Experimental Study On The Effect Of The GnRH-a On Vitrification And Autotransplantation Of Rabbit Ovarian Tissue

PartⅠExperimental Study on the Effect of the GnRH-a on Vitrification of Rabbit Ovarian TissueObjective:Observe differences of the injection of GnRH-a group and non-injection of GnRH-a group of the frozen-thawed ovarian tissue in rabbits at follicle morphology, quantity and apoptosis rate, to explore the possible effect of GnRHa pretreatment on rabbit ovarian tissue cryopreservation.Methods: (1) 18 female New Zealand white rabbits with normal estrous cycle were randomly divided into two groups:GnRH-a group(n=9) and non-injection of GnRH-a group (n=9).(2) GnRH-a group:injection of 2.5mg GnRH-a was treated every 15 days twice before ovarectomy. Then after ovarectomy ,one side of the ovary was vitrificated immediately after treatment(group A), the Other side of the ovary is not be frozen but drop into 4% paraformaldehyde fixed as control(group B); non-injection of GnRH-a group: injection of 0.3ml saline every 15 days,30 days later the ovarectomy was treated , one side of the ovary was vitrificated immediately after treatment(group C), the Other side of the ovary was not be frozen but drop into 4% paraformaldehyde fixed as control(groupD).(3) groupA and groupC have the same vitrification method using modified Noriko.K's . (4) Histologically examined the morphological of follicles and qauntity of various stage follicles;Detected the apoptosis positive follicles in ovarian tissue by TUNEL.Result: (1) Histological examination: the proportion of morphologically normal follicles in groupA, groupB, groupC and groupD were 71.37%, 84.85%, 66.57% and 82.93%, the proportion of morphologically normal follicles cryopreserved group were lower than those of fresh group(71.37%vs84.85%;66.57%vs82.93%;P<0.05), group A was significantly higher than group C on the proportion of morphologically normal follicles(71.37%vs66.57%,P<0.05);group B was higher than group D on the the proportion of morphologically normal follicles with no significant statistical difference(84.85%vs82.93%,P>0.05),The proportion of primordial follicle in GnRH-a group was higher than the non-injection GnRH-a group(P<0.05). (2) The percentages of TUNEL-positive follicles of group A, groupB, groupC and groupD were 13.91% ,9.21% ,12.58% and 8.16%. The percentages of TUNEL-positive follicles of frozen group was significantly higher than fresh group (13.91%vs9.21% ;12.58%vs8.16%,P<0 05). The percentages of TUNEL-positive follicles was not significant between groupA and groupC (P> 0.05).Conclusion: (1) Freezing and thawing process could cause a certain degree of damage on follicles;(2) The GnRHa pretreatment changed the proportion of the follicle pool with increasing percentage of primordial follicle, It is beneficial for ovarian tissue to diminish the frozen-thawed injury.(3) The freezing and thawing process induced apoptosis occurred. The GnRH-a pretreatment did not influence the apoptosis. PartⅡExperimental Study on the Effect of the GnRH-a on Autotransplantation of Rabbit Ovarian TissueObjective: To explore the possible effect of GnRH-a pretreatment on cryopreserved rabbit ovarian tissue after autotransplantation.Methods: (1) 22 female New Zealand white rabbits with normal estrous cycle were randomly divided into 3 groups: group A(n=8): injection of 2.5mg GnRH-a was treated every 15 days twice before ovarectomy;group B(n=8): injection of saline was treated as control;group C(n=6): Sham-operated group.(2) group A and group B were operated to remove both ovaries and then vitrification (same as partⅠ), After 4 weeks, a part of ovarian pieces after freezing and thawing were autografted at bilateral superficial muscles of groin area.Another part were fixed with 4% paraformaldehyde for histological examination. group C :Sham-operated group.(3) observed the restoration rate and the interval of regular oestrous cyclic by vaginal smear and detected the serum level of E2 and FSH .Result: (1) There were two rabbits in group A and one rabbit in group B died for infections, blood loss or nursing improper,the remaining rabbits in good health.(2) Observation of vaginal smear: there are four rabbits in group A resume estrous cyclic and there are five rabbits in group B resume estrous cyclic, Interval of regular estrous cyclic in group A(20.00±2.16 days)was significantly longer than group B(15.80±1.30 days)(P<0.01) (3) The serum level of E2 before ovarectomy was higher than those after transplantation (P <0.05) and the FSH level was lower than those after transplantation (P <0.05), The level of E2 after transplantation in groupA and groupB was significantly lower than groupC and the level of FSH after transplantation in groupA and groupB was significantly higher than groupC (P <0.05). The level of E2 and FSH between groupA and groupB after transplantation was not significant. (P> 0.05). (4) Histological examination: After freezing and thawing,the proportion of normal morphology follicle in GnRH-a group was higher than those in control,the difference between two groups was significant(P<0.05); the proportion of normal morphology follicle in grafts was not significant between GnRH-a group and control. (P>0.05), the proportion of normal morphology follicle in grafts was significantly lower than before transplantation (P <0.01).only a small number of primordial follicles and primary follicles were exist in grafts.Conclusion: (1) frozen-thawed ovarian tissue after autotransplantation could survival with the hormone secretion ability. (2) superficial muscle of groin area was a good implantation site for its convenience,plentiful blood supplying and easy monitoring.(3) the ovarian function in GnRH-a group need more time to recover, May be due to GnRH-a inhibit the follicle growth; (4) The GnRH-a pretreatment did not decrease the Ischemia - reperfusion injury.