Studys On Cryopreservation Of Human Ovarian Tissue

Objective: To compare the morphological alterations, ultrastructural characterization and DNA fragmentation in primordial and primary follicles from fresh and cryopreserved human ovarian tissue.Methods: Ovarian cortical tissue was obtained from women without ovarian diseases undergoing gynaecological investigations by either laparoscopy or laparotomy. The ovarian samples were performed cryopreservation with programmable freezing protocols using DMSO and sucrose as cryoprotectants. The morphology of primordial and primary follicles in ovarian tissue sections were assessed using both light and electron microscopy, and fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique in fresh and cryopreserved human ovarian tissue.Result:1. In the frozen fragments, the percentage of morphologically normal primordial follicles was not different compared with the case of fresh human ovarian fragments (P>0.05).The percentage of morphologically normal primary follicles in the frozen fragments was significantly lower than that of fresh human ovarian tissue (P<0.05). However, the percentage of morphologically normal both primordial and primary follicles was similar in between the non-cryopreserved and cryopreserved human ovarian tissue (P>0.05).2. Electron microscopy showed that the structural integrity of the primordial follicles were well preserved in cryopreserved ovarian tissue.The membranes of oocyte, cell nucleus and mitochondria ofprimordial follicles were intact, and the few mitochondria in oocytes were located mainly in the perinuclear cytoplasm.Some abnormal cytoplasm area in cryopreserved human primordial follicles was occupied by few small vesicles. The cryopreserved human primary follicles were found abnormal cytoplasm occupied by abnormal vesicles and some swollen mitochondria with or without abnormal vesicles.However, the membranes of oocyte ^ cell nucleus and mitochondria were intact in cryopreserved primary follicles.3. The TUNEL-positive cells were mainly located in the oocyte of primordial and primary follicles in fresh and cryopreserved human ovarian tissue, and apoptotic DNA fragmentation were detected in few granulose cells of human follicles.The percentage of the TUNEL-positive primordial follicles in the fresh and cryopreserved ovarian tissue showed no significant difference (P>0.05), and the primary follicles in cryopreserved human ovarian tissue were not found higher proportion of apoptotic follicles than in the fresh group(P>0.05).Conclusions:1. The primordial follicles are well preserved the structural integrity in cryopreserved human ovarian tissue.2. The proportion of morphologically primary follicle decreases significantly after programmable freezing.3. The freezing process has no effect on the apoptotic proportion of primordial or primary follicles in human ovarian tissue.Objective: By staining the isolated follicles using PI and CFSE, this study is to evaluate the effect of programmed freezing on the viability of two different stages of follicles and to assess the difference of the viability of primordial and primary follicles. To explore the difference between human primordial and primary follicular development from storaged human ovarian material in vitro culture. Methods:Human ovarian material was incubated with collagenase type I and deoxyribonuclease I, and was dissected manually under the stereoscope. The viability of fully or partially isolated primordial and primary follicles were studied with live-dead stains using PI and CFSE under a fluorescence microscope. The isolated primordial and primary follicles with good structure were transplanted into the three-dimension culture system including collagen gel solution. After 24h in vitro culture, those follicles maintaining three-dimension structure were examined under inverted microscope with a calibrated eyepiece micrometer for follicular diameter, the diameter of oocytes and the number of granulose cell layers. Results:1. By double staining method, the viability of whole primordial follicles from cryopreserved human ovarian tissue showed no difference compared to the rate of the fresh group (P>0.05). Both in fresh and frozen-thawed specimens, the viability rates of primary follicles were not different significantly (P>0.05).2. There was a similar percent viability between primordial and primary follicles from the fresh human ovarian tissue(P>0.05).In cryopreserved human ovarian cortex, the percent viability of primary follicles was not different significantly with the percent of primordial follicles (P>0.05).3. More increase in follicular size, the diameter of the oocytes andnumber of granulose cells layers were observed in the primordial follicles after culturing in three-dimension collagen system (PO.05), and primary follicles in vitro culture showed significantly increase of follicular size, the diameter of the oocytes and number of granulose cells layers than Oh culture (PO.01).4. In vitro culture, there was no difference in viable term preserving the structual intergrity of primordial and primary follicle isolated from cryopreserved human tissue (P>0.05), significantly lower than that of the viable nude oocytes (/><0.01). Conclusions:1. Programmed freezing has no effect on the viability of isolated primordial and primary follicles from human ovarian tissue.2. From the fresh and cryopreserved human ovarian tissue, there is no difference in the percent viability between primordial and primary follicles.3. Primordial and primary follicles isolated from cryopreserved human ovarian tisuse survive and grow in three-dimension culture system. The human primary follicles show higher developmental ability than primordial follicles.Objective: This study was to explore the effect of cryopreservation on the follicular development in human ovarian tissue culture. And to evaluate the effect of FSH and EGF on the growth and development of cryopreserved human ovarian tissue in vitro culture.Methods:Superficial pieces of ovarian cortex, from unfrozen and frozen human ovarian tissue, was cultured for 7 days in RPMI-1640 medium. The culturing medium of 400 micorliter were collected on the alternate day. Histological examination of cortical pieces were performed after the culture and in vitro culture was done to study hormone production ability. The remaining frozen human ovarian tissue was randomly distributed to three parallel cultures medium supplemented with FSH, EGF or FSH+EGF. Histological evaluation of each piece was performed after 7 days of culture and compared with the cryopreserved human ovarian in vitro culture.Results:1. After 7 days in culture, proportion of primordial follicle hadsignificantly decreased and proportion of growing follicles had increased significantly in all groups of culturing ovarian tissue, compared to the day 0 of culture (P<0.001). However, the cryopreserved ovarian tissue in vitro culture showed no difference in the follicular development compared to that of ovarian material from fresh cortical pieces (P>0.05).2. Cryopreservation had no effect on the levels of E2 secreted fromhuman ovarian cortexes in vitro (P>0.05), and there was no difference in the E2 level of conditional medium collected at 2,4 and 6 day in vitro culture (P>0.05).3. On day 7 of culture, the addition of FSH showed a significantlydecreasing rate of follicles and a significantly increasing the diameterof follicles of ovarian tissue relative to the control group (P<0.05). However, the addition of FSH had no effect on the follicular development (P>0.05). After 7 days in vitro culture, there was no significant difference in the follicular development, diameter of follicles and the rate of apoptotic follicles between the group of EGF and control group (P>0.05). In the conditional medium with FSH, the addition of EGF had no effect on the development of human follicles and diameter of follicles in culturing ovarian tissue compared to control group (P>0.05). Howerver, the rate of apoptotic follicles in cryopreserved ovarian cortexes with addition FSH and EGF was lower significantly than that of control group (P<0.05). Conclusions:1. Cryopreservation has no effect on the follicular development and the level of E2 secreted from human ovarian tissue in vitro culture.2. FSH and not EGF acts mainly as a survival factor as human earlyfollicular development in ovarian tissue culture.Objective: To develop a xenograft model in which fresh and cryopreserved human ovarian tissue was transplanted into nude mice s.c.and to study the effect of cryopreservation on the early stages of follicular growth in vivo and the effect of early administration of PMSG on follicular development of cryopreserved human ovarian xenografts. Methods:Fresh and cryopreserved human ovarian pieces were grafted into subtaneously female mice for 6 weeks before obtaining the xenografts. Histological observation was performed in xenografts from fresh and cryopreserved human ovarian tissue. The remaining cryopreserved ovarian tissue was grafted into subtaneously nude mice for 4 weeks with receiving 4IU of PMSG s.c. or saline solution on alternating days after one day of transplantation. The assessment of follicular development was performed by histological analysis. Results:1. There were a lower percentage of primordial follicles and higher proportion of growing follicles in the transplanted ovarian tissue than that of no-transplanted tissue (/><().05). However, between the fresh and cryopreserved ovarian xenografts, there was no difference in the follicular development after transplantation for 6 weeks of grafting.2. After 4 weeks of transplantation, ovarian tissue of receiving the administration of PMSG had significantly higher proportion of growing follicles compared the group with addition of saline solution (P<0.05). Moreover, the secondary follicles with beyond two-layer of granulose cells stage were observed only in the grafts with administration of PMSG.Conclusions:1. Cryopreservation has no effect on the follicular development in the transplanted human ovarian tissue.2. The early addition with PMSG is required for the further developmentof secondary follicles in human ovarian xenografts.