The Preliminary Study Of Human Ovarian Tissue Preservation By Vitrification

Early diagnosis and effective chemotherapy, as well as the application of bone marrow transplantation has significantly improved the therapeutic effect of cancer and greatly enhanced the survival rate of cancer patients. But for young female patients, raising the survival rate does not mean that the quality of life improved. Because ovarian tissue toxicity of the treatment of these cells, in particular, alkylating agent and radiotherapy are particularly sensitive, it may lead to the loss of female reproductive endocrine function. Various studies have confirmed that the implementation of these patients with ovarian tissue cryopreservation is effective. There are a lot ovarian follicles, if we can effectively save their frozen, thawed and be used when necessary, is bound to the further development of life sciences provides a wealth of resources and a strong research platform.In recent years, research and development ovarian tissue frozen rapidly, and has become a hot issue in the life sciences. Vitrification technology was developed in the 20th century,90 years of cold-storage technology has been in the embryo cryopreservation in a wide range of applications, some scholars have reported the successful vitrification preservation rats, rabbits, sheep, monkey, etc. Study of ovarian tissue and confirmed that vitrification can be stored ovarian tissue function in a more effective means.ObjectiveCurrent ovarian tissue by vitrification freezing still there is no uniform program, including cryoprotectants and frozen carrier. Domestic ovarian tissue vitrification is still at the exploratory stage, this study by observing the fresh and frozen-thawed ovarian tissue after certain physiological characteristics, focusing on the programs of different vitrification of ovarian tissue freezing effect, a prliminary comparison of different concentrations of Vitrification protecting agent and the different carriers of human ovarian tissue freezing vitrification effect initially established a human ovarian tissue vitrification program. Materials and methods1 MaterialsFrom August 2008 to March 2009, the present study because of raised 20 cases of ovarian cysts need surgery patients, aged 24-38 years (29.79±4.14). Laparoscopic ovarian tissue biopsies were obtained by carrying out spin-off after cyst cyst attached to the cut with no obvious thrombosis of ovarian tissue piece 1-2cm2 experiment.2 Methods2.1 The treatment of ovarian tissueWill achieve the ovarian tissue pieces placed in saline solution and placed in ice bucket can be preserved, sent to the laboratory within 30 minutes, the rinsing in PBS to remove the medulla, the ovarian cortical pieces cut into the size 2*2*1mm3 The tissue block, placed in PBS solution standby. Randomly selected part of the tissue placed in neutral formalin fixed as fresh control group.2.2 Ovarian tissues by vitrificationLow-concentration ratio of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for vitrification groupⅠ, a high concentration ratio of glass groupⅡ. Part of human ovarian tissue fluid balance and two vitrification solution in a certain time after exposure to each of the two micro-drop method, wheat-tube method and a hard surface vitrification (SSV) into liquid nitrogen for vitrification.2.3 Ovarian tissue cryopreservation and in vitro cultureUsing three-step recovery method will be the organization of cryopreservation recovery of particles one by one randomly selected part of the revival of the ovarian tissue pieces placed in CO2 incubator for 14 days every 48 hours to collect the supernatant centrifuged under test hormone levels. The remaining tissue and cultured tissue after the end of neutral fixation until after the histological examination.2.4 Histological examination and measurement of hormone levelsEach block of four organizations to be seized for a flat-line embedded in paraffin wax block in order to 4μm thickness of each slice, line HE staining. Supernatant measured by RIA of estradiol (estradiol, E2) levels, in order to assess ovarian tissue after freezing and thawing of the endocrine function.3 StatisticalUse SSPS 13.0 the date were analyzed statistically.Between the two groups the rate of morphologically normal primordial follicle useχ2 tests and analysis, comparing the rate of apoptosis between the two groups line single-factor analysis of variance, two weeks in vitro hormone secretion in each group were randomly paired line analysis of variance, withα=0.05 significance level.Results1 Freezing of human ovarian follicles, interstitial and all levels of the proportion and morphology.1.1 Frozen human ovarian tissue stromaFresh human ovarian mesenchymal cells arranged closely and the law will be close at all levels, wrapped in ovarian follicles within the cortex. Two kinds of vitrification solution, as well as three kinds of human ovarian tissue cryopreservation carrier recovery is seen after ovarian tissue in parts of mesenchymal cell separation, but the primordial follicle did not change significantly. After the ovarian tissue cultured in vitro stromal cell connections loose, or even fracture the formation of fissures, and disordered cell arrangement.1.2 Human ovarian tissue before and after freezing at all levels of follicle ratio and morphology Compared with the fresh, two frozen groups the number of primordial follicles and the normal form of the rate of decline in varying degrees, but the glass of the number of primordial follicles in groupⅠand the normal form of the rate of no statistically significant difference (P>0.05), glass transitionⅡgroup differences were statistically significant (P<0.05), two freezing difference between the groups was statistically significant (P<0.05). Frozen fresh group and the group the number of primary follicles and secondary follicles are small, this study and not subject to statistical analysis.2 Frozen human ovarian tissue of various types of cells in apoptosisHuman ovarian tissue in the primordial follicle, primary follicle oocytes and granulosa cells no apoptotic cells in both the positive expression, and ovarian stromal cells within the visible expression of the nucleus of apoptotic cells, brown, or brown color. With the fresh control group, each group vitrification of ovarian tissue mesenchymal cells was no significant difference in apoptosis rate (P>0.05).3 The frozen ovarian tissue after resuscitation group of E2 secretionDuring in vitro tissue culture, the constant increase in E2 secretion of freeze-thaw group, two freezing difference between the groups was statistically significant (P<0.05), the carrier was no significant difference between (P>0.05). Conlusions1 Solid-surface vitrification method is superior to micro-drop method and Mai-tube method, more suitable for primordial follicles in ovarian tissue cryopreservation.2 It is a better effect for the conservation of ovarian tissue low concentration of cryoprotective agents and solid-surface vitrification method.