The Experiment Of FSH And BFGF In Human Ovarian Tissue Culture In Vitro

Objective:The objective of this experiment was to evaluate the follicular development and Estradiol(E₂)secretion of human ovarian tissues deprived from fresh or frozen-thawed culture in vitro.Especially when culture medium was supplemented different concentration with follicle stimulating hormone(FSH)or basic fibroblast growth factor(bFGF).To observe and evaluate the influences of FSH or bFGF on frozen-thawed human ovarian tissues culture in vitro;to investigate the appropriate culture medium for human ovarian tissues and offer some necessary experiment data for organic replacement therapy of some gynecological diseases such as premature ovarian fallure(POF),ovariotomy and so on.Methods:Firstly,we set up the in vitro culture system of ovarian tissue as Scott's.The fresh or frozen-thawed human ovarian cortex tissues were obtained from 12 women(mean age±SEM:31.33±8.19years)undergoing gynaecological investigations either laparoscopically or by laparotomy.Secondly,the human ovarian tissues were cut into small pieces(1mm³)and randomly distributed them to 16 experiment groups.Every group included 12 pieces of ovarian cortex.There were 16 experiment groups from fresh or frozen-thawed human ovarian tissues according to different supplemented concentration with FSH(0IU/ml,5IU/ml,10IU/ml,15IU/ml) or bFGF(Ong/ml,1ng/m,10ng/ml,100ng/ml)in culture medium.Thirdly,the 96 culture plate wells had been precoated with artificial extracelluar matrix(ECM). Superficial pieces of ovarian cortex were cultured in vitro as organ culture for 14 days in a-minimum essential medium(a-MEM).The culture medium was replaced by the fresh medium and stored for E₂ measurememt by micro-particle enzyme immunoassay(CIA)methods every 48 hours.Some of the fresh or cultured ovarian tissues were sent for histological section and HE staining.Morphorlogic change of the cultured ovarian tissue and follicle count were analysised using SPSS11.5 among these 16 groups after cultured for 14 days.Result:1.Although there was no siginificantly Statistics different,the E₂ output of both fresh and frozen-thawed groups in culture time 8d were higher than culture time 2d and 14d in basic culture medium.At the same time,the output of E₂ and proportion of different stages of follicles both had no siginificantly statistics different between fresh and frozen-thawed groups(P＞0.05).2.When culture medium supplemented with FSH,the output of E₂ was increased following the increasing concentration of FSH.The output of E₂ in FSH15IU/mI group and control group had a siginifieantly statistics different,especially cultured for 14 days(P＜0.05).In contrast with the control and FSH5IU/ml groups,the proportion of the primary follicle and the secondary follicle in FSH10IU/ml group were increased while the proportion of primordial follicle and abnormal follicle were decreased.FSH15IU/ml group had a siginificantly statistics different with them(P＜0.05).3.When culture medium supplemented with bFGF for 8days,the output of E₂ in each bFGF group was higher than culture time 2 days and 14 days,but there was no siginificantly statistics different between them(P＞0.05).In the same culture time,the output of E₂ in bFGF100ng/ml group was higher than in bFGF1ng/ml,bFGF10ng/ml groups and control group.But there was no siginificantly statistics different between them (P＞0.05).At the same time,the proportion of the primordial follicle,primary follicle, secondary follicle and abnormal follicle had no siginificantly statistics different between each bFGF group in the same culture time(P＞0.05).4.When culture medium supplemented with FSH or bFGF after 14 days,the output of E₂ and proportion of different stages of follicles both had no siginifieantly statistics different between fresh and frozen-thawed ovarian tissue groups(P＞0.05).Conclution:1.When frozen-thawed ovarian tissues were cultured in vitro,the output of E₂ and proportion of different stages of follicles were the same as the fresh ones. This maybe good for the functional recovery of frozen-thawed ovarian tissues after transplantation.2.The culture medium containing FSH could enhance output of E₂, improve the proportion of the primary follicle,the secondary follicle and low the proportion of primordial follicle,abnormal follicle as well.When culture medium was supplemented with FSH10IU/ml for 14 days,FSH had the ability to improve the proportion of growing stage follicles.Therefore,the cultm'e medium containing FSH10IU/ml maybe can be put into culture media for ovarian tissues culture in vitro.3.When culture medium was supplemented with bFGF10ng/ml and bFGF 100ng/ml,these two bFGF groups could enhance the output of E₂ especial for bFGF100ng/ml group.But in all bFGF groups,the proportion of primary follicle and secondary follicle did not have an increase while the proportion of primordial follicle and abnormal follicle also did not have a decrease with the longer culturing duration after 14 days.Therefore,the impact of bFGF on the primordial follicle in ovarian tissue culture still need more reseach.4.Ovarian follicles can develop well when cultured in vivo.Maybe ovarian tissue loses control of the female body and frees from some inhibitors in the body,which make follicles begin to develop.5.In this experiment,FSH acts mainly as a survival factor as human early follicular development in ovarian tissue culture while bFGF same as no obvious impact on the human early follicular development and the priming of primordial follicle.