| Part Ⅰ The influence of age on pregnancy outcomes of embryo transfer cycles with normal chromosomesBackgroud and Objective: China is facing the crisis of low birth rate and high aging proportion.The main reason is that the scale of women of reproductive age continues to decline.It is significant to provide medical support for advanced maternal age women who want to get pregnant.Traditionally,it is believed that the main reason of the fertility decline in advanced maternal age women is the increase of embryonic chromosomes abnormalities and gene abnormalities caused by the decline in both quantity and quality of oocytes.There are few studies on the changes in the maternal state of advanced maternal age women.Using preimplantation genetic testing(PGT)to select normal embryos for transplantation can artificially reduce the influence of embryo abnormalities on pregnancy outcome.This study collected all the PGT cycles in our hospital’s reproductive center,then investigated and analyzed the age,basic conditions,pregnancy outcomes and other terms.So that we can explore the relationship between age and the pregnancy outcome of embryo transfer cycles with normal chromosomes and discuss the current status of fertility changes in advanced maternal age women after abnormal embryos were excluded,then provide basis for clinical strategy and further mechanism research.Methods: Clinical information of all 2023 PGT cycles performed in the Women’s Hospital School of Medicine Zhejiang University between 2009 and 2020 was retrospectively collected and described.Logistic regression was used for univariate and multivariate analysis to analyze the influence of age and other factors on pregnancy outcomes.Result: In all PGT cycles that were collected,the indications of PGT are mainly chromosomal structural abnormalities(74.5%),and the types of PGT used are: PGT-A(8.7%)? PGT-M(8.6%)? PGT-SR(82.7%).A total of 961 cycles performed embryos transfer,of which the average age of the older age group(≥35 years)was 36.57±1.64 years old,and the average age of the young age group(<35 years)was 28.95±2.81 years old.The older age group had more previous pregnancy(2.43±1.57 vs 1.26±1.43;p<0.001),more previous birth(0.50±0.59 vs 0.12±0.35;p<0.001),and more previous abortions(0.50±0.59 vs 0.12±0.35;p<0.001)than the young age group.The older age group had higher FSH(6.76 vs 6.17 m IU/m L;p=0.001),lower PRL(14.83 vs 16.70 ng/m L;p=0.020),lower progesterone(1.40 vs 1.60 nmol/L;p=0.017),and lower testosterone(0.70 vs 0.90 nmol/L;p<0.001)than the young age group.The older age group had a higher proportion of patients suffered from fallopian tube diseases(35.5% vs 23.2%;p-0.003),endometriosis(11.3% vs 4.1%;p=0.001)and uterine diseases(27.4%)vs 10.3%;p<0.001)than in the young age group.As for pregnancy outcomes,the older age group had lower implantation rate(31.0% vs 40.6%;p=0.033),lower clinical pregnancy rate(31.5% vs 43.7%;p=0.010),lower live birth rate(25.8% vs.34.8%;p=0.049),and higher rate of delivery-related complications(14.3% vs 2.9%;p=0.016).Using Logistic regression for multivariate analysis,and it is found that after all the confounding factors were adjusted,advanced maternal age women were less likely to get pregnant(AOR=0.656,95%CI: 0.434-0.927,p=0.048)and less likely to give live birth(AOR=0.462,95%CI: 0.420-0.987,p=0.043)than younger women.Cycles using cryopreserved embryos were more likely to get pregnant(AOR=1.371,95%CI: 1.058-1.776,p=0.017)and more likely to give live birth(AOR=1.328,95%CI: 1.014-1.739,p=0.039)than cycles using fresh embryos.Conclusion: After using PGT to choose normal embryos for transfer,women over 35 years old(the older age group)had lower implantation rate,lower clinical pregnancy rate,lower live birth rate,and higher rate of delivery-related complications.It showed that the trouble of fertility establishment in advanced maternal age women still exists after ruling out embryo abnormalities and other confounding factors,suggested that changes in the maternal status of advanced maternal age women plays a key role in age-related fertility decline.Part Ⅱ Exploration of key signaling pathways and hub genes involved in dysfunction of decidua in older women based on transcriptome sequencingBackgroud and Objective: In PartⅠwe found out that advanced maternal age women still have trouble in establishment and maintenance of pregnancy after ruling out embryo abnormalities and other confounding factors.In consideration of previous animal experiments which found that elderly mice had decidualization disorders and placental dysplasia secondary to decidual dysfunction,we focused on decidual function abnormalities in advanced maternal age women.We wanted to investigate the influence of decidual function abnormalities on fertility with an exploration of the key signaling pathways and hub genes involved in dysfunction of decidua in advanced maternal age women.Methods: Decidual tissue and chorionic tissue of elderly patients with inevitable abortion and young patients with social abortion were collected in the Women’s Hospital School of Medicine Zhejiang University.Chromosome detection was performed on the chorionic tissue,then total RNA is extracted from the decidual tissue for transcriptome sequencing after excluding embryonic chromosomal abnormalities.Quality control,comparison,correction,and annotation were performed on the raw sequencing data.Multiple bioinformatics methods including differentially expression gene analysis,gene set enrichment analysis,protein-protein interaction network analysis,functional module analysis and subnetwork analysis were performed to explore the key signaling pathways and hub genes involved in dysfunction of decidua in advanced maternal age women.Single cell sequencing data from public accessible database was used to confirm the expression of hub genes in decidual stromal cells.Reverse transcription-quantitative real time polymerase chain reaction was performed to verify gene expression results.Result: The transcriptome sequencing results of 5 cases of the elderly group and 6 cases of the control group were included.Differential expression analysis identified 423 differentially expressed genes.The results of gene ontology enrichment analysis found out the overexpression of cadherin-related genes,and suggested abnormal peptide release and decidual cell survival disorder.The results of KEGG signal pathway enrichment analysis showed that the c AMP signal pathway were abnormal.Protein-protein interaction network analysis results showed that AGT,SST,CXCL1 are hub genes in the interaction network composed of many abnormally-expressed proteins in the decidua of advanced maternal age women.Analysis of single-cell sequencing data showed that the hub genes identified by transcriptome sequencing were all expressed in decidual stromal cells.The reverse transcription-quantitative real time polymerase chain reaction got results that were consistent with the trends of transcriptome sequencing analysis results.Conclusion: The dysfunction of decidua in advanced maternal age women with fertility decline is caused by multiple factors,including molecular events such as overexpression of cadherin and protocadherin,abnormal polypeptide release function,decidual cell survival disorder,abnormal contraction of spiral arteries,immune disorders,etc.Key signaling pathways such as c AMP signaling pathway and hub genes such as AGT,SST,CXCL1 may play an important role in decidualization and normal decidual function supposed key point of further study and possible target treatment. |
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