Association Between Exposure To Phthalic Acid Esters And Missed Abortion In Early Pregnancy And Potential Mechanism Study

[Aim] To confirm the association between exposure to phthalic acid esters (PAEs) and missed abortion by clinical epidemiological studies and to explore the potential mechanisms by in vitro experimental study.[Methods] (1) Retrospective case-control study. The study was performed including 1945 cases of missed abortion and 1874 cases of normal pregnancy within 20 weeks of gestation. Chi-square test and logistic regressive analysis were applied to identify risk factors related to missed abortion with special attention to effect of environmental risk factor on the marked increase of missed abortion during past 20 years. (2) Analysis of exposure levels of PAEs. The urinary levels of 13 metabolites of PAEs were measured using high performance liquid chromatography-mass spectrometry (HPLC-MS) in 150 cases of missed abortion and 150 matched controls of normal pregnancy. (3) In vitro study. A trophoblastic cell line, HTR-8, was exposed to a series of concentrations of DEHP (one of the most commonly encountered PAEs). Changes in cell viability, apoptosis were studied to explore the potential mechanism of PAE-exposure caused missed abortion.[Results] (1) Result of logistic regressive analysis showed five independent risk factors of missed abortion, including hepatitis B virus carrier (OR 8.126, CI 4.688-14.084), anemia (OR 5.498, CI 2.243-13.473), spontaneous abortion (OR 2.359, CI 1.703-3.268), primipara (OR 2.305, CI 2.031-2.638) and outdoor profession (OR 1.777, CI 1.538-2.052). Further analysis of changes of proportions of the independent factors of indicated that the increased proportions of outdoor profession and environmental pollution were positively correlated to the increased number of missed abortion (P< 0.05) while no significant relationship between the increased numbers of missed abortion and changes of proportions of 3 or more times of induced abortion, spontaneous abortion, anemia and hepatitis B virus carrier (P>0.05). Primipara, a positive risk factor, was also found to have no contribution to the increase of missed abortion in recent years. (2) HPLC-MS measurement of urinary metabolites of DEHP indicated that four metabolites, MEHP, MEHHP, MEHOHP and DMP were significantly higher in missed abortion cases than those in controls (P<0.01). However, MEP were significantly higher in controls than those in cases(P<0.05). In correlation analysis, MEHP and MMP, respectively, had medium correlation with estradiol and progesterone. (3) In vitro study lied mainly in two parts: ① HTR-8 cells exposed to DEHP≤20ng/ml for 48h showed no significantly toxicity. Apoptosis of HTR-8 cells were significantly inhibited by DEHP concentration of 15ng/ml (P<0.05). The S-phase of HTR-8 cells were prolonged and GO/G1 phase arrest were reduced by DEHP at concentration of 15ng/ml (P<0.05). After 15ng/ml of DEHP treated HTR-8 cells for 6h, Bax, the apoptosis factor, expression decreased, what’s more, Bcl-2, as an inhabitor of apoptosis, expressed more. ② HTR-8 cells with 30ng/ml or higher concentrations of DEHP for 24-48 hours significantly inhibited cell viability (P<0.05). And DEHP at the concentration of 30ng/ml significantly induce the apoptosis of HTR-8 cells, simultaneously shorten S phase and increase the G0/G1 phase arrest (P<0.05).[Conclusion] (1) Outdoor profession, primipara, spontaneous abortion history, anemia and hepatitis B virus carrier are independent risk factors of missed abortion. The increase of proportions of outdoor profession and unknown factors among those risk factors are consistent in the increased numbers of missed abortion in recent years. We conclude that environmental pollution and other unknown risk factors rather than reproductive history and complications of pregnancy might contribute to the observed increase of missed abortion cases. (2) Certain urinary level of metabolites of PAEs in cases of missed abortion are significantly higher than that in controls of normal pregnancy, indicating exposure to PAEs, one of the common environmental pollutants, is related to the increased number of missed abortion. (3) DEHP at concentrations of 15ng/ml or less can inhibit apoptosis while increase S-phase and reduce G0/G1 phase arrest, which may relate to down-regulation of expression of Bax and up-regulation of expression of Bcl-2. This could be one of the mechanisms of DEHP-induced missed abortion.