Effect Of Phthalate Exposure In Follicular Fluid On Reproductive Outcomes And Related Mechanisms

Objective: To study the associations between phthalate acid esters(PAEs)metabolites in follicular fluid and the reproductive outcomes of in vitro fertilization/intracytoplasmic sperm injection(IVF/ICSI)and the mediating role of oxidative stress.The toxic mechanism of mono(2-ethylhexyl)phthalate(MEHP)on oocytes were investigated in an in vitro model,as well as the rescue effect of melatonin.Methods: This study included 641 infertile women who received IVF/ICSI treatment.On the day of oocyte retrieval,follicular fluid was collected and the concentrations of 8 phthalate metabolites and 2 markers of oxidative stress were measured.Generalized linear regression models and Bayesian kernel machine regression(BKMR)models were used to evaluate the associations between the concentrations of PAEs metabolites and IVF/ICSI outcomes.Mediation analysis was used to investigate the potential mediating role of oxidative stress markers.Finally,transcriptome analysis and in vitro maturation of oocytes were used to explore the toxicity mechanism of MEHP and the rescue effect of melatonin.Results: Monobenzyl phthalate(MBz P),mono(2-ethyl-5-hydroxyhexyl)phthalate(MEHHP),and MEHP in follicular fluid were suggestively associated with lower numbers of retrieved oocytes,metaphase Ⅱ(MⅡ)oocytes,and two distinct pronuclei(2PN)zygotes(all P for trends < 0.10).Oxidative stress plays a mediating role in the reduction of retrieved oocyte,MII,and 2PN number(P < 0.05).Exposure to MEHP significantly decreased the maturation rate of oocytes,the normal rate of spindles,and the mitochondrial membrane potential.In addition,it significantly increased the level of ROS and apoptosis level of oocytes.MEF2D-ND6 pathway may be involved in the toxicity mechanism of MEHP,and melatonin can reduce the toxicity effect of MEHP.Conclusions: PAEs in follicular fluid were associated with adverse reproductive outcomes.MEHP may damage oocyte quality through oxidative stress and the MEF2D-ND6 pathway.Melatonin can rescue the reproductive toxicity of MEHP.Part Ⅰ: Effects of Phthalate Exposure in Follicular Fluid on Reproductive OutcomeObjective: To explore the associations between phthalate metabolite concentrations in follicular fluid and IVF/ICSI outcomes among women recruited from a fertility clinic.Methods: We included 641 women undergoing IVF/ICSI treatment from December 2018 to November 2019 at the reproductive medicine center of Tongji Hospital.Follicular fluid collected on the day of oocyte retrieval were quantified for eight phthalate metabolites including monoethyl phthalate(MEP),mono-isobutyl phthalate(Mi BP),mono-n-butyl phthalate(MBP),MBz P,MEHP,MEHHP,mono(2-ethyl-5-oxohexyl)phthalate(MEOHP),and mono(2-ethyl-5-carboxypentyl)phthalate(MECPP).Generalized linear regression models were used to estimate the associations between quartiles of individual phthalate metabolite concentrations and IVF/ICSI outcomes.The effects of phthalate mixtures on IVF/ICSI outcomes were assessed by BKMR models.Results: We observed that quartiles of MBz P,MEHHP,and MEHP in follicular fluid were inversely associated with numbers of retrieved oocytes,MⅡ oocytes,and 2PN zygotes(all P for trends < 0.10).The highest quartile of molar sum of di(2-ethylhexyl)phthalate metabolites(∑DEHP)was also associated with 9.1%(95% CI: –17.1%,–0.37%)and 10.3%(95% CI: –18.8%,–0.94%)decreases in yielded oocyte and MⅡ oocyte numbers,respectively,compared with the lowest quartile.We generally found null results for implantation,clinical pregnancy,miscarriage,and live birth.The BKMR models also revealed inverse associations of phthalate mixtures with numbers of retrieved oocytes and MⅡ oocytes.Conclusions: Phthalate metabolites in follicular fluid were inversely associated with retrieved oocytes,MⅡ,and 2PN zygotes number among women undergoing IVF/ICSI treatment.Part Ⅱ: Mediating effect of oxidative stress in the associations between phthalates exposure in follicular fluid and adverse reproductive outcomesObjective: To explore the potential mediating role of oxidative stress in the associations of phthalate exposure in follicular fluid with adverse reproductive outcomes.Methods: Two oxidative stress markers,8-hydroxy-2-deoxyguanosine(8-OHd G)and 4-hydroxy-2-nonen-mercaptouric acid(HNE-MA)in the follicular fluid of infertile women undergoing IVF/ICSI were measured by high-performance liquid chromatography and tandem mass spectrometry.Generalized linear regression models were performed to evaluate the associations between the markers of oxidative stress in follicular fluid and the phthalate metabolites in follicular fluid and reproductive outcomes.We also explored the potential mediating role of the markers of oxidative stress of oxidative stress markers.Results: The concentrations of MBz P,MEHHP,MEHP and ΣDEHP in follicular fluid were positively correlated with the concentrations of 8-OHd G(P for trends < 0.001).The concentrations of MEP,Mi BP,MECPP,MEHP and ΣDEHP were significantly positively correlated with the concentrations of HNE-MA(P for trends < 0.001).Compared with the lowest quartile,8-OHd G and HNE-MA were significantly negatively associated with the number of retrieved oocytes,MⅡ oocytes,and 2PN zygotes(P for trends < 0.02).Mediation analysis showed that 8-OHd G significantly mediated the inverse associations of MBz P,MEHHP,and MEHP with oocyte yield and the number of MII.The estimated mediation proportion ranged from 21.54% to 91.3%.Also,HNE-MA significantly mediated the associations of MEHHP and MEHP with the number of retrieved oocytes,MⅡ oocytes,and 2PN zygotes.The estimated mediation proportion ranged from 13.89% to 24.58%.Conclusions: Our results suggest that oxidative stress may play a mediating role in the adverse reproductive outcome caused by phthalates exposure.Part Ⅲ: Effects of phthalate exposure on in vitro maturation of mouse oocytes and associated mechanismObjective: To investigate the effects of MEHP on in vitro maturation of mouse oocytes and related mechanism,as well as the rescue effect of antioxidant melatonin.Methods: In epidemiological,we found that DEHP metabolites are the main factors causing reproductive toxicity.Therefore,Transcriptome sequencing was performed on the ovaries of mice in the DEHP group and the control group to analyze potential toxicological mechanisms.MEHP is the secondary metabolite of DEHP,we used MEHP in vitro exposure to explore the effect of PAEs on oocyte maturation.The germinal vesicle(GV)oocytes of mice were randomly divided into control group and MEHP group.The polar body extrusion rate,ROS level,mitochondrial function and apoptosis level of the two groups were compared after 14-16 hours.Q-PCR and immunofluorescence staining were also used to analyze the change of expression levels of MEF2D-ND6 pathways.Finally,melatonin was added in MEHP exposed oocytes to observe its rescue effect.Results: Compared with the control group,MEHP exposure significantly reduced the maturation rate of mouse oocytes,interfered with spindle assembly,increased ROS and apoptosis levels,and decreased mitochondrial membrane potential.In addition,the protein and m RNA expression levels of MEF2 D and ND6 in oocytes treated with MEHP were also decreased.However,supplement of melatonin can significantly rescue the reduced maturation rate,disturbed assemble of meiotic spindle,increased ROS,decreased mitochondrial activity and increased apoptosis level.Moreover,the protein and m RNA expression levels of MEF2 D and ND6 were also significantly upregulated.Conclusions: Our results indicated that MEHP exposure may damage the quality of oocytes by oxidative stress and regulation of MEF2D-ND6 pathway.Melatonin can significantly alleviate the reproductive toxicity of MEHP.