| Part Ⅰ Research on Characteristics of TCM Syndrome in infertile patients with Diminished Ovarian Reserve Objective To explore the characteristics of TCM syndromes and the distribution of syndromes in DOR infertile patients with IVF-ET assisted pregnancy,and to analyze the relationship between clinical data and syndromes,so as to provide dialectical reference for the application of Chinese medicine to improve the pregnancy outcome of IVF-ET infertile patients.Methods To collect the diminished ovarian reverse infertile patients who were treated with IVF in the Reproductive Center of Anhui Provincial Hospital from January2016 to December 2016.They were divided into 3 groups according to age: 20-35,36-40 and 41-45 years.Designing Syndromes integral questionnaire by literature research and expert consultation,analysis and comparison of syndromes composition ratio and integral situation of infertile patients in each group according to the results of syndrome investigation and the clinical pregnancy rate of DOR infertile patients with different syndromes was also compared and analyzed.Results1、Distribution of different age syndromes in infertile patients with DOR The distribution of TCM syndromes in DOR infertile patients of different ages was mainly liver depression.The difference of kidney yin deficiency syndrome,blood stasis syndrome and kidney yang deficiency syndrome was statistically significant between 20-35 years old and over 36 years old(P<0.05 or P<0.01),there was no significant difference between liver stagnation syndrome,spleen deficiency syndrome,phlegm dampness syndrome and others(P > 0.05).2、Comparison of syndromes of different ages in infertile patients with DOR The comparative results of TCM syndromes integral in three groups showed that there was no significant difference between the different age groups of liver stagnation syndrome,kidney yang deficiency syndrome,spleen deficiency syndrome,phlegm dampness syndrome and other syndromes(P>0.05).The over 41 years old infertile patients compared with under 35 years old,the TCM syndromes integral of kidney yin deficiency and blood stasis were higher,the difference was statistically significant(P <0.05).3、Distribution of complex syndromes at different ages in infertile patients with DOR Complex syndrome was the most common syndrome in infertile patients with DOR,the analysis and comparison of the composition ratio of TCM syndromes in different ages showed that there was no significant difference between kidney deficiency and liver depression,spleen and kidney deficiency and spleen deficiency phlegm dampness,and there was no significant difference between them(P > 0.05).But there was significant difference between 20-35 years old and 41-45 years old in the syndrome of kidney deficiency and blood stasis 、liver depression and blood stasis,kidney deficiency and liver stagnation and blood stasis(P < 0.05).The syndrome of kidney deficiency and liver depression and blood stasis was the main syndromes in the elderly infertile patients with DOR.4 、 Comparison of clinical pregnancy rate in infertile patients with different syndromes For DOR infertile patients under the age of 35 years old,the clinical pregnancy rate of liver depression is the highest and the kidney yin deficiency is the lowest,but the difference is not statistically significant(X~2=1.41,P=0.234>0.05).The clinical pregnancy rate of kidney yin deficiency syndrome,kidney yang deficiency syndrome and spleen deficiency syndrome was lower than that of liver stagnation syndrome,blood stasis syndrome and phlegm dampness syndrome,but the difference was not statistically significant(P < 0.05).But for those over 35 years old,the clinical pregnancy rate is low of kidney yin deficiency and kidney yang deficiency syndrome.Compared with kidney yin deficiency syndrome,only blood stasis syndrome,liver stagnation syndrome,phlegm dampness syndrome clinical pregnancy rate significantly increased.(P < 0.05).There was no significant difference in clinical pregnancy rate between kidney yang deficiency and other syndromes(P>0.05).Conculsion1.Kidney deficiency and liver depression is the most common syndrome of DOR infertile patients,followed by kidney yin deficiency syndrome,blood stasis syndrome,kidney yang deficiency syndrome,spleen deficiency syndrome and phlegm dampness syndrome from high to low.2.The distribution of TCM syndromes in infertile patients with DOR of different ages has a high proportion of liver depression syndrome.The proportion of kidney yin deficiency,blood stasis and kidney yang deficiency syndrome in elderly patients with DOR was significantly higher than that in young patients with DOR.3.Compound syndromes were more common in infertile patients with DOR,common complex syndromes include kidney deficiency and liver stagnation,kidney deficiency and blood stasis,liver stagnation and blood stasis,kidney deficiency and liver depression and blood stasis,spleen and kidney deficiency,spleen deficiency and phlegm dampness,etc.Compared with young DOR,the ratio and syndrome integral of kidney deficiency and blood stasis syndromes increased significantly in elderly infertile patients.With the increase of age,kidney deficiency,blood stasis ratio and syndromes integral showed an increasing trend.4.There is no significant difference in clinical pregnancy rate of IVF with different syndromes in DOR infertile patients aged less than 35 years.But the clinical pregnancy rate of senile DOR with kidney yin deficiency syndrome was significantly lower than that of liver stagnation syndrome,blood stasis syndrome and phlegm dampness syndrome.The pregnancy rate of deficiency syndrome was lower than that of sthenia syndromes.Keywwords: Diminished ovarian reserve(DOR);TCM syndrome;In vitro fertilization and embryo transferPart Ⅱ Research on Pregnancy Outcome and its Mechanism of IVF in DOR infertile patients of Different Ages Objective To explore the difference of IVF pregnancy outcome in DOR infertile patients of different ages and to provide evidence for prognosis of DOR infertile patients.At the same time,transcriptome sequencing technique was used to analyze the changes of m RNA expression profile of ovarian granulosa cells in infertile patients with DOR of different ages,and to explore its mechanism at gene level.Methods1.Retrospective analysis of clinical data Retrospective analysis of the clinical data of DOR infertile patients receiving IVF microstimulation program in reproductive center of Anhui Provincial Hospital from January 2015 to December 2016.According to age,they were divided into 3 groups: group A: 100 cases of 20-35 years old,group B: 118 cases of 36-40 years old,group C: 222 cases of 41-45 years old.The body mass index(BMI),infertile years,basic FSH、LH、E2 level,basic antral follicle number(AFC),the number of eggs obtained,the number of high quality embryos,embryo implantation rate,clinical pregnancy rate and so on were analyzed and compared among the infertile patients of different age groups.2.m RNA experimental study of granulosa cells(1)Ovarian granulosa cells were collected from 10 infertile patients under 35 years of age with normal ovarian reserve(a)and from 30 infertile patients with DOR of different ages.Among them,infertile patients with DOR were divided into three age groups according to their age:b1:≤35years old;b2:36-40 years old;b3:41-45.(2)The total RNA of granulosa cells was extracted from infertile patients in each group,and m RNA c DNA library was prepared.The total RNA was sequenced and analyzed by RNA-sequencing and CPSS tools.(3)The m RNA expression profiles of granulosa cells in each group were detected,and the m RNAs of differential expression were com Pared.(4)Application of RT-qPCR to verify the expression level of differentially expression genes.Results1.Comparison of IVF pregnancy outcome in DOR infertile patients of different ages There was no significant difference in body mass index(BMI),infertile years,basic FSH 、 LH 、 E2 and basic antral follicle count(AFC)in infertile patients of different ages(P > 0.05).There was no significant difference in the total dose and days of gonadotropin used,the number of embryos available,number of high-quality embryos,rate of cycle cancellation too.The embryo implantation rate,clinical pregnancy rate and live delivery rate were different between the two groups,but there was no significant difference between the two groups(P > 0.05).But the embryo implantation rate,clinical pregnancy rate and live birth rate in group C were significantly decreased,while the rate of abortion was significantly increased(P <0.01).2.Experimental study on granulosa cell mRNA Comparative analysis of m RNA expression in granulosa cells of each group by RNA-sequencing,599 up-regulated genes and 724 down-regulated genes were screened.Comparison of group a with group b1、group b2 and grou P b3,3、188、284differentially expression genes were screened respectively.There were 1 Coexpression of differentially genes between a VSb1、a VSb2 and a VSb3(1VS2,1VS3,1VS4),while There were 11 Coexpression of differentially genes between b1VSb2、b1VSb3 and b1VSb3(2VS3,2VS4,3VS4).It mainly includes the down-regulated IGFBP5 、IGFBP3、INHBB、TNFSF10,etc.And the up-regulation of HLA-DR5.The results of RT-q PCR verification of differentially expression genes are consistent with those of sequencing.Through the enrichment analysis of KEGG signaling pathway,it was found that the differentially expression genes were enriched into Wnt signaling pathway,complement and coagulation cascade,phagocytosome,phospholipase d signal pathway,chemokine signal pathway and platelet activation.protein digestion and absorption,cytokine receptor interaction,etc.Conculsion1.The embryo implantation rate,clinical pregnancy rate,live delivery rate of DOR infertile patients decreased with the increase of age,and the abortion rate increased with the increase of age.Compared with the elderly infertile patients with dor infertility,the young infertile patients with dor infertility still have a better clinical pregnancy outcome.2.The expression of m RNA was different between infertile infertile patients with normal ovarian function and infertile patients with DOR,there are also many differentially expression genes in DOR between different ages.The down-regulation of IGF、INH family and other related genes and the up-regulation of HLA-DR5 may be related to the occurrence and development of DOR.The down-regulation of SPRR2 F gene expression may be associated with the low clinical pregnancy rate in infertile patients with DOR.The down-regulation of SPARCL1、CITED1 and the up-regulation of TNFSF10 gene may be related to the difference of IVF pregnancy outcome in DOR infertile patients of different ages.These differentially expression genes are related to cell activation,immune system regulation,immune response,stress response,defense response,growth factor binding,protein complex binding,etc.Thus,TGF-β signaling pathway and cytokine receptor interaction affect the ovarian reserve function of infertile patients through Wnt signaling pathway,and participate in the occurrence and development of DOR. |
