| Female cancer patients release and/or chemotherapy may seriously affect the function of ovarian reserve which led to ovarian failure. The improvement of treatment techniques in recent years makes women cancer patients, especially young patients with long-term survival have been greatly improved, but release and/or chemotherapy, the damage caused by the female gonads has aroused great concern, so scholars have begun working on How do cancer patients after treatment of cancer can still save the reproductive functions. In 2006, Chen proposed a new type of vitrification, which direct cover vitrification method (DCV), this approach can speed up the largest freezer and easier to form the glass state to reduce ice crystal The formation of damage is also smaller. Within the mammalian ovarian follicle is the basic functional unit, which has oocyte growth and maturity to provide micro-environment. Mammalian ovarian cortex stored a large number of preantral follicles, including primordial follicles, primary follicles and secondary follicles, they are small, the large number of difficult to damage during freezing is suitable for cryopreservation. To explore its in vitro culture technique could be observe the folliclgenesis and mechanisms, not only for the regulation of human oocytes have a new understanding of women's reproductive capacity and the preservation of endangered species also has great significance. As mimesis in vivio cultivation is very difficult, so current research focuses mainly on the cultivation of the preantral follicles.ObjectiveIn this study using direct cover vitrification human ovarian tissue after thawing and isolated preantral follicles cultured in vitro. Using three-dimensional culture systems and adding activin A in the culture medium, by comparing before and after follicle diameter and estradiol levels in order to explore the human preantral follicles growth and development, Thus to establish an experimental basis for building a database of human oocyte.MethodsThe subjects are eighteen patients who were treated by ovarian tumorectomy during normal curative activity in our hospital. The ovarian tissue was dissected into lmmxlmmxlmm under stereoscopic microscope, and the ovarian tissue was divided into fresh group,direct cover vitrification group. After thawing combined with enzyme digestion and mechanical separation to isolate a single huamn preantral follicles. Respectively, the use of alginate gel system, and adding activin A in culture medium, measurement of follicular diameter and half the amount of fluidon on alternate daysfor, and the collected culture medium was stored in refrigerator at-20℃. The level of estradiol was measured with the method of electrochemiluminescence immunoassay (ECLIA). Statistical analysis:Statistical analysis on the experimental data was done by SPSS 13.0 software package.Result1. The frozen ovarian tissue biopsies before and after HE staining, freeze-thaw group were similar as contral, follicular morphology was normal and distribution at all levels of follicle was no significant difference between the fresh group. It does not observed significant changes in follicle morphology. The survival rate of follicle before and after freezing was no significant difference (P>0.05), the survival rate of follicle after frozen was also not statistically significant. All levels of follicle after frozen compared with the fresh, granular cells grew well, by the trypan blue after 1 minute is not colored, indicating that the follicles was survive(P<0.05). 2. Using different concentrations of calcium alginate gel culture, we thought in which the normal follicles can growth and development, after 8 days cultured follicular diameter was significantly increased (P<0.05). However, follicles in the concentration of 0.25% calcium alginate gel in the fastest growing trend, an increase of follicle diameter and survival rate was significant higher. The other two experimental groups compared with follicular diameter increases, the difference was not statistically significant.3. When cultured 8 days, single follicular in the control group and different concentrations of calcium alginate three-dimensional culture medium can secret estradiol The E2 release in each experimental group were higher, the difference was significant(P>0.05).The follicle in a concentration of 0.25% calcium alginate gel culture release E2 was most, the difference was statistically significant(P<0.05).4. Using 0.25% concentration of alginate gel were cultured different diameters of follicles, the survival rate of two groups were no difference(P>0.05). However, the increase in diameter compared between two groups,the difference was statistically significant(P<0.05).5. Using the concentration of 0.25% alginate gel to culture, add with different concentrations of activin A in the culture medium,after 8 days,in addition to lng/ml group had no significant growth in follicle diameter (P>0.05), the other two groups were significantly increased follicular diameter(P<0.05). During in vitro culture, the control group and different experimental group can secrete E2, and E2 in different experimental group was significant higher in control group(P< 0.05).Adding 100ng/ml of activin A in the medium release of E2 was higher than 10 ng/ml group, the difference was statistically significant.Conclusions1. Direct cover vitrification cryoapplication does not damaging human primordial, primary follicles,can be used for ovarian tissue cryopreservation.2. The Alginate system is conducive to the growth of human preantral follicles, especially the concentration of 0.25% calcium alginate gel.3. Larger diameter follicle growth rate and potential of developmental greater than smaller. 4. Adding activin A in the culture medium are conducive to human preantral follicles growth and development...
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