A Study On The Relationship Of Exposure To Multiple Metals And The Risk Of Early Embryonic Arrest Among Assisted Reproductive Cycle Women

Background During assisted reproduction,early embryonic arrest(EEA)is a phenomenon that a mature oocytes successfully fertilized but stops cleavage during2～8 cell stage when was in vitro cultured,providing a great challenge to assisted reproductive technology(ART).Environmental factors is one of the most important factors that can affect reproductive development.Therefore,it is scientifically important to explore the risk factors of EEA from the perspective of environmental factors in order to improve the clinical pregnancy and live birth rate of infertility patients.Objective Based on a case-control study we measured blood levels of arsenic(As),mercury(Hg),lead(Pb),and barium(Ba)of infertile women who have undergone in vitro fertilization(IVF)treatment and analyzed the association between combined exposure levels of As,Hg,Pb and Ba and the risk of EEA as well as explored which metal(s)played a dominant role in this association.Additionally,we screened gene polymorphism site(s)of mt DNA in the displacement loop region(D-loop)to explore which gene polymorphisms were statistically significantly associated with the risk of EEA,and analyzed the strength of the association between the selected gene polymorphism(s)with the risk of EEA.Finally,we assessed whether the selected gene polymorphism(s)of mt DNA mediated the association between the harmful metal exposure and the risk of EEA among infertile women;and assessed whether there is an interaction between the selected gene polymorphism(s)of mt DNA and the harmful metal exposure.Method In this study,an unmatched case-control study design was used.From June2017 to March 2020,we recruited 74 infertile women with EEA in their fresh(oocyte retrieval)cycles as cases(123 cycles in total)and 157 infertile women whose proportions of high quality blastocysts in each fresh cycle were more than 70% were treated as controls(180 cycles in total)from the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University.No statistically significant differences of age and body mass index(BMI)between two groups of infertile women were observed at the time of inclusion.Demographic and clinical information of the study subjects were collected from the case system of our reproductive medicine center.The blood levels of four metals(As,Hg,Pb and Ba)collected on the day of oocyte retrieval during their first fresh cycles of infertile women in both groups were measured by inductively coupled plasma-mass spectrometry(ICP-MS);PCR(polymerase chain reaction,PCR)amplification and sequencing were used to screen the gene polymorphisms of mt DNA in D-loop region.Bayesian kernel machine regression(BKMR)was used to analyze the association between the combined exposure levels of four metals and the risk of EEA,and selected which metal(s)played a dominant role in the association via BKMR.The generalized estimating equation(GEE)model was used to analyze the association of the selected metal(s)with indicators related to oogenesis and embryonic developmental processes;chi-square test was used to explore gene polymorphism site(s)of mt DNA in D-loop region that statistically associated with the risk of EEA,and analyzed the strength of the association of selected gene polymorphism(s)of mt DNA with the risk of EEA.Mediating model was used to assess whether the selected gene polymorphism(s)mediated the association between the harmful metal exposure and the risk of EEA among infertile women.GEE model was used to analyze the association between the interaction of the selected gene polymorphism(s)and the harmful metal exposure;and calculate the relative excess risk due to interaction(RERI),the attributable proportion due to interaction(AP)and the synergy index(S)and their 95% confidence interval(CI)using Andersson excel calculator.Results The geometric mean(GM)concentrations of As,Hg,Pb and Ba in blood of infertile women in the case group were 1.35 μg/L,1.51 μg/L,23.45 μg/L and 34.15μg/L,respectively.Ba level in blood of infertile women in the case group was significantly higher than that of infertile women in the control group(P = 0.009).The BKMR results showed that Ba and Hg were positively correlated with the risk of EEA,As and Pb were negatively correlated with the risk of EEA;the posterior inclusion probabilities(PIP)values of As,Hg,Pb and Ba were 0.5902,0.6374,0.5548,0.9322,respectively,and Ba had the highest value of PIP among the four metals and Ba may play a dominant role in the combined exposure.A significantly increased risk of EEA in the medium(13.36 ～ 46.29 μg/L)and high(≥ 46.29 μg/L)exposure groups compared to low Ba exposure level group(< 13.36 μg/L)of infertile women was observed,respectively;and adjusted odds ratio(OR)and 95% confidence interval(CI)were 2.61(1.27,5.36)and 3.88(1.68,8.95),respectively(P trend = 0.005);when we further analyzed the association of Ba level and oogenesis and embryonic development-related indicators of infertile women,we found that the level of Ba was significantly and negatively correlated with the above indicators before and after adjusted for covariates.Among the 268 measured gene polymorphism sites of mt DNA in D-loop region,27 gene sites with mutant frequency being >10% in both groups of infertile women.After chi-square test was performed and P values were corrected via the method of false discovery rate(FDR),we only found the mutant frequency of the site of mt DNA 16519 gene(T16519T vs.T16519C)was significantly different in both groups of infertile women(P = 0.027).Infertile women with mutant type of mt DNA16519 gene(T16519C)had a significantly increased risk of EEA compared to those with wild type(T16519T)((OR(95% CI): 3.30(1.58,6.79)).The results were robust when sensitivity analyses were performed.The results of mediating model showed that in this study,after adjusted for covariates,mt DNA 16519 gene polymorphism had no mediation effect in the association between blood Ba exposure level and the risk of EEA among infertile women.The results of the interaction showed that compared with the infertile women with low Ba exposure and carried the wild type 16519 gene(Ba ≤ 21.74 μg/L & T16519T),the adjusted OR(95% CI)for the risk of EEA in infertile women with high Ba exposure and carried the mutant type 16519 gene(Ba >21.74 μg/L & T16519C)was 8.83(2.93,26.65)(P < 0.001),it suggested that the both factors had a multiplicative interaction.At the same time,the estimated values and95% CI for REPI,AP,and S were 4.07(-2.70,10.84),0.46(-0.02,0.95),and 2.08(0.68,6.37),respectively,when analyzed the additive interaction associated with mt DNA 16519 gene polymorphism and Ba exposure,it suggested that the both factors had no additive interactions.Conclusion In this study,we found the combined exposure levels of four metals(As,Hg,Pb and Ba)in blood of infertile women was positively associated with the risk of EEA risk,and Ba played a dominant role in the combined exposure;there were significantly negative correlations between Ba exposure level and indicators related to oogenesis and embryonic development progression.Infertile women with mutant type of mt DNA 16519 gene(T16519C)had a significantly increased risk of EEA compared to those with wild type(T16519T),but mt DNA 16519 gene polymorphism had no mediation effect in the association between blood Ba exposure level and the risk of EEA among infertile women after adjusted for covariates in the present study;there was a multiplicative interaction between Ba exposure levels and mt DNA 16519 gene polymorphisms and the risk of EEA in infertile women,rather than additive interactions.