The Effect And Its Mechanism Of Carbon Disulfide On The Early Period Of Pregnancy Of Occupationally Exposed Women

Many deleterious factors in occupational environment have reproductive toxicity, among which carbon disulfide (CS₂) is a case in point. CS₂ is an organic solvent widely used in industry and also acts as material for the production of synthetic fibers (rayon and synthetic feather), where there are a large number of female employees exposed to it. It has been found that the incidences of spontaneous abortion and congenital malformation are relatively high, and menstrual disorders often occur among female employees occupationally exposed to CS₂, which indicate that CS₂ bears significant reproductive toxicity. It has also been indicated by animal experiments that the rate of chromosome aberration in oocytes and oosperms in exposed mice increased markedly. Although its reproductive toxicity has attracted great concern of investigators, so far there has been extremely limited information about the harm of CS₂ to the early period of pregnancy, especially its influence on the implantation period of embryo. Meanwhile, there is a lack of sensitive indicators to screen and evaluate reproductive toxicity of toxicants. Has the reproductive toxicity of CS₂ disappeared or been negligible because of the gradual reduction of the CS₂ concentration in workshop as the improvement of working conditions in occupational environment? The answers to the questions above are the keys to take feasible and effective material labor protection measures for women employees exposed to CS₂.Objective: To investigate the effect on the earlier period of pregnancy in women employees exposed to CS₂ and to probe the mechanism of developmenttoxicity of CS2 in order to provide the theoretical basis for emending and carrying out labor protection measures of female employees.Methods:1. Cohort study. Prospective epidemiological methods were employed. Samples of CS2 were collected three times per month on the working spots, where the women who want to have a baby were exposed to CS2. The time-to-pregnancy of the women was observed by following up their menstrual cycles when they stopped using any means for contraception. At the same time, urine samples of volunteer workwomen in embryonic implanting period were collected in order to investigate the very early loss of pregnancy. Meanwhile, the pregnancy outcome of woman employee whose pregnancy was detected clinically was observed prospectively (refer to the first part of the article).2. Animal experiments: (Animal: KM female mouse; Dosage of CS2: 631.4mg/kg body weight; Way of exposure: intraperitoneal administration; Volume of injection: 0.1 ml/1 Og body weight):Model I : The exposure was continuous for three days at different three periods, the follicle development period (before mating), the embryonic implantation period (the fourth to sixth days of gestation, the plug day was defined as day 1 of pregnancy), and the period of post-implantation (the seventh to ninth days of gestation). Each group received one disposal and there was a solvent control group for CS2 with plant oil. On the fourteenth day of gestation, the maternal status and the uterus contents were examined in order to observe, compare and analyze the different influences of CS2 on embryonic development and implantation.Model II :The exposure was continuous for two days at the follicle development period and the embryonic implantation period. The implantation was divided into two periods, the earlier-implantation period (the fourth and fifth day of gestation) and the later-implantation period (the sixth and seventh day of gestation). Superovulation was used in all female mice to get more specimen at the end point of examination by using hCG and PMSG. On the ninth day of pregnancy, the statusof embryonic implantation and development were examined in order to observe, compare and analyze the different influences of CS2 on the earlier embryonic development (refer to the second part of article).3. Mechanism research. The expression of implantation-associated factors in embryos and uterus tissues at the ninth day of gestation for exposed and control mice were detected. The expression of FN and integrin Pi were detected with SDS-PAGE and western blotting; the active expression of MMP-9 and MMP-2 were determined with protein zymogram analysis; and the change in expression of ICAM-1 and p-selectin on the cell surface of tissues were analyzed with Flow Cytometry (refer to the third part of article).Results:1. In the cohort, there were 623 subjects who planned to and were pregnant. The results of follow-up study coming from 257 women employees exposed to CS2 and 366 unexposed women at the same factory were as follows.?The concentration of CS2 at the working spots for the 257 women was 8.67mg/m3, the relatively lower level.?The time-to-pregnancy of workwomen occupationally exposed to CS2 was prolonged markedly and the non-pregnant probability in each menstrual cycle decreased significantly (P=0.002, versus control). The ratio of pregnancy for the first menstrual cycle were 27.2% and 35.8% in exposed and control groups respectively (P=0.025), and the ratio of cumulative pregnancy for the first three month were 58.7% and 69.1% respectively in the two groups (P=0.008). The numbers of menstrual cycle needed for pregnancy were positively correlation with the level of CS2 they exposed at their working spots (P=0.024) and their exposure time (P=0.000). The ratios of cumulative pregnancy for exposure couples were 23.0%, 58.6%, and 90.8% respectively for the first month, the first three month, and one year, which were significantly lower compared with that for unexposed couples (38.1%, 70.0%, 97.4%, P value were 0.010, 0.049, and 0.008, respectively).(3)ln the menstrual cycles of those women with desire of procreation, theincidence of very early loss of pregnancy reached 48.7% with that of only 26.3% in control group (P=0.004); and the risk of earlier loss of embryo was increased by 2.5 times (RR=2.513, 95%CI=1.279⁴.938, P=0.007) after adjustment of history of abortion, husbands exposure to CS2, self-reported working pressure, and their education level. It was found that the incidence of very early loss of pregnancy was in positive correlation with the level of CS2 they exposed at their working spots (P=0.041) and the cumulative exposure index ( x 2 =25.59, P=0.001; r=0.867, P=0.002). The incidence of very early loss of pregnancy among exposure couples was 52.5%, which was significantly higher than that of unexposed couples (26.2%, P=0.007).@The rates of spontaneous abortion, fetal death and stillbirth, neonate death and congenital malformation in exposed women and controls were 8.55% and 5.12% (P=0.146), 3.42% and 0.60% (P=0.029), 1.46% and 0.64% (P=0.634), 9.7%o and 6.4%o, respectively. There were no significant differences in spontaneous abortion, neonate death, congenital malformation, the low birth weight and the sex ratio of neonatal babies between exposure group and control. Induced abortion was excluded.2. Results of animal experimentsModel I : In the follicle development and embryonic implantation exposed groups, the weights of the embryos in every fossa of fourteenth day were significantly decreased by 41% and 39% (P=0.000 and 0.068, versus control), respectively, and the average weights per embryo were decreased by 41% and 39% (P=0.082 and 0.045), respectively. The amount of blastocyst loss was markedly higher by 4.7 times in implantation group than that in control (P=0.036). There were no significant difference between oil groups and each CS2 exposed groups in the maternal body weights of the first day and the fourteenth day of gestation when the trial ended except for the body weigh gain and the liver weight.Model II: In the follicle development and later-implantation exposed groups, the weights for the embryos in every fossa and the uterus of ninth day weresignificantly decreased by 37% and 46% (P=0.049 and 0.029), respectively. The average weight for each embryo in follicle development group was decreased by 36% (P=0.018). The amount of implanted blastocysts in the earlier and later-implantation groups were significantly decreased by 28.7% and 47.5% (P=0.043 and 0.001), respectively, and the average weight and the coefficient of uterus in the two groups above were decreased evidently (P<0.01 in each of the two compared groups).3. In the tissues of embryo and uterus of mice at ninth day of gestation, the expressions of the factors related to implanting were as follows.?The expressions of FN in embryo and uterus tissue were lowered in each exposure group (PO.01, versus control). The levels of expression in embryo tissues were decreased by 45%, 60% and 50%, respectively, in the follicle, the earlier and the later-implantation exposed groups, and that in uterus tissues were decreased by 70%, 40%, and 64% compared with relative controls (PO.001 in each of the six compared groups).?Integrin Pi were weakly expressed in uterus tissues of three exposed groups above, and the expression levels decreased by 16%, 20% and 22%, respectively (P<0.001 in each of the three compared groups). In embryo tissues the levels decreased significantly in follicle and later-implantation exposed groups (P=0.058, 0.009, respectively).(3)MMPs: In embryo tissues, the activity of MMP-9 and MMP-2 was reduced markedly, by 25% in follicle development exposed group and 27% in later-implanting exposed group for MMP-9, and 24% and 33% in earlier and later-implanting exposed groups for MMP-2 (P<0.001 in each of the four compared groups), respectively. In uterus tissues, the active expressions of MMP-9 and MMP-2 were increased, of which the activity of MMP-9 in later-implantation exposed group was elevated 5 times compared with that in control group (P=0.001).?Expression of ICAM-1 was down-regulated in cell surface of embryos and uterus tissues after exposure, especially in group of later-implantation periodexposed and in uterus tissues.?P-selectin in embryo and uterus tissue was weakly expressed in all exposed groups.In addition, in uterus tissues of normal non-pregnant mice, the expression level of FN was rather low (hardly to be detected), while that of integrin Pi was quite high. Moreover, the expression level of integrin |3] was relatively high in follicular development period (identical to that on the ninth day of pregnancy) and that in ovulation period and luteal period were relatively low (P=0.000 and 0.072, respectively).Conclusions:1. Lower level of CS2 in the working spots of women employees who want to bear and was pregnant would result in the significant damage of embryo at its early period of development.2. The incidence of embryo loss in early period was obviously higher than that in middle and late periods of pregnancy for women employees occupationally exposed to CS2.3. The risk for longer time-to-pregnancy and very early loss of pregnancy was higher when couples exposure to CS2. Time-to-pregnancy and very early loss of pregnancy might be employed to screen and evaluate the reproductive injury effect caused by environment toxicants.4. Implanting period of blastocysts may be another sensitive toxic point for development toxicity caused by CS2 except for oocytes, and the development toxicity of CS2 on embryo implanting period was more obvious than that on oocytes when cutting down the dose of CS2 exposure.5. The exposure to CS2 could result in abnormal expression of implanting factors, reduce the ability for adhesion and invasion of blastocysts, and represent the disorder of implantation and embryo development in the earlier period of pregnancy.Suggestions:1.Women employees should prevent from being exposed to CS2 before and when planning a pregnancy. In particular, they should be banned from contacting CS2 in the embryo peri-implantation period of pregnancy.2.The mechanism of inducing early pregnancy loss by CS2 should make further study.The innovative discovery from the study:Based on the cohort study on the women employees occupationally exposed to CS2, who attempt to have gestation, it was first reported to deal with embryo loss at peri-implantation period. It was shown that exposure to CS2 could result in embryo damage, and the degree of pregnancy loss in earlier period of embryo development was more serious than that in medium and terminal period of pregnancy. The risk of earlier loss of embryo was increased by 2.5 times (RR=2.513, 95%CI=1.279 4.938) after adjustment of history of abortion, husbands exposure to CS2, self-reported working pressure, and their education level. Very early loss of pregnancy and time-to-pregnancy would be more sensitive to indicate the embryos development disorder in earlier period of pregnancy to screen the productive toxicants.Based on the animal experiment working on the female mice exposed to CS2, it was the first report to divide the pregnancy period into three main period: the follicle development period, the implantation period and the post-implantation period in order to decompound the manner of workwomen exposure and to compare the difference with or without the effect of oocyte exposed. Further more, decompounding the implantation into two parts, the earlier and the later-implanfation to validate the effects of developmental toxicity in implantation period exposure. It was shown that the normal implantation period of mouse embryo could be affected by CS2. Implanting was another sensitive toxic point for developmental toxicity caused by CS2 except for oocytes, and the developmental toxicity was more obvious in embryo implantation period exposure than in oocytesdevelopment period exposure when cutting down the dose of CS2.Based on the molecular biology experiment on the ninth embryos and uterus tissues of female mice exposed to CS2, it was the first time to mensurate the expression of molecules which were involving in the ability of adhesion and invasion of the blastocysts to reveal the mechanism of baffling the implanting of the blastocysts for maternal exposed to CS2. It was shown that abnormity of adhesion and invasion of the blastocysts would be an important reason for the difficulty of embryo implantation in CS2-exposed mouse.