| Ovarian tissue cryopreservation has already became a hot spot in reproduction medical domain.Ovarian after thawing is used for reimplantation or culture in vitro.As for patients with malignant tumor,autoplastic transplantation is not suitable,and xenoplastic transplantation is limited by ethics and animal source disease.Culture in vitro includes ovarian tissue in vitro culture and follicle in vitro culture. Many animal empirical studys demonstrate that ovarian tissue in vitro culture can maintain follicular integrity and cell-cell interaction, becoming an important study model to explore follicle growth in fresh and thaw ovarian tissue. But because of follicular maldistribution in ovarian tissue,it is difficult to evaluate the quantity of follicles in freezing tissue piece only by histomorphology before cryopreservation.The indeterminateness of the quantity of follicles in freezing tissue degrades the value of ovarian tissue in vitro culture. Isolated follicles in vitro culture surpass ovarian tissue in vitro culture because of providing source of oocytes for assisted reproductive techniques. ObjectiveIn this study, we preserved human ovarian tissue with the method of direct cover vitrification (DCV), follicles were isolated after ovarian tissue thawed. Integrity survival follicles were cultured in vitro,using an alginate matrix as three-dimensional (3-D) culture system to maintain cell-cell communication and support follicle development. According to the study requirement, stem cell factor and leukaemia inhibitory factor were added to explore the effects of these on human primordial follicles in vitro culture and to find optimal condition.The final aim of this study is to provide fundamental research on constructing human oocyte storeroom.MethodsWe preserved human ovarian tissue as Chen's.1. The 18 ovarian tissue examples were dissected into Imm×1mm×2mm, and the ovarian tissue pieces were divided into there parts randomly,one for histomorphology analysis by 10% neutral formalin fixation,one for isolating follicles, the rest for direct cover vitrification.2.2～3 pieces of thawed ovarian tissue were fixed by10% neutral formalin for histomorphology analysis, the rest for isolating follicles.3. Integrity survival follicles divided into five groups randomly were cultured for 8 days in three-dimensional (3-D) culture system added some different components.Half of the cultured medium was replaced with fresh on the alternate day, and the collected cultured medium was stored in refrigerator at-20℃. The level of estradiol was measured with the method of electrochemiluminescence immunoassay (ECLIA). Follicular diameter was measured on the first day,the fourth day and the last day in vitro culture.Trypan blue dyeing was used to evaluate the follicular survival rate.4. Statistical analysis on the experimental data was done by SPSS13.0 software package.Result1. The percentage of follicles in different developmental stages was no significant difference before and after ovarian tissue cryopreservation (P>0.05).The percentage of follicles in different developmental stages was significant different within group(P<0.05).The most predominant type of follicle in the ovarian tissue was the primordial follicle, then primary follicles and secondary follicles,no antral follicles,moreover,follicles in the ovarian tissue were maldistribution.2. The rate of abnormal primordial follicles was no difference before and after cryopreservation(P>0.05).The rate of abnormal primordial follicles was lower than that of primary and secondary follicles within group(P<0.05).The rate of abnormal primary and secondary follicles after cryopreservation was higher than before cryopreservation,but there was no significant difference(P>0.05).3. A lot of follicles isolated from ovarian tissue before and after cryopreservation were survival.The effect of freeze-thaw process on the survival rate of isolated follicles in different developmental stages was no difference between them(P> 0.05).But,the survival rate of primary and secondary follicles was lower compared with primordial follicles.4. After several days cultured in vitro,the follicular diameter in control group increased slowly,and was obviously smaller than experiment groups(P<0.05).The follicular diameter in SCF and LIF co-culture groups increased quickly, and there was significant difference compared with solo-culture groups (P<0.05). In co-culture groups,there was no difference with different density of SCF(P>0.05). There was no difference in solo-culture groups(P＞0.05).5. Follicles in all groups have function of hormone secretion after several days cultured in vitro. After four days,the level of estradiol in control group increased slowly,and was lower than experiment groups(P<0.05).The level of estradiol in SCF and LIF co-culture groups increased quickly,and was higher than solo-culture groups(P<0.05).In co-culture groups,there was no difference with different density of SCF,but, there was significant difference on the fourth day(P<0.05).There was no difference in solo-culture groups(P>0.05).6. Trypan blue dyeing was used to evaluate the survival rate of follicles in each group after cultured for 8 days.The survival rate of follicles in control group was lower than experiment groups(P< 0.05),but there was no difference among experiment groups(P>0.05).Conclusions1. Direct cover vitrification cryoapplication,manipulated simplely and little time consuming,can be used for ovarian tissue cryopreservation for conserving follicles in human ovarian tissue very well.2. Freeze-thaw process has no obvious damage on primordial follicle,but has some damage on primary and secondary follicles.3. Alginate matrix, maintaining follicular integrity construction and cell-cell communication,can be used as three-dimensional (3-D) culture system to culture isolated follicles in vitro.4. The method of collagenase combined with machine isolating follicles can relieve damage of collagenase alone on basement membrane and theca of follicle, maintaining satisfactory activity of isolated follicles.5. SCF and(or) LIF added to culture medium in vitro can promote follicular growth and development, raise the survival rate of follicles and the level of estradiol secretion,moreover,the joint action of SCF and LIF was more manifest. |
PDF Full Text Request