| BackgroundPremature ovarian failure usually refers to women who have amenorrhea,along with continuous decreased estrogen level?increased gonadotropin level and other endocrine disorders under 40.A worldwide epidemiological survey shows the morbidity of POF is 1-2%,but there is an increasing trend year by year.At present,the etiology of most patients with POF remains unclear;for those with no fertility requirements,hormone replacement therapy can relieve the symptoms of menopause in advance.Yet those who have not yet been given birth or are still in demand,High quality eggs can't be obtained through conventional assisted reproductive techniques,or extremely difficult.This has become the most difficult problem of POF assisted reproductive treatment.For the majority of POF patients,the most effective method is IVF-ET with donated oocytes.But under the limitation of law?ethics and ideas,it is hard to satisfy these patients' fertility requirements.Some study shows some primordial follicles in a resting stage remain in the cortex,but these follicles show no response to gonadotropin.So they can't nomarlly develop and mature.How to preserve these follicles with latent fertility,and activte them to develop,so as to acquire effective eggs become a new breakthrough of assisted reproductive treatment for POF patients.At present,the common used methods of fertility preservation include embryo cryopreservation ? cryopreservation of unfertilized oocytes ? in vitro maturation of unfertilized oocytes and ovarian tissue cryopreservation.However for the patients of POF.Either mature or immature eggs can't be acauired,not to mention use mature eggs to fertilization with sperm in vitro.Therefore,these methods of feretility preservation such as embryo cryopreservation?cryopreservation of unfertilized oocytes and in vitro maturation of unfertilized oocytes are not appropriate for POF patients,which made ovarian tissue cryopreservation become the unique way for fertility preservation of POF patients.Ovarian tissue cryopreservation include vitrification and slow freezing.More and more researches reveal that vitrification is more simple an effective than slow freezing.In recent years,How to activate primordial follicles in a dormant stage becomes a hot spot of POF assisted reproductive treatment.Because there is a risk of tumorigenesis after the use of activator in vivo,more and more researches focus on in vitro activation,In vitro activation needs to take out ovarian tissue from patients,after some managements or using activators,stimulate follicles in a resting stage to development,and reimplant to get mature eggs and finally pregnant.The researches of ovarian tissue cryopreservation reveal that PTEN inhibitors can activate PI3K-AKT signal pahway and the development of primordial follicle.Researches about human ovarian xenoplastic transplantation also verificate this role.The Hippo signaling pathway is also widespread in mammals,and highly conservative,It's major function is limiting the excessive growth of tissue and organ.A scholar from Japan found that fragment processing the ovary of mice into small fragments can disrupt Hippo signal pathway,and accelerate follicle development.Our research by means of vitrification freezing of human ovarian and revive them,so as to evaluate the effectiveness of vitrification freezing and revive technology,and we fragment processing huaman ovarian tissue,and test the content change of YAP?p-YAP protein ?CCN growth factor and the inhibitor of apoptosis of BIRC.Thus,we can verify whether the ovarian tissue after fragment processing can block Hippo signal pathway or not.After the treated ovarian tissue was cultured in vitro by AKT excitant,the fragments were cultured in vitro,we transplant it to the dorsal broad muscle of nude mice,and investigate whether it can activate follicle development.Materials and methodsIn this research,ovarians were collected from transgender patients(female to male)originated from department of plastic surgery in our hospital or from ovarian resection patients due to many problems other than ovarian problems in gynaecology and obstetrics department and the obtained fresh ovarian tissue were vitrification frozen.A period of time later the frozen ovarian tissue were frozen-thawed,and the HE dyeing method was used to compare the ovarian tissue morphology before and after frozen.Then the frozen-thawed ovarian were exposed to fragmentation and immunohistochemical method was used to detect the YAP protein orientation changes and CCN3 expression levels before and after fragmentation treatment.Besides,the mRNA expression levels of CCN and BIRC were detected by RNA extraction,reverse transcription and qPCR method at different time points after fragmentation.Furthermore,Western blotting analysis was used to detect the protein levels of phosphorylated YAP and CCN.At last,the fragmented ovarian tissue were cultured with AKT stimulation in vitro.After that the tissue was transplanted to the latissimus dorsi of nude mice and the development of follicular was detected.Results1.The ovarian tissue has no obvious morphological changes before and after vitrification freezing.The HE dyeing results showed that both the fresh and the vitrification froze ovarian section contains ovarian stromal cells which are arranged in spindle,and the primordium follicular scattered in the former,which contains clearly visible circular follicular oocytes.The oocytes were surrounded by a layer of spindle cells-the particles.There is no obvious morphological difference between the fresh and the vitrification froze ovarian.2.The fragmentation of ovarian tissue can decrease the p-YAP protein levels,and increase the YAP protein levels in the cell nucleus of particle cells.The frozen thawed ovarian tissue were fragmented and the protein levels of YAP and p-YAP of the ovarian tissues before and after fragmentation were detected by western blotting analysis.The result showed that the p-YAP protein levels is significantly lower 1 hour after treatment in ovarian than before treatment,and there was no significant changes in YAP protein before and after treatment.Accordingly,p-YAP/YAP ratio decreased obviously.At the same time,the immunohistochemical analysis was used to detect the the YAP protein location changes in particles cells of ovarian tissues before and after treatment and the results showed that YAP protein content was increased in the nuclei of particle cells.3.The expression levels of CCN growth factors and BIRC apoptosis inhibiting factors were increased after the fragmentation of ovarian tissue.The mRNA expression levels of CCN and BIRC were detected by RNA extraction,reverse transcription and qPCR method at different time points after fragmentation and the results showed that the expression levels of CCN2,CCN3 and BIRC1,BIRC7 were increased,with statistical significance.Western blotting analysis were used to detect the CCN3 protein level changes before and after treatment,suggesting the CCN3 protein level was significantly increased after fragmentation treatment.At the same time,the immunohistochemical results suggested a similar trend,that CCN3 expression levels was increased significantly after fragmentation treatment.4.Applying AKT stimulation to the in vitro fragmented ovarian tissue cultivation system can increase the p-AKT expression levels,no obvious necrosis was detected after transplanting the ovarian tissue to the latissimus dorsi of nude mice.The fragmented ovarian tissue were cultured with AKT stimulation in vitro,and western blotting analysis was used to detect the p-AKT protein levels,and the results showed that the expression levels of p-AKT was significantly increased in the AKT stimulation group.The fragmented ovarian tissue were cultured with AKT stimuli in vitro.After that the tissue was transplanted to the latissimus dorsi of nude mice and new vessels was detected by HE staining.Conclusion1.Ovarian tissue vitrification freezing can be used for fertility preservation;2.The fragmentation process human ovarian tissue may block the Hippo signaling pathway;3.AKT stimulation in the in vitro fragmented ovarian tissue cultivation system can increase the p-AKT expression;4.Ovarian tissue can survival after transplanted to the latissimus dorsi of nude mice by fragmentation and AKT stimulation. |
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