A simple method to quantify follicle survival in cryopreserved human ovarian tissue

Abstract STUDY QUESTION Can follicle survival in frozen-thawed human ovarian tissue be quantified in situ using the dye Neutral Red (NR) to stain viable follicles specifically? SUMMARY ANSWER A follicle survival rate within ovarian tissue can be calculated using NR followed by histological evaluatio...

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Published in:Human reproduction (Oxford) 2018-12, Vol.33 (12), p.2276-2284
Main Authors: Kristensen, S G, Liu, Q, Mamsen, L S, Greve, T, Pors, S E, Bjørn, A B, Ernst, E, Macklon, K T, Andersen, C Y
Format: Article
Language:English
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Summary:Abstract STUDY QUESTION Can follicle survival in frozen-thawed human ovarian tissue be quantified in situ using the dye Neutral Red (NR) to stain viable follicles specifically? SUMMARY ANSWER A follicle survival rate within ovarian tissue can be calculated using NR followed by histological evaluation and evidence for a consistently high follicle survival in a series of ovarian tissue from 25 Danish girls and women undergoing ovarian tissue cryopreservation (OTC) was obtained. WHAT IS KNOWN ALREADY Securing follicle survival in cryopreserved ovarian tissue is crucial for proper quality control when centers wish to implement OTC. The only established technique for validation of follicle survival is xenografting of thawed ovarian tissue to immunodeficient mice. However, this functional test is expensive, time consuming, requires animal facilities and only provides a qualitative-not quantitative-measure for follicle survival. STUDY DESIGN SIZE, DURATION Quantification of follicle survival in human ovarian tissue donated from 30 girls and women having tissue cryopreserved for fertility preservation from 2000 to 2015 at the Laboratory of Reproductive Biology in Copenhagen, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS Cryopreserved ovarian cortex was donated from 25 girls and young women aged 10-36 years (mean age: 25 years) and the average storage time in liquid nitrogen was 9.1 ± 5.6 years, ranging from 1.6 to 17.9 years. In 12 of the cases, the ovarian tissue was collected from the local hospital and in the other 13 cases the ovarian tissue was transported on ice up to 6 h prior to freezing. Donated fresh ovarian surplus tissue was obtained from five women aged 23-34 years (mean age: 27 years). Ovarian tissues were chopped into small fragments and incubated in culture medium containing 50 mg/ml NR for 3-4 h. Fragments of ovarian tissue containing clearly NR-stained follicles were selected for counting, encapsulated in 4% agar and were processed for histology to calculate a follicular survival rate. MAIN RESULTS AND THE ROLE OF CHANCE The mean follicle survival rate in the 25 patients after freezing and thawing was 84% ± 11 (mean ±SD), ranging from 50% to 98%. The high follicle survival rate in this clinical series of patients reflects a constant high-quality service performed in our center and confirms the robustness of the slow freezing protocol. No significant association between follicle survival rates and storage time was found using linear regression
ISSN:0268-1161
1460-2350
DOI:10.1093/humrep/dey318