| Objective:Since the 1970s,the incidence of cancer has continued to increase at an annual rate of 2.9%.The incidence of cancer in children,adolescents and young people is on the rise.Advances in cancer treatment have increased the cure rate of malignant tumors,but may significantly induce gonadal damage in young women.Fertility preservation of female tumor patients is a matter of concern.The commonly used methods of female fertility preservation in clinic,embryo cryopreservation and mature oocyte cryopreservation have their limitations,especially for prepubertal,adult unaccompanied cancer patients who need emergency radiotherapy and chemotherapy and can not tolerate ovarian stimulation.There is sufficient evidence to support the clinical application of ovarian tissue cryopreservation as an effective technique.The purpose of this study was to evaluate the effect of normal ovarian cortex cryopreservation on follicular activity in patients with gynecological tumors,and to evaluate the significance of ovarian tissue cryopreservation in patients with gynecological tumors of different ages and stages.More scientific program guidance for patients who need to preserve fertility.Methods:Selection of research objects:this study was a retrospective study.The ovarian tissue came from female patients who needed to remove all or part of the ovarian tissue in the department of gynecology of Henan people’s Hospital from December2018 to January 2020 for various reasons.A total of 34 cases of residual ovarian tissue on the wall of ovarian cyst were removed,and the discarded tissue removed by the patient was used for study.The ovarian tissue was obtained by laparotomy or laparoscopy.There were no cancer cells in the frozen section and pathological examination of the ovarian tissue during the operation.The patients aged from 8 to 42years old signed the informed consent form.Case inclusion criteria:patients with regular menstruation(except for children),normal sex hormone levels,no history of hormone use in the past 3 months,no other diseases.Case exclusion criteria:those who had a history of radiotherapy and chemotherapy before operation.Methods:The obtained ovarian tissues were numbered,and the ovarian cortical slices were vitrified and remelted,and the ratio of normal follicles to abnormal follicles was calculated by H&E staining.The tissue activity of ovarian tissue was evaluated by immunohistochemical detection of cleaved-caspase-3 expression,AMH expression and CD31 expression.The tissue activity was counted under×400inverted microscope.The immunohistochemical results were scored by staining intensity combined with staining distribution in positive cells.The positive cell rate was calculated according to the sum of the above two scores,and the positive cell rate in every ten visual fields in each section was calculated.The experiment was divided into two parts,each of which was grouped in a different way.The first part:the study of female age on the freezing effect of ovarian tissue.According to the age of the patients,the patients were divided into:(1)childhood female(4-<12 years old);(2)adolescent female(12-19 years old);(3)Women in the best childbearing period(20-<35 years old);(4)Women in the period of declining ovarian function(35-45 years old).Because the ovarian reserve function of patients in different age groups is different,women aged 20-35 years old are selected for the second part of the study.According to the type of tumor,the patients were divided into(A)ovarian chocolate cyst patients;(B)teratoma patients and;(C)patients with other gynecological tumors.The measurement data were expressed by mean±standard deviation,the independent sample t test was used,the test level wasα=0.05,and the counting data was measured byχ~2 test,the test level wasα=0.05,and the significant difference was judged by P<0.05.Results:Results of histomorphological analysis of ovary:1.There was no significant difference in the morphology of primordial follicles between frozen ovarian tissue and fresh ovarian tissue,but the cryopreservation damage of primary follicles was significantly increased.2.The follicle density in ovarian tissue of children after cryoresuscitation was higher than that of adult patients,and the follicle density decreased significantly with the increase of age.The ovarian tissue of childhood contains a high proportion of abnormal anaplastic follicles.3.In the fresh and frozen resuscitated ovarian tissues,there was no significant difference in the abnormal rate of primordial follicles between the childhood female group and the>35 years old female group,but it was significantly higher than that in the other two groups(P<0.05).There was no significant difference in the abnormal rate of primordial follicles between the other two groups,and there was no significant difference in the abnormal rate of primary follicles between the other two groups.4.The follicular density of patients with ovarian chocolate cyst was lower than that of patients with teratoma and other gynecological tumors.Histological analysis of fresh and thawed ovarian tissues showed extensive damage to stroma and follicles.5.In fresh and frozen resuscitated ovarian tissues,there was no significant difference in the abnormal rate of primordial follicles and primary follicles among different disease groups.Results of immunohistochemical staining:1.The expression of Cleaved-caspase-3 in fresh and frozen resuscitated ovarian tissues was significantly different among four age groups.The expression of Cleaved-caspase-3 in ovarian tissue of childhood female group was significantly lower than that of the other three groups.The expression of Cleaved-caspase-3 in ovarian tissue of>35-year-old female group was significantly higher than that of the other three groups.2.The positive rate of AMH in ovarian tissue cells decreased with the increase of age.There was significant difference in the expression of AMH between fresh and frozen resuscitated ovarian tissues among the four age groups.Further pairwise comparison showed that there was significant difference in the expression of AMH in ovarian tissues between each two groups(P<0.05).3.The positive rate of CD31 in ovarian tissue cells decreased with the increase of age.There was significant difference in the expression of CD31 between fresh and frozen resuscitated ovarian tissues among the four age groups.Further pairwise comparison showed that there was significant difference in the expression of CD31 in ovarian tissues between each two groups(P<0.05).4.There was no significant difference in the expression of Cleaved-caspase-3,AMH and CD31 in fresh and frozen resuscitated ovarian tissues among the three disease groups.Conclusion:1.The cryopreservation of children’s ovarian tissue has its particularity.The density of follicles in children’s ovarian tissue is high,although it contains a high proportion of abnormal follicles,but there are a large number of blood vessels in children’s ovarian tissue,which may be helpful for vascular reconstruction after transplantation.For children with malignant tumors,it is necessary to adopt ovarian tissue freezing technique to preserve fertility.2.The ovarian function of women over 35 years old decreased significantly,the abnormal rates of primordial follicles and primary follicles in fresh and remelted ovarian tissues increased significantly,and the benefits of ovarian tissue freezing were limited.it is recommended that female patients with high risk of gonad injury should use fertility preservation technology to protect fertility as soon as possible.3.There is no significant difference in the abnormal rate of primordial follicles and primary follicles among different disease groups,suggesting that the type of disease has less impact on ovarian function,and ovarian tissue cryopreservation is suitable for fertility preservation of patients with various tumor types. |
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