Effectiveness Of Two Freezing Methods On The Follicular Viability Of Large Pieces Of Human Ovarian Tissues

ObjectiveTo investigate the effects of cryopreservation of large pieces of human ovarian tissues(about15mm2×2-3mm) by two freezing methods and assess the follicular viability after in vitro culture of fresh and cryopreserved-thawed ovarian tissues.MethodsOvarian biopsies were obtained from15women who were suffered from neoplasm except of ovarian tumor during gynecologic operations in Gynecology and Obstetrics department of Qilu Hospital of Shandong University from June2011to June2012. The ovarian tissues were dissected from the connective tissues around by sterilized scissors. The cuted tissue accounting for1/2-1/3volume of ovary was transferred to10ml PBS, and transported in tube to the laboratory on ice in10min. The ovarian medullary tissues were removed by scraping with a eye scissor and a scalpel in the laminar airflow on the aseptic laboratory table. The ovarian cortex was cut into three pieces under a steremicroscope. Each pieces were2-3mm thick and area of1.5cm2that were randomly distributed into fresh group(A), vitrification group(B), and two-step freezing group(C). Two thirds of tissue in group A was for in vitro culture and rest of it was for histological analysis. Pieces from cryopreserved group A and B were cultured in vitro after thawing, then compared the morphological change of follicles and counted the ratio of normal follicles in three groups. The viability and proliferative capacity of tissues were evaluated respectively by in vitro production of estrogen, progestogen, histological analysis and immunohistochemical staining of nuclear-associated antigen Ki67and B cell lymphoma/lewkmia-2. Besides, detection of apoptotic cells was assessed by TUNEL Assay (TdT-mediated dUTP-biotin nick end labeling).Results1. The ratio of normal follicles in group A(91.9%) was significant higher than group B(71.3%) and group C(82.9%)(P<0.05). The ratio from group C was higher than group B(P<0.05). The ratio of morphologically normal primordial and primary follicles from group C was86.8%and54.4%which was respectively higher than73.8%and44.4%from group B(P<0.05).2. Difference between three groups was not found as regard to hormonal activity(P>0.05).3. Positive expression of Ki67of group C(73%) was higher than group A(50%)and group B(55%)(P<0.05). The ratio of positive expression of group A was lower than group B(P<0.05).There was no difference of positive expression of Bcl-2staining between group A(57%)and group C(61%)(P>0.05), but the ratio of positive expression of group B(42%)was lower than group A and C respectively(P<0.05).4. TUNEL assay indicates that no TUNEL staining was found in any primordial follicles, primary follicles and secondary follicles of fresh and frozen/thawed ovarian tissue, although there was an indication of apoptosis of stromal cells in frozen/thawed ovarian tissue. The percentage of apoptotic cells in the vitrification(0.12%) and two-step freezing(0.05%)groups was higher than the fresh group(0.02%), but there was no siginificant difference between fresh group and two-step freezing group(P=0.058). The percentage of apoptotic cells in vitrification group was higher than other two groups(P<0.05). Conclusions1. The frozen/thawed large pieces of human ovarian tissues still reserved good morphology of follicles and follicular proliferation and anti-apoptosis of follicles is apparent after in vitro culture of ovarian tissues. It demonstrates that cryopreservation of large pieses of human ovarian tissues is feasible.2. There’s no significant influnce for the endocrine function of the human ovarian tissue after vitrification or two-step freezing.3. Ovarian tissues with two-step freezing is better preserved than with vitrification.