Study On The Status Of Time To Pregnancy And Its Association With Vaginal Microbiome Based On The Pregnancy-attempting Cohort

Background and Objectives:With the aging of the population and the increasing prevelance of female infertility,the problems of reproductive health and the reduction of human fecundability have been widely concerned by the international community.Public health scholars proposed an indicator of "time-to-pregnancy"(TTP),which reflects the time taken by a couple for attempting their pregnancy.The shorter the TTP,the higher the fecundability among population.Recent studies have shown that TTP is associated with the age of couples,female blood lipid levels,diabetes mellitus and related complications.In addition,reproductive tract infection is considered to be one of the major factors causing female infertility,but most of the previous studies were designed based on case-control studies and only focused on the pathogenic bacteria,however,the structure of the normal vaginal microbiome and its association with fecundability have not been elucidated.Meanwhile,the psychological stress suffered by women before pregnancy may affect the outcome of pregnancy attempting.In order to objectively evaluate the balance state between "stress" and "individual adaptation",scholars put forward the "Allostatic load"(AL)index,which can reflect the multi-system physiological changes caused by the body’s response to stress.However,no association between AL and TTP has been reported.Therefore,this program carried out a cohort study of pregnancy-attempting couples,to comprehensively explor the influencing factors of TTP,using high-throughput sequencing technology to reveal the structure and potential function of female vaginal microbime,and elucidate its association with TTP.At the same time,AL index was used to explore the effect of psychological stress on fecundability of women.It provides a solid foundation for further promoting the early prediction,evaluation and intervention of fertility,and provides a new idea for the development of targeted health management programs for pregnancy-attempting couples.Methods:1.Based on the National Free Pre-pregnancy Checkups Project(NFPCP)database,the TTP of54737 couples in Jiangsu Province from 2015 to 2018 was evaluated.The vaginal microenvironments were evaluated through using female leucorrhea examination indices(vaginal cleanliness,cue cell,pH value,ammonia odor test,etc.)and reproductive tract pathogenic bacteria detection indices.The association between female vaginal microenvironment and TTP was explored by propensity score matching method.Cox proportional risk model was used to analyze the influencing factors of TTP in combination with the basic demographic characteristics,physical examination and lifestyle factors of the couples.On this basis,the prediction model of one year pregnancy rate for couples was established by using the method of histogram,and the model was evaluated.2.Under the premise of informed consent,454 eligible couples who are attempting for pregnancy in Nanjing were included.(1)Survey and collect the basic demographic characteristics,living habits,pre-pregnancy health examination and other information,collect female vaginal swabs and blood samples;(2)All subjects in the cohort were followed up for one year,with successful conception as the outcome.The time of confirmed pregnancy was recorded in detail,and TTP of the couples was evaluated.(3)Bacterial DNA was extracted from vaginal swabs,16 S rRNA gene V3-V4 region was sequenced on Illumina platform,and some samples were selected for metagenomic sequencing;(4)Bioinformatics method was used to annotate the sequencing results of vaginal microbiome,Qiime2 software was used to calculate the α diversity of the flora,principal coordinate analysis was used to reduce the dimension of the flora data,and PERMANOVA was used to compare the β diversity of the flora between groups according to the distance matrix between samples.Cluster analysis was used to explore the bacterial types of vaginal microbiome,and according to the dominant genus of different bacterial types,they were divided into different types of community structure.Lefse analysis was conducted to search for biomarker genus that might be different between groups,and to dig out different species and different functional genes.(5)The absolute abundance of five marker bacteria in the female vaginal microbiome was detected by q PCR technique,including Lactobacillus crispatus,Lactobacillus iners,Lactobacillus gasseri,Gardnella vaginalis,and Atopobium vaginae.(6)enzyme-linked immunosorbent method was used to test cortisol,noradrenaline,interleukin 6,C-reactive protein and high density lipoprotein cholesterol in blood samples.Combined with pregnancy health examination data,including body mass index,systolic pressure,diastolic blood pressure and fasting glucose index,to assess AL index.3.Statistical analysis methods: the mean ± standard deviation was used to describe the continuous data of normal distribution,and the independent sample t test was used for comparison between groups.Non-normal distribution continuous data were described by median(interquaternary interval),and comparison between groups was performed by rank sum test.The classification data were described by frequency(rate).Chi-square test or Fisher’s exact method were used for comparison between groups.Cox proportional risk model was used to explore the relationship between vaginal microbiome type,AL and TTP in women.Sequencing data annotation was realized with the help of Biamark cloud platform(www.biocloud.net).The remaining statistical analysis was performed by R software,and the test level α=0.05.Results:1.From 2015 to 2018,the median TTP of 54,737 couples in Jiangsu province was 5 months,and the average age of women was 27.41±4.52 years old.In general,the correlation between female age and fecundability has a plateau period(20-24 years old),and then the fecundability decreases with age,while the fecundability of male always decreases with age.Female with a history of stillbirth had lower fertility;However,female with a history of induced abortion had higher fertility;Couples with higher blood pressure before pregnancy,as well as couples who regularly drink alcohol,have lower fecundability.Based on these factors,preliminary prediction model for one-year pregnancy rate was built,and the discrimination effect of this model was0.706.2.The fecundability of women with poor vaginal microenvironment(without the diagnosis of bacterial vaginitis)decreased by 8%(Fecundability Ratio,FR=0.92,95%CI: 0.84-0.99).Fecundability was reduced by 18% in women with vaginal cleanliness of grade IV(FR=0.82,95%CI: 0.73-0.91).Those who reported "relatively high/great" life/work stress before pregnancy showed a decrease in fecundability by 41%(FR=0.59,95%CI: 0.41-0.84).There was an interaction between the vaginal microenvironment and the life stress on the fecundability of women.Women with poor vaginal microenvironment and high life stress had the greatest decline in fecundability.3.After the cohort follow-up,there were 214 couples achived pregnancy,accounting for47.14%.There were significant differences in the pre-pregnancy structure of vaginal microbiome between the two groups of pregnant or non-pregnant women.The abundance of Lactobacillus crispatus in the pregnant group was higher,while the abundances of Gardnella vaginalis and Atopobium vaginae in the non-pregnant group were higher.Functional analysis of the flora indicated that the metabolic pathways of phenylalanine,phosphoinositol and arachidonic acid in the vaginal microbiome of non-pregnant women were up-regulated,while the pathways of lactose metabolism were down-regulated,which may be one of the reasons for the effect of vaginal microbiome on fecundability.4.Through cohort validation,it was found that the absolute abundance of Lactobacillus gasseri in the vagina was positively correlated with fecundability,while the abundance of Atopobium vaginae was negatively correlated with fecundability.Compared with the female vaginal microbiome type A(dominated by three kinds of Lactobacillus,with lower abundances of Gardnella vaginalis and Atopobium vaginae),the women with type D(dominated by Lactobacillus iners,while the other four kinds of bacteria abundance lower)had a significant lower fecundability(FR = 0.45,95% CI: 0.26-0.76).5.The fecundability of women with higher AL index reduced to a certain extent.The fecundability of women with AL index in group 5-6 was reduced by 59% compared with that in group 0(FR=0.41,95%CI: 0.20-0.82).There was an interaction between AL and vaginal microbiome on fertility.Women with greater AL and "poor" vaginal bacteria type had a greater decrease in fecundability.Conclusions and Recommendations:1.The fecundability of couples attempting for pregnancy is associated with several health indicators before pregnancy,including basic demographic characteristics,physical examination results and life habits factors,etc.These factors can be used to evaluate the fecundability of couples.It is necessary to improve the indicators of pre-pregnancy examination and improve the accuracy of fertility evaluation model.2.The structure and function of vaginal microbiome of women before pregnancy have potential influence on fecundability.Among them,Lactobacillus crispatus,Lactobacillus iners,Lactobacillus gasseri,Gardnella vaginalis,and Atopobium vaginae may be used as potential markers.According to the abundance of these 5 kinds of bacteria,vaginal microbiome can be classified.At the same time,the potential molecular mechanism of the effect of female vaginal flora on fecundability needs to be further explored.3.There was a correlation between pre-pregnancy women’s AL score and fecundability,and the women with high psychological pressure and "poor" vaginal microbiome type had the greatest fertility reduction.It is suggested that in the pre-pregnancy health examination,the vaginal microbime and allostatic load should be assessed pertinently,the early evaluation and intervention of fecundability should be promoted,and the effective health management strategy for pregnancy-attempting couples should be formulated.