Associations Of Exposure To Disinfection Byproducts With In Vitro Fertilization And Embryo Transfer Outcomes And The Mediating Role Of Ovarian Reserve

Reproductive health is the foundation for human flourishing and also is an important guarantee for the sustainable development of the economy and society.According to the World Health Organization(WHO),infertility and sterility will be followed by cancer and cardiovascular diseases as the third-most serious disease worldwide in the 21st century.Assisted reproductive technology(ART)has become the most effective treatment method for the infertility and sterility.It has been shown that there is an increasing trend in the numbers of ART cycles with the increasing occurrence of infertility and sterility worldwide.However,the certain indicators of in vitro fertilization and embryo transfer(IVF-ET)outcomes such as successful rates of clinical pregnancy and live birth per ART cycle remain still around 30%.Therefore,exploring potential risk factors for IVF-ET outcomes has been a public health issue in the field of clinical and public health,which has important implications for taking targeted preventive measures to improve the quality of pregnancy outcomes and guarantee the health of newly-born population.Recent studies have shown that exposure to environmental pollutants with reproductive and developmental toxicity may adversely affect IVF-ET outcomes.Disinfection by-products(DBPs)are inadvertent widespread contaminants in drinking water that are formed when oxidizing disinfectants react with natural and synthetic organic matter during the treatment of drinking water.DBPs can be detected in human biological samples such as blood,urine and exhaled breath.Among those,urinary trichloroacetic acid(TCAA)and dichloroacetic acid(DCAA),the two most abundant haloacetic acids(HAAs)class,have been suggested as candidate biomarkers for chronic ingestion of drinking water DBPs.Experimental studies have shown that DBPs can interfere with reproductive hormone levels,inhibit follicle growth and development,reduce IVF ability,induce abnormal embryo development,and lead to adverse pregnancy outcomes.However,there is a paucity of epidemiological studies on DBP exposures and female reproductive health,and also no epidemiological studies have examined the effects of DBP exposures on IVF-ET outcomes.Furthermore,the mechanisms of action underlying the associations between exposure to DBPs and adverse IVF-ET outcomes remain unclear.Therefore,we recruited women who presented to the Reproductive Medicine Center of Tongji Hospital in Wuhan,Hubei Province to seek ART treatment.We measured trichloroacetic acid(TCAA)and dichloroacetic acid(DCAA)concentrations in urine samples as biomarkers of DBP exposures.Total oocytes,total mature(MII)oocytes,the rates of fertilization,the rates of good-quality embryos,implantation,clinical pregnancy,and live birth were used as indicators of IVF-ET outcomes.We examined the associations between DBP exposures and IVF-ET outcomes.In addition,we measured antral follicle count(AFC)and basal serum reproductive hormones as markers of ovarian reserve and examined the associations of DBP exposures with ovarian reserve.Furthermore,we explored the mediating role of ovarian reserve in the associations between DBP exposures and IVF-ET outcomes.Our results would provide the evidence for adverse reproductive and developmental health outcomes exerted by DBP exposures and the related mechanisms of action,as well as a reference for taking preventive measures to improve female fecundity and IVF-ET pregnancy quality.Part One Associations between exposure to DBPs and IVF-ET outcomesObjectives:To evaluate the associations between exposure to DBPs and IVF-ET outcomes,using repeated urinary DCAA and TCAA concentrations as internal biomarkers.Methods:A total of 1048 women were selected from the Tongji Reproductive and Environmental(TREE)study,an ongoing cohort recruiting women who presented to the Reproductive Medicine Center of Tongji Hospital in Wuhan,Hubei Province to seek ART treatment during December 2018 and January 2020 and were followed up to fetal birth outcomes.The concentrations of DCAA and TCAA in the urine samples as a biomarker of ingestion of DBP exposures were measured by liquid-liquid extraction and gas chromatography.Total oocytes,total MII oocytes,the proportion of fertilization and goodquality embryos,implantation,clinical pregnancy,and live birth were assessed as indicators of IVF-ET outcomes.Generalized linear mixed models were applied to evaluate the associations of urinary DCAA and TCAA concentrations with IVF-ET outcomes.In addition,stratified analyses were also performed to explore whether age,body mass index(BMI),treatment protocol,fertilization protocol,and type of embryos transferred modified the associations between urinary HAA concentrations and IVF-ET outcomes.Results:The mean age and BMI of the participants in this part of the study were 30.96 years and 22.17 kg/m2,respectively.The 1048 women underwent a total of 1136 IVF cycles,and more than half of the women used long gonadotropin-releasing hormone(GnRH)agonist as treatment protocol(57.13%),IVF as fertilization protocol(64.88%),and frozen-thawed embryos transferred as type of embryos transferred(53.17%).The geometric mean(interquartile range)specific gravity-adjusted concentrations were 6.06(4.58-7.53)μg/L for DCAA and 5.34(4.02-6.84)μg/L for TCAA,respectively.After adjustment for age,BMI,infertility causes,education level,household income,passive smoking status,and alcohol use,elevated urinary DCAA concentrations were negatively associated with the numbers of total oocytes and MII oocytes,the proportion of good-quality embryos,as well as the odds of successful implantation,clinical pregnancy,and live birth.Compared with women in the lowest quartile of urinary DCAA concentrations,women in the highest quartile had decreases of 12.00%(RR=0.88;95%CI:0.79,0.97;P for trend<0.01)in total oocyte numbers,14.00%(RR=0.86;95%CI:0.78,0.96;P for trend<0.01)in total MII oocyte numbers,and 20.00%(RR=0.80;95%CI:0.62,1.03;P for trend=0.05)in the proportion of good-quality embryos,as well as had 36.00%(RR=0.64;95%CI:0.44,0.94;P for trend=0.02),36.00%(RR=0.64;95%CI:0.44,0.94;P for trend<0.01),and 31.00%(RR=0.69;95%CI:0.48,1.00;P for trend=0.04)lower probabilities of successful implantation,clinical pregnancy,and live birth,respectively.After adjustment for the same potential confounders,elevated urinary TCAA concentrations were negatively associated with the numbers of total oocytes and MII oocytes,as well as the odds of successful implantation and clinical pregnancy.Compared with women in the lowest quartile of urinary TCAA concentrations,women in the highest quartile had decreases of 15.00%(RR=0.85;95%CI:0.76,0.94;P for trend<0.01)in total oocyte numbers,and 13.00%(RR=0.87;95%CI:0.78,0.96;P for trend=0.01)in total MII oocyte numbers,as well as had 34.00%(RR=0.66;95%CI:0.45,0.96;P for trend=0.08)and 32.00%(RR=0.68;95%CI:0.47,0.99;P for trend=0.09)lower probabilities of successful implantation and clinical pregnancy,respectively.The stratified analysis showed that the negative associations of urinary TCAA concentrations with total oocytes and MII oocytes were stronger among women aged>35 years(all P for interactions<0.01,respectively).In addition,the negative associations between urinary HAAs concentration and IVF-ET outcomes were only observed among women with BMI<24 kg/m2,non-long GnRH agonist,IVF,and frozen-thawed embryos transferred,while there were not statistically signifcant differences(all P for interactions>0.10).Conclusions:Exposure to DBPs was inversely associated with total oocytes,total MII oocytes,the proportion of good-quality embryos,as well as successful implantation,clinical pregnancy,and live birth among women undergoing IVF-ET treatment.Part Two Associations between exposure to DBPs and ovarian reserveObjectives:To investigate the associations between exposure to DBPs and ovarian reserve measured as AFC and basal reproductive hormones.Methods:Based on the study population of part one,we additionally excluded those women who had not the available measurement data for indicators of ovarian reserve and were diagnosed with polycystic ovary syndrome,and so on,leaving 865 women in the current analysis.AFC and serum reproductive hormones including anti-Mullerian hormone(AMH),luteinizing hormone(LH),estradiol(E2)and follicle-stimulating hormone(FSH)were measured as indicators of ovarian reserve.Generalized linear models(GLM)were applied to estimate the associations of urinary DCAA and TCAA concentrations with ovarian reserve.In addition,stratified analyses were also performed to explore whether age and BMI modified the associations between urinary HAA concentrations and ovarian reserve.Results:There were no signifcant differences between the subgroup population of this part(n=865)and the overall population(n=1048)in demographic characteristics(all P>0.05).After adjustment for age,BMI,education level,household income,passive smoking status,and alcohol use,elevated urinary DCAA concentrations were negatively associated with AFC and AMH,but were positively associated with LH and E2.Compared with women in the lowest quartile of urinary DCAA concentrations,women in the highest quartile had decreases of 8.00%(95%CI:-13.00%,-3.00%;P for trend<0.01)in total AFC and 14.55%(95%CI:-27.82%,1.16%;P for trend=0.03)in AMH,but had increases of 8.09%(95%CI:-0.78%,17.76%;P for trend=0.10)in LH and 10.22%(95%CI:1.25%,19.98%;P for trend=0.01)in E2.After adjustment for the same potential confounders,elevated urinary TCAA concentrations were negatively associated with AFC and AMH,but were positively associated with FSH.Compared with women in the lowest quartile of urinary TCAA concentrations,women in the highest quartile had decreases of 12.00%(95%CI:-17.00%,-7.00%;P for trend<0.01)in total AFC and 13.16%(95%CI:-26.68%,2.86%;P for trend=0.09)in AMH,but had an increase of 6.33%(95%CI:0.15%,12.89%;P for trend=0.04)in FSH.The age-stratifed analysis showed that the associations of elevated urinary DCAA concentrations with reduced left AFC and elevated LH,as well as of elevated urinary TCAA concentrations with reduced left AFC and elevated E2 were stronger among women aged>35 years(all P for interactions<0.10).In the analyses stratifed by BMI,the positive associations between urinary DCAA concentrations and E2 were stronger among women with BMI<24 kg/m2(P for interaction=0.03),while the negative associations between urinary TCAA concentrations and AMH were stronger among women with BMI>24 kg/m2(P for interaction=0.03)Conclusions:Exposure to DBPs was associated with decreased ovarian reserve among women undergoing IVF-ET treatment.Part Three The mediating role of ovarian reserve in the associations between exposure to DBPs and IVF-ET outcomesObjectives:To investigate the potential mediation effect of ovarian reserve on the associations of exposure to DBPs with IVF-ET outcomesMethods:Based on the study population of part two(n=865),GLM models were applied to evaluate the associations of ovarian reserve with IVF-ET outcomes.We further performed a mediation analysis to test the mediating role of ovarian reserve in the associations between exposure to DBPs and IVF-ET outcomes.Results:AFC and AMH were positively associated with the numbers of total oocytes and MII oocytes,the proportion of fertilization,as well as the odds of successful implantation,clinical pregnancy,and live birth,whereas LH was positively associated with the numbers of total oocytes and MII oocytes(all P values<0.05).For example,each one-unit increase in total AFC was associated with increases of 5.00%(RR=1.05;95%CI:1.04,1.05)in total oocyte numbers,4.00%(RR=1.04;95%CI:1.04,1.05)in total MII oocyte numbers,1.00%(RR=1.01;95%CI:1.00,1.02)in the proportion of fertilization,as well as 8.00%(RR=1.08;95%CI:1.05,1.11),7.00%(RR=1.07;95%CI:1.04,1.10),and 6.00%(RR=1.06;95%CI:1.03,1.09)in the odds of successful implantation,clinical pregnancy,and live birth,respectively.Moreover,FSH was negatively associated with the numbers of total oocytes and MII oocytes,the proportion of fertilization,as well as the odds of successful implantation,clinical pregnancy,and live birth,whereas E2 was negatively associated with the numbers of total oocytes and MII oocytes(all P values<0.05).For example,each oneunit increase in natural logarithm-transformed FSH was associated with decreases of 45.00%(RR=0.55;95%CI:0.51,0.59)in total oocyte numbers,46.00%(RR=0.54;95%CI:0.50,0.58)in total MII oocyte numbers,as well as 56.00%(RR=0.44;95%CI:0.27,0.71),60.00%(RR=0.40;95%CI:0.25,0.65),and 54.00%(RR=0.46;95%CI:0.29,0.73)in the odds of successful implantation,clinical pregnancy,and live birth,respectively.The mediation analysis showed that AFC mediated the negative associations of urinary DCAA and TCAA concentrations with IVF-ET outcomes including successful implantation,clinical pregnancy,live birth,total oocytes,and total MII oocytes,with the mediation proportions ranging from 7.10%to 53.40%.Among them,total AFC accounted for the highest mediating effect(53.40%)in the associations of urinary DCAA concentrations with reduced numbers of total oocytes(β=-0.83,95%CI:-1.86,-0.04).Also,basal reproductive hormones including AMH,FSH,and E2 mediated the negative associations of urinary DCAA and TCAA concentrations with IVF-ET outcomes including successful implantation,clinical pregnancy,live birth,total oocytes,and total MII oocytes,with the mediation proportions ranging from 5.00%to 44.69%.Among them,AMH accounted for the highest mediating effect(44.69%)in the associations of urinary DCAA concentrations with reduced numbers of total oocytes(β=-0.79,95%CI:-1.72,0.07).Conclusions:Ovarian reserve played a mediating role in the negative associations between exposure to DBPs and IVF-ET outcomes.