbers recovered were expressed as a percentage of the numbers of primordial follicles in comparable blocks of tissue and the viability of the whole follicle and oocyte were scored separately.


RESULTS
On average, 18.0 +/- 3.8 and 15.9 +/- 2.2 (mean +/- SEM) follicles per block were recovered from fresh and cryopreserved ovarian tissue, respectively, corresponding to recovery rates of 57.9% +/- 8.8% and 56.2% +/- 16.7%. In the fresh group, the percent viability of whole follicles and oocytes were 71.6% +/- 2.4% and 91.3% +/- 2% compared with 71.5% +/- 4.7% and 95% +/- 4.3% in the frozen-thawed group. Electron microscopy confirmed that the majority of the cells lacked ultrastructural signs of damage after isolation and cryopreservation.


CONCLUSIONS
Primordial follicles can be isolated from fresh and cryopreserved human ovarian tissue with similar high efficiency and viability rates.
Oktay
K
K
University of Leeds School of Clinical Medicine, West Yorkshire, United Kingdom. koktay@netmail.hscbklyn.edu
Nugent
D
D
Newton
H
H
Salha
O
O
Chatterjee
P
P
Gosden
R G
RG
D000328:Adult; D002470:Cell Survival; D002585:Cesarean Section; D017364:Collagenases; D015925:Cryopreservation; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D006080:Ovarian Follicle / Q000166:cytology* / Q000648:ultrastructure; D010053:Ovary; D011247:Pregnancy; D011446:Prospective Studies
D005456:Fluorescent Dyes; D017364:Collagenases