title: Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue.
abstract: At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.
authors.affiliation: Department of Obstetrics and Gynecology, Cologne University, Cologne, Germany. v.isachenko@yahoo.com
authors.forename: Vladimir
authors.initials: V
authors.lastname: Isachenko
authors.forename: Peter
authors.initials: P
authors.lastname: Mallmann
authors.forename: Anna M
authors.initials: AM
authors.lastname: Petrunkina
authors.forename: Gohar
authors.initials: G
authors.lastname: Rahimi
authors.forename: Frank
authors.initials: F
authors.lastname: Nawroth
authors.forename: Katharina
authors.initials: K
authors.lastname: Hancke
authors.forename: Ricardo
authors.initials: R
authors.lastname: Felberbaum
authors.forename: Felicitas
authors.initials: F
authors.lastname: Genze
authors.forename: Ilija
authors.initials: I
authors.lastname: Damjanoski
authors.forename: Evgenia
authors.initials: E
authors.lastname: Isachenko
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods*; D003893:Desmin / Q000032:analysis; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016513:Mice, SCID; D018919:Neovascularization, Physiologic; D006080:Ovarian Follicle / Q000254:growth & development / Q000378:metabolism / Q000637:transplantation; D010053:Ovary / Q000098:blood supply / Q000502:physiology* / Q000637:transplantation; D015203:Reproducibility of Results; D013997:Time Factors; D046509:Tissue Culture Techniques / Q000379:methods*; D014183:Transplantation, Heterologous; D055815:Young Adult; D014841:von Willebrand Factor / Q000032:analysis
chemical_list: D003893:Desmin; D014841:von Willebrand Factor