Effect Of Human Amniotic Epithelial Cells On Ovarian Function,fertility And Ovarian Reserve In Primary Ovarian Insufficiency Rats And Analysis Of Underlying Mechanisms By Mrna Sequencing

Every year,millions of women are diagnosed with cancer worldwide,of which about 10% are in their reproductive years.The application of chemotherapy has significantly improved the survival rate of cancer patients,and about 90% patients with early-diagnosed cancer can survive after treatment.However,the risk of long-term complications due to chemotherapy cannot be ignored.Among them,early menopause and infertility secondary to primary ovarian insufficiency(POI)or premature ovarian failure(POF)after chemotherapy greatly affect their lives.Therefore,protecting ovarian function and fertility of these patients has become a common issue of physicians and patients.Animal experiments and clinical studies have showed that many chemotherapy drugs can damage ovarian function or structure,cause ovarian fibrosis,and affect the initiation,development and maturation of follicles,however,the specific mechanism remains unclear.Current approaches for female fertility preservation when exposure to chemotherapy drugs include hormone replacement therapy(HRT),gonadotropin releasing hormone analog therapy and fertility cryopreservation.HRT can only relieve perimenopausal symptoms but not restore ovarian function and fertility.It is now clear that gonadotropin releasing hormone analogs does reduce the incidence of POI in women with breast cancer in clinic studies.Cryopreservation technology is restricted by patient age,marital status,etc.Therefore,each method has its limitations and cannot be widely used in clinical practice.With the rise and development of regenerative medicine,stem cells play an important role in tissue repair and regeneration which have been used in a variety of diseases.Until now,several studies have indicated that stem cell transplantation has a certain role in improving ovarian function,repairing ovarian tissue structure and promoting fertility,but its mechanism is still unclear.Nowadays,the discovery and research of human amniotic epithelial cells(h AECs)have guided new directions for regenerative medicine.As amniotic membranes are medical waste,rich in sources,easy to obtain,and without ethical constraints.Moreover,h AECs exhibit stem cell characteristics,low immunogenicity and no tumorigenicity,which have broad application prospects in the field of regenerative medicine and are promising new method to repair ovarian insufficiency caused by chemotherapy.According to previous reports,h AECs can secrete a variety of cytokines,which may be involved in the process of proliferation,immune regulation,apoptosis and angiogenesis,and can improve ovarian function by intravenous transplantation,in situ injection or intraperitoneal injection of h AECs or h AECs conditioned medium,but the underlying mechanism remains unclear.Part one: isolation,culture,identification and characterization of h AECsObjective To study the extraction and biological characteristics of h AECs.Methods(1)Amniotic membranes were collected from pregnant women who underwent uncomplicated caesarean sections after testing negative for HIVI,hepatitis B and C with written and informed consents,h AECs were obtained by trypsin digestion method;(2)To identify the isolated cells,the cell morphology was observed by optical microscope and the expression of h AECs surface markers was detected by flow cytometry;(3)FSH receptor(FSHR)expression of HAECs was detected by cellular immunofluorescence.Results(1)The isolated h AECs were paving stone like in morphology,the cells were uniform in size and morphology,round or polygonal,and colony-like growth;after 4 passages,the cells gradually became spindle shaped,and cell proliferation slowed down;(2)The h AECs were highly expressed stem cell markers(SSEA4,Nanog),epithelial cell markers(CD324,CD326);while lowly expressed mesenchymal markers(CD105),hematopoietic stem cell markers(CD34,CD45),and immunologic(HLA-DR)marker;(3)A portion of h AECs were positive of FSHR which was well known as a specific marker of ovarian granulosa cells.Conclusion h AECs have abundant sources,show characteristics of stem cells and epithelial cells,with low immunogenicity and tumorigenicity,thus have broad application prospects in the field of stem cell therapy.Part two: Study on establishment of CTX induced ovarian injury in ratsObjective To establish a stable cyclophosphamide(CTX)induced ovarian injury model in rats.Methods 8-10 weeks old SD rats with regular estrous cycle were randomly divided into 4 groups and the first intraperitoneal injection of CTX or saline was recorded as the first day(D1): control group(D1-D15 0.9% saline);low dose CTX treatment group(D1 50 mg / kg,D2-D15 8mg / kg);middle dose CTX treatment group(D1 100 mg / kg,D2-D15 8mg / kg);high dose CTX treatment group(D1 200 mg / kg,D2-D15 8mg / kg);rat blood samples,vaginal smears and ovarian tissue were obtained for the subsequent experiments.(1)The general state of rats was observed,the body and ovarian weight were recorded,the survival curve was drawn;(2)The estrous cycles were obtained to assess the toxic effects of CTX on rat ovaries;(3)The serum estradiol(E2)level was detected 3 days and 2 weeks after the end of chemotherapy by chemiluminescence method to analyze the effect of CTX on endocrine function in rats;(4)The serum AMH and FSH levels were measured 2 weeks after CTX exposure by Elisa method to assess the effect of CTX on endocrine function in rats;(5)The histological changes and follicle counts of ovaries were observed and recorded to evaluate the damage of ovarian histology by CTX after HE staining;Results(1)The rats in the low dose CTX treatment group showed lower body weight and ovarian weight,and the mortality was low.The rats in the middle and high dose CTX treatment group had high mortality rates.Therefore,low dose CTX treatment strategy was used to construct the model of ovarian insufficiency;(2)Serum E2 level in the model group was lower than that in the control group 3 days after the end of chemotherapy,and decreased significantly 2 weeks after the end of chemotherapy;(3)Compared with the control group,the serum AMH levels of the rats in the model group decreased significantly 2 weeks after the end of chemotherapy,while the FSH levels of the rats in the model group increased significantly 2 weeks after the end of chemotherapy;(4)The rats in the model group and the control group had regular estrous cycles before chemotherapy.One-week exposure to CTX,about fifty-four percent of the rats in the model group had abnormal estrous cycles.However,forty-six percent of the model rats still showed abnormal estrous cycles one week after CTX injection.The control group rats had normal estrous cycle during this process;(5)CTX administration led to obvious ovarian damage and ovarian fibrosis.Compared with the control group,the preantral follicles and antral follicles in the model group were significantly reduced,and the atretic follicles were significantly increased.Conclusion Intraperitoneal injection of low dose cyclophosphamide(D1 50 mg / kg,D2-D15 8mg / kg)can establish a stable model of ovarian insufficiency in rats.Part three: Effectiveness study of h AECs in repairing ovarian damage,promoting fertility in POI ratsObjective To study the effectiveness of h AECs in repairing ovarian damage,promoting fertility in POI rats and the preliminary mechanisms.Methods The first generation of h AECs was successfully labeled with PHK26 fluorescent dye for in vivo tracking of h AECs transplantation.SD rats aged 8-10 weeks were included in the experiment.The rats were randomly assigned to 4 groups,and the first intraperitoneal injection of CTX or saline was recorded as the first day(D1).Control group(D1-15 intraperitoneal injection of 0.9% saline,D16 tail vein injection of PBS and in situ ovarian injection of PBS);model group(D1-15 intraperitoneal injection of CTX,D16 tail vein injection of PBS,in situ ovarian injection of PBS);intravenous h AECs injection group(D1-15 intraperitoneal injection of CTX,D16 tail vein injection of h AECs and in situ ovarian injection of PBS);ovarian h AECs injection group(D1-15 intraperitoneal injection CTX,D16 tail vein injection of PBS and in situ ovarian injection of h AECs).Rat blood samples and ovaries were collected 2 weeks after cell transplantation.Vaginal smears were done before CTX injection,during CTX injection and after cell transplantation.(1)The general state of the rats was observed,and the weight of body and ovary was recorded;(2)The survival and distribution of PKH26 labeled h AECs in the ovaries 24 hours and 2weeks after cell transplantation were checked by frozen sections of ovaries;(3)The serum AMH and FSH levels were measured by Elisa method to analyze the effect of h AECs transplantation on ovarian endocrine function in rats;(4)Vaginal smears were applied to detect changes in the estrous cycle of rats,and to assess the effect of h AECs transplantation on the integrity of the hypothalamus-pituitary-gonadal axis;(5)Ovarian histological changes were observed,and follicle counts were performed to evaluate the effect of h AECs on repairing ovarian structure in rats;(6)The distribution and expression of AMH,FSHR and Klotho were detected in the ovaries by immunohistochemical method,to explore the role of h AECs transplantation on proteins that are important for follicular growth and development,and to preliminarily explore their possible mechanisms;(7)After mating test,implanted fetus number was recorded to assess the effect of h AECs transplantation on rat fertility.Results(1)Twenty-four hours after cell injection,the labeled cells were mainly detected in the interstitial area of ovaries rather than in follicles both in the intravenous and in situ h AECs groups.Moreover,the red fluorescent signal could still be weakly observed 2 weeks after cell transplantation in these groups;(2)Both intravenous or in situ injection of h AECs can increase the body and ovary weight of POI rats to some extent;(3)CTX injection caused irregular estrous cycles because of ovarian toxicity while h AECs transplantation could facilitate decreased the irregular estrous cycles;(4)After h AECs treatment,serum AMH level was significantly higher and FSH level was significantly lower in the intravenous and in situ injection h AECs group than that in the model group;(5)Following h AECs transplantation,there were more atretic follicles in the model group than those in the treatment group.In addition,there were fewer preantral follicles(primordial follicle,primary follicle)in the model group than those in the h AECs treatment group.Similarly,there were more antral follicles(secondary follicle,mature follicle)in treatment group than those in the model group;(6)Compared with the model group,h AECs transplantation increased ovarian expression of AMH,FSHR and Klotho proteins,which played important roles in regulating follicle fate;(7)Number of implanted fetuses in the intravenous h AECs injection group,ovarian h AECs injection group,and model group was significantly lower than that in the control group.However,the fetuses in the intravenous h AECs injection group were significantly higher than in the model group while a slight increase was observed in the direct ovarian injection group compared with the model group.Conclusion h AECs transplantation can repair the ovarian function and structure induced by cyclophosphamide,increase the number of preantral/antral follicles,and reduce the number of atresia follicles;h AECs intravenous transplantation can promote fertility;h AECs transplantation upregulates ovarian expression of AMH,FHSR and Klotho which may be involve in preventing overactivation of primordial follicles and promoting the development and maturation of growing follicles.Part four: Mechanism study of h AECs in repairing ovarian damage in ratsObjective To explore the potential mechanisms of h AECs in repairing ovarian damage due to CTX.It is hoped to provide relevant theoretical basis for the treatment of ovarian injury caused by chemotherapy drugs,and to provide new ideas and strategies for clinically preservation of ovarian function.Methods After the feasibility of h AECs in repairing ovarian damage caused by cyclophosphamide in rats has been confirmed,RNA SEQ was carried out to sequence 3 groups of ovarian tissues,each group containing 3 rat ovaries.Three groups including:intravenous h AECs injection group,ovarian h AECs injection group and model group.(1)Total RNA extraction,reverse transcription,double-stranded c DNA synthesis,amplification and sequencing of ovarian tissue,bioinformatics analysis of differentially expressed genes(DEGs)in the h AECs treatment group and model group,and GO and KEGG analysis to understand the function and involved pathways of DEGs were performed orderly;(2)RT-q PCR was applied to validate some differentially expressed genes;(3)The protein levels of steroid hormone synthesis pathway were detected by immunohistochemistry and western blotting,to explore the underlying mechanism of h AECs treatment from the gene and protein levels.Results(1)Differentially expressed genes between groups were screened out according to the adjusted p value and fold change of gene expression.Totally,783 DEGs were found between intravenous h AECs and model group,including 619 upregulated and 164 downregulated genes.Similarly,116 upregulated DEGs and 52 downregulated DEGs were obtained between in situ h AECs and model group;(2)GO analysis showed that the DEGs(intravenous h AECs group vs model group)were mainly enriched in channel activity and transmembrane transporter activity while the DEGs(in situ h AECs group vs model group)were significantly enriched in sterol biosynthetic process and sterol metabolic process.KEGG analysis displayed that the DEGs(intravenous h AECs group vs model group)were significantly enriched in ribosome,protein digestion and absorption,neuroactive ligand-receptor interaction and c AMP signaling pathway;whereas the DEGs(in situ h AECs vs model group)were mainly participated in steroid biosynthesis pathway;(3)Moreover,4 DEGs simultaneously changed both in intravenous h AECs group and in situ h AECs group compared with model group,including 2 upregulated and 2 downregulated genes.Four commonly expressed genes were verified by real-time PCR analysis.IRF7 and Mx1 were remarkably upregulated both in intravenous h AECs and in situ h AECs group compared with the model group,which is consistent with the m RNA screening result.However,the other 2 simultaneously DEGs were not significantly changed as the m RNA sequencing analysis;(4)Eight enzymes significantly enriched in steroid biosynthesis pathway were confirmed by real-time PCR,immunohistochemical analysis and western blotting.Five(Msmo1,SQLE,NSDH1,FDFT1,TM7SF2)of them were significantly upregulated in the in situ h AECs group while 3(DHCR24,CYP51,HSD17B7)of them were still increased compared with the model group by real-time PCR analysis.FPKM values of Star,Cyp11a1,Cyp19a1 and Hsd17b1,which are key enzymes for estrogen and progesterone production,were also increased in the in situ h AECs group compared with model group by m RNA sequencing result.Besides,the expression of VEGFR1,VEGFR2 and VEGFR3 were also observed among 3 groups.VEGFR1(in situ h AECs group)and VEGFR2(intravenous h AECs group)expression were significantly increased compared with the model group respectively while no difference of VEGFR3 was noticed among 3 groups;(5)SQLE and FDFT1,2 members of steroid biosynthesis pathway,were mainly expressed in granulosa cells and dramatically increased in the ovarianh AECs treatment group compared with the model group by immunohistochemical and western blotting analysis.Conclusion h AECs transplantation can increase the ovarian expression of AMH,FSHR and KL which played important roles in preventing overactive of primordial follicles and maintaining the development of growing follicles;Finally,high-throughput sequencing showed that h AEC transplantation had an effect on ribosomes,protein digestion,protein absorption,neuroactive ligand-receptor interaction,c AMP signaling pathway and steroid biosynthesis pathways,which are involved in steroidogenesis,follicle development and ovulation.h AEC treatment also increased the expression of the vascular and anti-inflammatory factors IRF7,Mx1,VEGFR1 and VEGFR2,which participate in angiogenesis and inflammation.