| ObjectivesTo profile the current status of sleep and circadian rhythm disturbances in couples of childbearing age.To investigate the associations of sleep problems and infertility in couples of childbearing age;and to further explore the mediating effects of DNA methylation between sleep problems and infertility in females of reproductive age.MethodsPart 1:Based on the Reproductive Health of Childbearing Couples-Anhui Cohort（RHCC-AC）sub-cohort of newlywed couples cohort,and a total of 33 483 couples were recruited at their premarital health check-ups/preconception health examination project.Participants complete an electronic questionnaire（including the demographic,dietary and sleep behavior,and more）on the We Chat public account,and provide 10m L urine and 5 m L blood sample.All participants were followed up every 6 months until the occurrence of the primary endpoint（pregnancy ended or infertility）.According to the inclusion and exclusion criteria,30 729 couples of childbearing age were included in the final analysis.Qualitative data such as difficulty in falling asleep were described in terms of the number（constitution ratio）,while quantitative data such as BMI were expressed as mean±standard deviation（±s）;The comparison of sleep characteristics between couples was performed using theχ~2test.In order to explore whether sleep behavior and circadian rhythm disturbances affect infertility in couples of childbearing age,a 1:1 nested case-control study was established,and 407 couples of childbearing age were finally included.First,the conditional logistic regression method was used to investigate the effect of sleep problems on infertility in the total couples/wives/husbands,respectively.Secondly,an actor-partner interdependence model（APIM）was constructed by generalized estimation equation（GEE）to test the effect of sleep problems on infertility in couples.Finally,structural equation model（SEM）path analysis was used to further verify the effect of sleep problems on infertility.Part 2:Based on the RHCC-AC study included 2 sub-cohort（infertility cohort and newlywed couple cohort）.Infertility cohort recruited infertile couples who visited the reproductive center outpatient clinic,and collected biological samples after completing the electronic questionnaire.The pregnancy conditions and obstetric outcomes were followed up every 6 months.To further clarify the effects of sleep behavior and circadian rhythm disturbance on female infertility and male semen quality,a 1:1 case-control design was used.The cases were from infertility cohort of345 infertility female and 172 male with abnormal semen;the controls were from newlyweds cohort.χ~2test was used to compare characteristics between cases and controls,and the effect sizeφwas used to compare the effect of each sleep index on infertility.Conditional logistic regression was also used to analyze the effect of sleep characteristics on female infertility and male semen quality.Part 3:Based on the two sub-cohorts of the RHCC-AC study,a 1:1 case-control study was established.The studied group consisted of 43 infertile females with ovulation disorders as the case group and individually-matched 43 fertile females as the control group.DNA from blood samples was extracted,and DNA methylation analysis was performed using the Illumina Human 850 K EPIC Infinium Methylation Bead Chip.Theχ~2test and conditional logistic regression were used to explore the effect of female sleep characteristics on infertility.Ch AMP was used to perform quality control,standardization,and calculation of methylation sites and regions,and the associated loci were visualized with a Manhattan plot.Then,GO analysis and KEGG enrichment pathway analysis were performed on the obtained differential genes.and we compared the enrichment results and graphically displayed the top 10most significant enrichment results.Causal mediation analysis was applied to study the average causal mediation effect（ACME）and the average direct（unmediated）effect（ADE）of DNA methylation linking female sleep problems to infertility.ResultsPart 1:Of the 61 458 subjects,18.2%reported frequent snoring,and the reporting rates of husbands and wives were 28.9%and 7.4%,respectively.The reporting rate of husbands was significantly higher than that of wives,and the difference was statistically significant（χ~2=7790.06,P<0.01）.Approximately 32.2%of couples of childbearing age had excessive daytime sleepiness,and the reported rates of husband and wife were 27.7%and 36.6%,respectively.The reported rate of daytime sleepiness of wife was significantly higher than that of husband,and the difference was statistically significant（χ~2=559.63,P<0.01）.6.8%of the couples had circadian rhythm disturbances;The prevalence of sleep problem scores≥4 was 11.2%.Correlation analysis found that the husband’s sleep characteristics were positively correlated with the wife’s sleep characteristics.Conditional logistic regression analysis showed that the wife’s workday sleep midpoint delay（OR=1.70,P<0.05）,average sleep midpoint delay（OR=2.25,P<0.01）,no nap（OR=1.39,P<0.05）and sleep problem score≥4（OR=2.00,P<0.05）were risk factor for infertility of couple;husband’s work was shift（OR=1.65,P<0.05）and the weekend sleep duration is 7-8 h（OR=1.57,P<0.05）were risk factors for infertility of couple.GEE results showed that the wife’s workday sleep midpoint delay（OR=1.76,P<0.05）and average sleep midpoint delay（OR=2.29,P<0.01）were risk factors for infertility of couple.The husband’s work was shift（OR=1.75,P<0.05）and weekend sleep duration was 7-8 hours（OR=1.62,P<0.05）were risk factors for couple infertility.Part 2:The results of conditional logistic regression analysis showed that weekday sleep duration was≥8 h（OR=8.23,P<0.01）,weekend sleep duration was≥9 h（OR=3.39,P<0.01）,and the average sleep duration was≥8 h（OR=6.06,P<0.01）,large social jet lag（OR=2.29,P<0.05）,difficulty falling asleep（OR=4.15,P<0.01）and circadian rhythm disturbance（OR=3.34,P<0.05）were risk factors for female infertility.Weekday sleep duration<7 h（OR=2.46,P<0.01）,weekday sleep duration of 7-8 h（OR=2.06,P<0.05）,and average sleep duration<7 h（OR=2.36,P<0.05）and average sleep duration of 7-8 hours（OR=2.02,P<0.05）were risk factors for abnormal semen quality of male.Part 3:A total of 476 differentially methylated genes such as PER3 and CRY2 were screened,in which 763 differentially methylated sites,and 60 groups of differentially methylated regions were identified through 850 K Illumina Infinium Methylation EPIC Bead Chip array data analyses.The results of GO and KEGG enrichment analysis found that biological processes mainly involve neuron development,hormone secretion and circadian rhythm regulation,etc.,and the cellular components mainly involve neuronal cell body and plasma membrane protein complex,etc.,the molecular functions mainly involve phosphatidylinositol binding and neurotransmitter transporter activity,etc.;also involve pathways such as endocytosis and focal adhesion.Causal mediation analysis found that a total of 3 methylation sites（including cg07675898,cg08298632 and cg15500907）can be used as mediator connecting female difficulty falling asleep and infertility.At the same time,33methylation sites（including cg01535674,cg14254882 and cg0216771733）can be used as mediator connecting female sleep duration and infertility.ConclusionsSleep problems are widespread within couples of childbearing age,and the sleep characteristics between couples are unique and interdependent.wife’s workday sleep midpoint delay,lack of naps,and multiple sleep problems may be risk factors for couple infertility.The husband’s work type is shift,and short sleep duration on weekends may be risk factors for couple infertility.Longer sleep duration,greater social jet lag,difficulty falling asleep,and circadian rhythm disturbances are associated with female infertility.Short workday and mean sleep duration in males were associated with poorer semen quality.The sleep characteristics of women with different types and causes of infertility are unique.For example,difficulty in falling asleep in females was associated with primary infertility,Longer average sleep duration in females was associated with secondary infertility,while females with circadian rhythm disturbances and prolonged weekend sleep duration were associated with unexplained and ovulatory infertility.There were not only different methylation sites but also different methylation regions in infertile women compared with the control group.The sterility related genes identified in this study are mainly involved in biological processes and molecular functions such as neuronal development,hormone secretion,circadian rhythm regulation and neurotransmitter transporter activity.Our study also found that difficulty falling asleep and sleep duration may affect female infertility through DNA methylation levels. |
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