title: In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility.
abstract: BACKGROUND
  Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential.
  
  
  METHODS
  In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 μg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section.
  
  
  RESULTS
  In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3.1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age.
  
  
  CONCLUSIONS
  NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.
authors.affiliation: Division of Reproduction and Early Development, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, The LIGHT Laboratories, Clarendon Way, Leeds LS2 9JT, UK.
authors.forename: E L
authors.initials: EL
authors.lastname: Chambers
authors.forename: R G
authors.initials: RG
authors.lastname: Gosden
authors.forename: C
authors.initials: C
authors.lastname: Yap
authors.forename: H M
authors.initials: HM
authors.lastname: Picton
mesh_terms: D000328:Adult; D000818:Animals; D002452:Cell Count / Q000379:methods; D002470:Cell Survival; D015925:Cryopreservation; D005260:Female; D005452:Fluoresceins / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D007247:Infertility, Female / Q000517:prevention & control*; D009499:Neutral Red / Q000032:analysis; D006080:Ovarian Follicle / Q000166:cytology* / Q000254:growth & development / Q000637:transplantation; D012756:Sheep; D013194:Staining and Labeling; D013388:Succinimides / Q000032:analysis
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides; D009499:Neutral Red