Ficoll density gradient method for recovery of isolated human ovarian primordial follicles.
OBJECTIVE
To develop a simple and efficient technique to allow rapid recovery of a maximum number of good quality isolated follicles.


METHODS
Prospective experimental study.


METHODS
Academic research unit of the department of gynecology in a university hospital.


METHODS
Biopsies were obtained from five women (between 26 and 31 years of age).


METHODS
Biopsies were cut with a tissue sectioner. Enzymatic digestion was performed in a collagenase solution for 90 min at 37 degrees C. The follicles were recovered using a discontinuous Ficoll density gradient method.


METHODS
The number of follicles present in the interface layers of Ficoll gradient was quantified. Follicular viability of these recovered follicles was assessed with live-dead stains, using calcein-AM and ethidium homodimer-I.


RESULTS
Out of a total of 6,811 recovered follicles, we found 63% (n = 4,201) at the medium-1.06 Ficoll interface and 36.9% (n = 2,590) at the 1.06-1.09 Ficoll interface, which represents 99.9% of total recovered follicles. Analysis by vital fluorescent staining showed that 95.8% of the follicles treated with Ficoll were totally viable.


CONCLUSIONS
The Ficoll density gradient method allows us to maximize the recovery of isolated human ovarian follicles and minimize the manipulation time while maintaining high follicular viability.
Martinez-Madrid
Belen
B
Department of Gynecology, Université Catholique de Louvain, Brussels, Belgium.
Dolmans
Marie-Madeleine
MM
Langendonckt
Anne Van
AV
Defrère
Sylvie
S
Van Eyck
Anne-Sophie
AS
Donnez
Jacques
J
D000328:Adult; D001706:Biopsy; D002499:Centrifugation, Density Gradient; D017364:Collagenases / Q000494:pharmacology; D005260:Female; D005362:Ficoll; D006801:Humans; D008856:Microscopy, Fluorescence; D006080:Ovarian Follicle / Q000033:anatomy & histology / Q000502:physiology; D010053:Ovary / Q000187:drug effects / Q000473:pathology; D011446:Prospective Studies; D013048:Specimen Handling / Q000379:methods*; D014022:Tissue Survival
D005362:Ficoll; D017364:Collagenases