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Ficoll density gradient method for recovery of isolated human ovarian primordial follicles
Belen Martinez-Madrid1, Marie-Madeleine Dolmans, Anne Van Langendonckt
1Department of Gynecology, Université Catholique de Louvain, Brussels, Belgium.
Objective:
To develop a simple and efficient technique to allow rapid recovery of a maximum number of good quality isolated follicles.
Design:
Prospective experimental study.
Setting:
Academic research unit of the department of gynecology in a university hospital.
Patient(S):
Biopsies were obtained from five women (between 26 and 31 years of age).
Intervention(S):
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.
Main Outcome Measure(S):
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.
Result(S):
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.
Conclusion(S):
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.
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