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Environmental exposure to endocrine-disrupting chemicals and miscarriage
ENVIRONMENTAL EXPOSURES: PREGNANCY, PLACENTA, AND MISCARRIAGE
Environmental exposure to endocrine-disrupting chemicals and miscarriage Sacha A. Krieg, M.D., Ph.D.,a Lora K. Shahine, M.D.,b and Ruth B. Lathi, M.D.c a Oregon Health Sciences University, Portland, Oregon; b Pacific Northwest Fertility, Seattle, Washington; and c Stanford University, Stanford, California
Establishment of early pregnancy is the result of complex biochemical interactions between the decidua and blastocyst. Any alteration in this chemical dialogue has the potential to result in adverse pregnancy outcomes including miscarriage. Sporadic miscarriage is the most common complication of pregnancy and can be caused by multiple factors. While the most common cause of miscarriage is genetic abnormalities in the fetus, other contributing factors certainly can play a role in early loss. One such factor is environmental exposure, in particular to endocrine-disrupting chemicals, which has the potential to interfere with endogenous hormone action. These effects can be deleterious, especially in early pregnancy when the hormonal milieu surrounding implantation is in delicate balance. The purpose of this paper is to review the current evidence on the role of environmental toxins in reproduction. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) Key Words: Miscarriage, endocrine disrupting chemicals, environment Discuss: You can discuss this article with its authors and with other ASRM members at
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poradic miscarriage, the loss of an embryo or fetus before 20 weeks of pregnancy, is the most common complication of pregnancy, affecting approximately 15% of all clinically recognized pregnancies (1). A much larger percentage of pregnancies are impacted if preclinical losses are included. Of these clinical pregnancy losses, a large majority occur during the first trimester. A majority of these first trimester losses are secondary to genetic abnormalities in the conceptus. However, sporadic, euploid losses do occur. Risk factors for pregnancy loss include but are not limited to advanced maternal age, hormonal imbalances, immunological interactions, and uterine anatomic abnormalities (1–4). Certainly, lifestyle factors such as tobacco smoke and alcohol usage are generally well accepted as contributing causes of miscarriage (5–7). Exposure to other
chemicals such as endocrine disru ptors, heavy metals, or occupational chemicals has also been implicated in miscarriage. The sensitivity of embryonic tissue or fetal tissue to such compounds is far greater than that of adult tissue. This is in part because of the small size and totipotent nature of many of the cells in the embryo or fetus (8, 9). A single insult at these early stages of development can have deleterious effects on development. Not only can environmental toxins impact the developing embryo, there is also a potential for alteration of the endometrium of pregnancy, that is, the decidua. Successful implantation requires a complex biochemical dialogue to occur between the blastocyst and decidua. These early interactions allow for implantation to occur and ultimately for establishment of pregnancy (10). Environmental alteration
Received May 13, 2016; revised and accepted June 29, 2016. S.A.K. has nothing to disclose. L.K.S. has nothing to disclose. R.B.L. has nothing to disclose. Reprint requests: Sacha A. Krieg, M.D., Ph.D., University Fertility Consultants, Oregon Health Sciences University, CH10F, 3303 SW Bond Avenue, Portland, Oregon 97239 (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.06.043
of these early uterine events can also disrupt an early pregnancy or potentially result in miscarriage or other adverse pregnancy outcomes. In particular, abnormal hormone signaling pathways could potentially affect not only the ability of a blastocyst to implant in the endometrium but also the ability of the decidua to communicate with fetoplacental unit in a normal way. Multiple industrial contaminants have the potential for endocrine disruption; these chemicals can impact the ability to become pregnant and sustain a healthy pregnancy. Other environmental contaminants have also been implied to have a causal effect on pregnancy loss; these include radiation exposure, heavy metals, agricultural chemicals, and industrial solvents. Given the scope of this review, our discussion will be limited largely to endocrine-disrupting chemicals (EDCs). EDC exposure may represent an additional cause(s) of sporadic or recurrent loss.
DDT Perhaps one of the most well-known EDCs is DDT, or 1,1,1-trichloro–
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ENVIRONMENTAL EXPOSURES: PREGNANCY, PLACENTA, AND MISCARRIAGE 2,20 bis(p-chorophenyl) ethane. DDT was first synthesized in 1874. However, it was not recognized as a pesticide until 1939, when it was first used during World War II for protecting the military from malaria. It then became commercially available in 1945. DDT had widespread use and is credited for eradicating malaria from the United States and Europe. While other pesticides began to emerge, DDT was still the most widely used pesticide until the mid-1960s, when environmental impacts started to become apparent. Unfortunately, part of its efficacy as an insecticide is due to its persistence in the environment. Because of its chemical stability and lipophilic nature, DDT persists in the environment and results in accumulation and even bioconcentration within the food chain. Even today, DDT is widespread in the environment; in fact some countries are still using DDT in vector control (8, 11). By the mid-1950s animal studies were beginning to suggest that DDT was causing poor reproductive outcomes. Effects were first noted in birds, with plummeting of some bird populations. The birds most affected are those that are at the top of the food chain, in particular those that are fish eating. One specific example is the Californian brown pelican. They were found to have 3,000 breeding pairs in 1960, but by 1969 there were only 300 pairs and only five chicks were viable (12, 13). This decline in bird populations became the impetus for Rachel Carson's Silent Spring. Other animal populations were affected such as fetal resorption in rabbits and miscarriage and stillbirth in rats. Ultimately, DDT was restricted or banned in most developing countries by 1970 (11, 14). Early human studies of DDT suggested a possible correlation with cancer in general. However, with time, reproductive disorders became apparent. Similar to the bird population, human population declines were secondary to reproductive failures. In vitro studies have shown that DDT has estrogenic activity (15). Dichlorodiphenyl dichloroethene (DDE), a metabolite of DDT, has been shown to be an androgen receptor antagonist. The reproductive repercussions of DDT use have been multiple; these include a suggested role in decline in sperm counts, increase in time to conception, and even intrauterine growth restriction (11, 16, 17). Spontaneous abortion has been correlated with DDT exposure in multiple studies. Given the stability of DDT and its metabolites (in particular DDE), it is possible to correlate serum levels with pregnancy history. Most human studies on miscarriage and DDT have correlated DDT or DDT metabolites with pregnancy outcomes. Some studies show limited impact of DDT on miscarriage (11, 18, 19). These studies are limited by the timing of collection of samples and the timing of observation of pregnancy losses. In these studies, DDT and/ or its metabolites were measured in blood or serum that was collected after the pregnancies had completed. The levels of DDT would have the greatest impact during the pregnancy, that is, during the exposure to the fetus. In addition, many of the samples were collected in the postpartum period when women were nursing their infants. Since breast feeding decreases the DDT levels in the serum (20), levels could be lower than what they were in pregnancy. In addition to timing of sample collections, many pregnancy
losses could have gone unaccounted for since these studies are retrospective in nature. Only prospective studies that collect samples and data throughout menstrual cycles and detect early preclinical losses with bhCG levels can account for all the pregnancy losses associated with high levels of serum DDT. However, these studies have been in patients with lower serum levels of DDT or DDE. Several studies have shown that there is also an increase in spontaneous miscarriage at higher concentrations of DDE (>15 mg/L) (11, 18, 19). One of the largest studies of DDT and DDE in relation to miscarriage to date was done by Longnecker and coworkers (20). They used serum from enrollees in the Collaborative Perinatal Project (1959–1965). These women were asked about their pregnancy history, and serum samples were frozen. Serum from 1,717 women who had a pregnancy history was analyzed for concentrations of DDT and DDE. Women were compared to a group who had serum DDE levels of <15 mg/L; exposures greater than 15 mg/L were divided into quartiles of increased exposure. Longnecker et al. found an adjusted odds ratio (OR) of miscarriage per 60 mg/L increase in DDE of 1.4 (95% confidence interval [CI], 1.1–1.6) (20). One flaw of these studies is that they are retrospective in nature and therefore are subject to recall bias. There are two studies that are prospective in nature that found similar effects. Korrick and coworkers found an OR for miscarriage of 1.13 (95% CI, 1.02–1.26) in a casecontrol study matching patients by age, smoking status, body mass index, and workplace exposures; they were then followed prospectively through pregnancy (18). Control patients had an ongoing pregnancy, whereas the cases had miscarried. Serum levels of DDT and DDT metabolites were measured. Patients who miscarried were found to have higher levels of DDE in their serum (22 vs. 12 ng/g) than controls (P¼ .025) (18). Another prospective study by Venners et al. followed Chinese textile workers prospectively with serum hCG levels (19). In this study DDT levels were drawn before conception. Venners et al. found that there was a relative odds of pregnancy loss of 1.17 (95% CI, 1.05–1.29) associated with a 10 ng/g increase in serum total DDT (19). Overall, these studies consistently show an increase in spontaneous abortion in patients with higher serum levels of DDT or DDT metabolites (Table 1) (11, 18, 20).
BISPHENOL A (BPA) BPA [2,2-bis(4-hydroxyphyenyl) propane] was first synthesized in 1891 as a synthetic estrogen for the pharmaceutical industry. It was taken out of production once a much more potent estrogen was developed, diethylstilbestrol. Then, in the 1950s, its production resumed as it was found to be useful as an epoxy resin and the basis for polycarbonate plastics. It is now one of the highest-volume chemicals produced worldwide. BPA is an estrogenic compound used in the manufacture of polycarbonate plastic and epoxy resins. The use of BPA is so widespread that it is estimated that 100 tons of this chemical are released into the atmosphere annually (24, 34). While BPA is used to manufacture plastics frequently used for food storage, studies have shown that VOL. - NO. - / - 2016
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TABLE 1 Endocrine disrupting chemicals and their reproductive outcomes. Compound
Mechanism
Oocyte effect
Endometrial effect
DDT
Estrogen receptor agonist, androgen receptor antagonist
Decrease in mature oocytes (zebrafish), increased granulosa aromatase activity (human)
Decreased proliferation and increased apoptosis
BPA
Estrogen agonist
Meiotic errors, epigenetic changes, early apoptosis
Altered gene expression of HOXA10, Era, ERb, B3integrin, and ITGB3
PCBs and dioxin
Mixed estrogen agonist/antagonist, androgen receptor agonist
Inhibit meiotic spindle assembly, prevent normal oocyte maturation
Altered expression of estrogen responsive genes
Phthalates
P receptor agonist in silico, interacts with PPAR with decrease in aromatase activity
Anovulation, abnormal granulosa steroidogenesis
Abnormal expression of Era, PR, and E-cadherin
Miscarriage
Reference
OR, 1.4 (1.1–1.6) per 60 mg/L increase (prospective, n ¼ 1,717); OR, 1.13 (1.02–1.26) (case control study); OR, 1.17 (1.05–1.29) prospective (n ¼ 372) Sporadic: OR, 1.97; recurrent: OR, 3.33 in highest quartile (n ¼ 115); highest concentration: recurrent spontaneous abortion OR, 9.34 (3.06–28.44; n ¼ 264) OR, 1.6–2.52 depending on type of PCB; Agent Orange relative risk, 0.99 (0.85– 1.16) Increased miscarriage OR, 2.87 (1.09– 7.57)
(18–23)
(24–27)
(28–30)
(31–33)
Krieg. Environmental toxins and miscarriage. Fertil Steril 2016.
this compound can leach into the food and, accordingly, be ingested. BPA is also used in thermal receipt paper and some dental sealants. Given that BPA is used in so many frequently used products it is not surprising that detectable levels of this compound are found in 90% of urine samples in a representative U.S. population that was tested (24, 34). The ubiquitous nature of BPA exposure is particularly concerning as it is a known EDC. Indeed, in the 1970s it was found to be a reproductive toxin by the national toxicity program. In 2008 Canada placed a limited ban on BPA, classifying it as a toxic substance. While the Food and Drug Administration declared that BPA is safe at current estimated levels of human exposure, retailers began pulling baby bottles and children's food containers that contained BPA in 2008. Meanwhile, the impact of continuous lowdose exposures remains in debate (34). BPA has been linked to reproductive disorders such as polycystic ovarian syndrome, endometriosis, infertility, and thyroid disease (24, 34, 35). Detectable concentrations of BPA have also been identified in reproductive tissues such as follicular fluid and amniotic fluid, suggesting that exposure can occur during the time of implantation and establishment of early pregnancy (24, 35). Alteration of expression of genes associated with implantation has been associated with BPA as BPA is able to weakly bind the estrogen receptor (24, 36). As a result of its estrogen agonist properties, an increase in expression of estrogen-regulated
genes is found with BPA. This includes genes such as HOXA10, B3integrin, and ITGB3. These genes are known to be important for normal implantation. BPA has also been shown to impact endocrine signaling by downregulating expression of ERa and PR. Endometrial angiogenesis is also impacted by BPA by downregulating vascular endothelial growth factor expression (24, 36). These gene expression data, taken together, suggest that BPA has the potential to disrupt implantation, with the potential downstream effects of miscarriage, antenatal complications, and even implications in adult health and disease. While many have questioned the impact of BPA upon fertility, development, and adult disease, the focus of this review will be upon miscarriage. It is known that mice exposed to BPA have a lower number of implantation sites (37). In fact, when mice are exposed to BPA during pregnancy, their offspring have abnormal endometrial stromal signaling pathways. BPA has also been shown to result in thinner endometrial lining in human studies, further supporting a negative impact on the deciduae (35). BPA has been associated with an increase in aneuploidy oocytes in murine studies, which could also be a cause of miscarriage. BPA has been shown in several animal models to affect oocyte meiosis, leading to aneuploidy (38). A similar result has been found in human oocytes during culture (39). This increase in oocyte aneuploidy can certainly lead to miscarriage. In fact, a recent study by Lathi and coworkers demonstrated a statistically
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ENVIRONMENTAL EXPOSURES: PREGNANCY, PLACENTA, AND MISCARRIAGE significant increase in both euploid and aneuploid losses in patients in the fourth quartile of urinary BPA concentration (25). The relative risk of loss for sporadic miscarriage was 1.97 (95% CI, 1.08–3.59) and for recurrent loss was 3.33 (95% CI, 1.04–10.71) in the highest quartile of serum BPA concentration (25). Similarly, another group has demonstrated an increase in idiopathic miscarriage in patients with higher urinary BPA levels, but the sample size was small (n ¼ 45) and there was no fetal karyotyping performed (40). Shen and coworkers found that a higher urinary BPA level is associated with recurrent miscarriage with an OR of 3.91 (95% CI, 1.23–12.45) in the 0.4–0.93 mg/g Cr group and an OR of 9.34 (95% CI, 3.06–28.44) in the .93 mg/g Cr group adjusting for confounding factors; however, these samples were not collected during pregnancy for the miscarriage group (26). Taken together, BPA has been shown in laboratory models to increase the risk of miscarriage at the level of both the endometrium and the oocyte. These molecular changes as a result of BPA exposure do appear to confer an increased risk of miscarriage in patients with higher BPA accumulation. These preliminary data suggest a critical need for larger epidemiological studies to confirm an association between BPA exposure and miscarriage.
PCBs Dioxin-related toxins including polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans are ubiquitous contaminants of industrial combustion processes. PCBs were first introduced in the late 1920s, but by the early 1970s many countries began banning or restricting their use. Dioxins are perhaps one of the most infamous members of this group, having a well-accepted role in carcinogenesis. These compounds are highly lipophilic, resulting in accumulation and concentration through the food chain. Unfortunately, because of their resistance to degradation, these compounds still persist in the environment, despite their use being banned (28, 41). These compounds can act not only as estrogen agonists or antagonists but also can have androgenic properties. PCBs have been shown to inhibit meiotic spindle assembly in vitro (42). These chemicals also hamper maturation of oocytes in vitro. Both of these effects have the potential to cause miscarriage. The endometrium also appears to be impacted by PCBs. In vitro endometrial cultures show an alteration in estrogen-responsive genes upon exposure to PCBs (29). Given that the PCBs including dioxin have the potential to alter both normal oogenesis and endometrial function, how is pregnancy impacted? Dioxin exposure in pregnancy has been associated with fetal loss and low birth weight in rodents and monkeys. PCB exposure has also been associated with a developmental syndrome involving hydronephrosis, cleft palate, and thymic atrophy in mice (43). Monkeys exposed to PCBs have an increased number of miscarriages and higher rates of fetal demise (44). Human studies generally show similar findings. One example of PCB toxicity is Yusho oil disease. In 1968 in western Japan, rice oil was contaminated with PCBs and other dioxin-
related compounds. As a result over 1,900 people presented with clinical symptoms including acne-like eruptions, pigmentation changes, eye discharge, and paresthesias. More than 500 people died as a result. The levels of PCBs and dioxins remained elevated for a prolonged period of time in these individuals. In a study done by Tsukimori et al., a statistically significant increase in miscarriage was noted, with OR ranging from 1.6 (95% CI, 1.1–2.33) to 2.52 (95% CI, 0.92–6.87) depending on the type of PCB measured (28). This study included interviews with 214 women (512 pregnancies) regarding pregnancy outcomes over 36 years; these patients had also had blood levels of PCBs measured. Other groups have shown an increase in low birth weight and growth restriction in this population. The Yusho oil incident is not isolated; similarly the Yu-sheng accident and Vietnamese exposure to Agent Orange have shown an increase in spontaneous abortion (30, 45). After an explosion at a chemical factory in Seveso, Italy, that involved dioxin, no increase in miscarriage after evaluating pregnancy outcomes in 510 women was observed (41). This could be explained by different concentrations of dioxin, PCBs, and dibenzofurans in these exposures and the timing of exposure. Another dioxin-related compound is Agent Orange, a dioxin-contaminated defoliant used during the Vietnam War. Agent Orange is a mixture of the herbicides 2.4-D [(2,4-dichlorophenoxy)acetic acid] and 2,4,5-T [(2,4,5-trichlorophenoxy)acetic acid]. During the Vietnam War, Agent Orange was contaminated with 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). A systematic review by Ngo et al. showed that Agent Orange appeared to be associated with an increased risk of birth defects (46). Later epidemiological studies demonstrated an increase in spina bifida after paternal exposure to Agent Orange. There are conflicting data regarding Agent Orange and risk of spontaneous abortion. While many studies have been limited by small sample size or inadequate exposure, the data are inconsistent. Most studies have evaluated paternal exposure; however, studies of exposed Vietnamese women have shown an increase in miscarriage and premature deliveries (47). Larger studies have found a trend toward increased risk of spontaneous abortion in wives of veterans with low exposure but not at high exposure levels (48). A larger crosssectional study of 281 workers exposed to TCDD during production of Agent Orange, showed primary outcomes of increase in stillbirth, spontaneous abortion, and sex ratio. In this study no increase was seen in any of these outcomes (45). However, this study was vulnerable to recall bias, and it occurred over a time period when early pregnancy tests were not available (1950–1960s), so early losses would not be captured. While PCBs represent a large class of chemicals, it appears from largely retrospective studies that they are associated with an increase miscarriage and other adverse reproductive outcomes. Further longitudinal studies are needed to further understand the reproductive repercussions of PCB use not only on pregnancy complication but also on the effect on offspring. VOL. - NO. - / - 2016
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PHTHALATE AND PHTHALATE METABOLITES Phthalates are frequently used in plastic manufacturing and are used to describe multiple phthalate monoesters. This group of plasticizers is used to make plastic more flexible and is found in many personal care products as well as medical supplies. In fact, exposures during medical interventions have been found to be up to 150 times the baseline exposure risks per day as phthalates are used in medical supplies such as IV tubing and bags. Globally, more than 18 billion pounds of phthalates are used annually. Phthalates have been associated with developmental abnormalities of the male reproductive system, miscarriage, endometriosis, and low sperm counts. The mechanism by which phthalates cause adverse reproductive outcomes includes P-like effects; in fact, di-(2ethyl)phthalate (DEHP) has been modeled in silico to interact with the P receptor. Other studies have shown in vitro that some forms of phthalates activate the PPARs and ultimately decrease aromatase activity (31, 32, 49). These possible P-like effects and decrease in aromatase activity have been theorized to contribute to adverse reproductive outcomes. In rat models, multiple studies have shown ovarian toxicity stemming from DEHP exposure. While rodent studies may be suggestive of human health risks, they allow the observation of titrated effects. Administration of DEHP to female rats has shown a decrease in serum E2 levels and an increase in anovulation (50). Histologically, these rats showed cystic follicles with abnormal granulosa cells and smaller follicle size. In human studies of patients undergoing IVF, a lower number of mature oocytes are found in patients with higher phthalate concentrations (51). These patients are also found to have lower implantation rates. In the mouse endometrium, titrated phthalate dosages are found to cause abnormal gene expression of ERa, PR, and E-cadherin. These gene products are important for normal implantation, and, concordantly, these mice had a lower number of implantation sites (52). In rodents that did become pregnant, a decrease in the number of litters, litter size, and proportion of live-born offspring was observed after DEHP exposure (53). Another form of phthalate, di-n-butyl phthalate, was found to confer an increase in midpregnancy miscarriage in rats. Chronically high exposure levels associated with manufacture have been linked to miscarriage and lower fertility rates in humans (54). Similarly, in humans, elevated urinary concentration of the phthalate monoethylhexyl phthalate around the time of conception was associated with an increase in miscarriage, with an OR of 2.87 (95% CI, 1.09, 7.57) in the highest quartile of exposure (54). Mu and coworkers also found an increase in miscarriage rates after measuring eight different urinary phthalate metabolites, dividing patients into four different quartiles of exposure. Participants falling into the first and fourth quartile of exposure had an increase in miscarriage (33). Exposure to phthalates has other effects in pregnancy. Elevated concentrations of phthalates have also been associated with adverse antenatal complications such as preeclampsia and gestational anemias (55). In utero exposure to DEHP in particular is associated with abnormal reproductive development (56, 57). These developmental abnormalities include
early breast development, shortened anogenital distance, and cryptorchidism, among others (33). Many of these effects are thought to be secondary to aromatase inhibition or other hormonal mimicry. As EDCs that have widespread use, phthalates appear to have an impact not only on miscarriage rates but also on offspring. Further studies with larger sample sizes are needed to delineate these effects.
CONCLUSIONS Environmental contaminants have been suggested to play a role in spontaneous miscarriage. The focus of this review was limited to endocrine disruptors. This group of chemicals, which are able to interact with hormone receptors, is known to impact wildlife, laboratory animals, and humans. Some of the effects on wildlife are profound, including marked decreases in population, inability to reproduce, physical changes in the reproductive organs, birth defects, and miscarriage. These exposures may occur in the workplace, from ingesting contaminated food, while receiving medical treatment (i.e., IV tubing), or from the pesticides/herbicides used in agriculture or for pest control. Many of these exposures are cumulative and as a result can be bioconcentrated in the food chain. The reproductive outcomes impacted by endocrine disruptors are many, but the focus of this review paper is on miscarriage. Many studies have shown a dose-dependent increase in miscarriage with these endocrine disrupters (Table 1). There are some considerations when interpreting these data. Many of these studies rely on small sample sizes and often rely on recall of pregnancy history. These are obvious confounders in interpreting the data. That being said, many of these compounds are extremely stable, allowing researchers to measure serum or urinary levels reliably. These studies suggest that women of reproductive age should exercise caution in exposure to these endocrine disruptors. Unfortunately, these compounds are ubiquitous in the environment and are often difficult to avoid. One concern with the ubiquitous nature of these chemicals is that many of these studies could be confounded by the presence of multiple chemicals. Ideally, multiple endocrine disruptors would be assayed for, permitting an assessment of the actual compound(s) causing an effect. Also there is the potential that these EDCs could behave in a synergistic manner, exacerbating a preexisting effect. These studies show the need and importance of more prospective studies of adequate sample size and design so that we can understand the full extent of the impact of these hormone-like compounds on the establishment and maintenance of pregnancy.
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Environmental exposure to endocrine-disrupting chemicals and miscarriage Sacha A. Krieg, M.D., Ph.D.,a Lora K. Shahine, M.D.,b and Ruth B. Lathi, M.D.c a Oregon Health Sciences University, Portland, Oregon; b Pacific Northwest Fertility, Seattle, Washington; and c Stanford University, Stanford, California
Establishment of early pregnancy is the result of complex biochemical interactions between the decidua and blastocyst. Any alteration in this chemical dialogue has the potential to result in adverse pregnancy outcomes including miscarriage. Sporadic miscarriage is the most common complication of pregnancy and can be caused by multiple factors. While the most common cause of miscarriage is genetic abnormalities in the fetus, other contributing factors certainly can play a role in early loss. One such factor is environmental exposure, in particular to endocrine-disrupting chemicals, which has the potential to interfere with endogenous hormone action. These effects can be deleterious, especially in early pregnancy when the hormonal milieu surrounding implantation is in delicate balance. The purpose of this paper is to review the current evidence on the role of environmental toxins in reproduction. (Fertil SterilÒ 2016;-:-–-. Ó2016 by American Society for Reproductive Medicine.) Key Words: Miscarriage, endocrine disrupting chemicals, environment Discuss: You can discuss this article with its authors and with other ASRM members at
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poradic miscarriage, the loss of an embryo or fetus before 20 weeks of pregnancy, is the most common complication of pregnancy, affecting approximately 15% of all clinically recognized pregnancies (1). A much larger percentage of pregnancies are impacted if preclinical losses are included. Of these clinical pregnancy losses, a large majority occur during the first trimester. A majority of these first trimester losses are secondary to genetic abnormalities in the conceptus. However, sporadic, euploid losses do occur. Risk factors for pregnancy loss include but are not limited to advanced maternal age, hormonal imbalances, immunological interactions, and uterine anatomic abnormalities (1–4). Certainly, lifestyle factors such as tobacco smoke and alcohol usage are generally well accepted as contributing causes of miscarriage (5–7). Exposure to other
chemicals such as endocrine disru ptors, heavy metals, or occupational chemicals has also been implicated in miscarriage. The sensitivity of embryonic tissue or fetal tissue to such compounds is far greater than that of adult tissue. This is in part because of the small size and totipotent nature of many of the cells in the embryo or fetus (8, 9). A single insult at these early stages of development can have deleterious effects on development. Not only can environmental toxins impact the developing embryo, there is also a potential for alteration of the endometrium of pregnancy, that is, the decidua. Successful implantation requires a complex biochemical dialogue to occur between the blastocyst and decidua. These early interactions allow for implantation to occur and ultimately for establishment of pregnancy (10). Environmental alteration
Received May 13, 2016; revised and accepted June 29, 2016. S.A.K. has nothing to disclose. L.K.S. has nothing to disclose. R.B.L. has nothing to disclose. Reprint requests: Sacha A. Krieg, M.D., Ph.D., University Fertility Consultants, Oregon Health Sciences University, CH10F, 3303 SW Bond Avenue, Portland, Oregon 97239 (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.06.043
of these early uterine events can also disrupt an early pregnancy or potentially result in miscarriage or other adverse pregnancy outcomes. In particular, abnormal hormone signaling pathways could potentially affect not only the ability of a blastocyst to implant in the endometrium but also the ability of the decidua to communicate with fetoplacental unit in a normal way. Multiple industrial contaminants have the potential for endocrine disruption; these chemicals can impact the ability to become pregnant and sustain a healthy pregnancy. Other environmental contaminants have also been implied to have a causal effect on pregnancy loss; these include radiation exposure, heavy metals, agricultural chemicals, and industrial solvents. Given the scope of this review, our discussion will be limited largely to endocrine-disrupting chemicals (EDCs). EDC exposure may represent an additional cause(s) of sporadic or recurrent loss.
DDT Perhaps one of the most well-known EDCs is DDT, or 1,1,1-trichloro–
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ENVIRONMENTAL EXPOSURES: PREGNANCY, PLACENTA, AND MISCARRIAGE 2,20 bis(p-chorophenyl) ethane. DDT was first synthesized in 1874. However, it was not recognized as a pesticide until 1939, when it was first used during World War II for protecting the military from malaria. It then became commercially available in 1945. DDT had widespread use and is credited for eradicating malaria from the United States and Europe. While other pesticides began to emerge, DDT was still the most widely used pesticide until the mid-1960s, when environmental impacts started to become apparent. Unfortunately, part of its efficacy as an insecticide is due to its persistence in the environment. Because of its chemical stability and lipophilic nature, DDT persists in the environment and results in accumulation and even bioconcentration within the food chain. Even today, DDT is widespread in the environment; in fact some countries are still using DDT in vector control (8, 11). By the mid-1950s animal studies were beginning to suggest that DDT was causing poor reproductive outcomes. Effects were first noted in birds, with plummeting of some bird populations. The birds most affected are those that are at the top of the food chain, in particular those that are fish eating. One specific example is the Californian brown pelican. They were found to have 3,000 breeding pairs in 1960, but by 1969 there were only 300 pairs and only five chicks were viable (12, 13). This decline in bird populations became the impetus for Rachel Carson's Silent Spring. Other animal populations were affected such as fetal resorption in rabbits and miscarriage and stillbirth in rats. Ultimately, DDT was restricted or banned in most developing countries by 1970 (11, 14). Early human studies of DDT suggested a possible correlation with cancer in general. However, with time, reproductive disorders became apparent. Similar to the bird population, human population declines were secondary to reproductive failures. In vitro studies have shown that DDT has estrogenic activity (15). Dichlorodiphenyl dichloroethene (DDE), a metabolite of DDT, has been shown to be an androgen receptor antagonist. The reproductive repercussions of DDT use have been multiple; these include a suggested role in decline in sperm counts, increase in time to conception, and even intrauterine growth restriction (11, 16, 17). Spontaneous abortion has been correlated with DDT exposure in multiple studies. Given the stability of DDT and its metabolites (in particular DDE), it is possible to correlate serum levels with pregnancy history. Most human studies on miscarriage and DDT have correlated DDT or DDT metabolites with pregnancy outcomes. Some studies show limited impact of DDT on miscarriage (11, 18, 19). These studies are limited by the timing of collection of samples and the timing of observation of pregnancy losses. In these studies, DDT and/ or its metabolites were measured in blood or serum that was collected after the pregnancies had completed. The levels of DDT would have the greatest impact during the pregnancy, that is, during the exposure to the fetus. In addition, many of the samples were collected in the postpartum period when women were nursing their infants. Since breast feeding decreases the DDT levels in the serum (20), levels could be lower than what they were in pregnancy. In addition to timing of sample collections, many pregnancy
losses could have gone unaccounted for since these studies are retrospective in nature. Only prospective studies that collect samples and data throughout menstrual cycles and detect early preclinical losses with bhCG levels can account for all the pregnancy losses associated with high levels of serum DDT. However, these studies have been in patients with lower serum levels of DDT or DDE. Several studies have shown that there is also an increase in spontaneous miscarriage at higher concentrations of DDE (>15 mg/L) (11, 18, 19). One of the largest studies of DDT and DDE in relation to miscarriage to date was done by Longnecker and coworkers (20). They used serum from enrollees in the Collaborative Perinatal Project (1959–1965). These women were asked about their pregnancy history, and serum samples were frozen. Serum from 1,717 women who had a pregnancy history was analyzed for concentrations of DDT and DDE. Women were compared to a group who had serum DDE levels of <15 mg/L; exposures greater than 15 mg/L were divided into quartiles of increased exposure. Longnecker et al. found an adjusted odds ratio (OR) of miscarriage per 60 mg/L increase in DDE of 1.4 (95% confidence interval [CI], 1.1–1.6) (20). One flaw of these studies is that they are retrospective in nature and therefore are subject to recall bias. There are two studies that are prospective in nature that found similar effects. Korrick and coworkers found an OR for miscarriage of 1.13 (95% CI, 1.02–1.26) in a casecontrol study matching patients by age, smoking status, body mass index, and workplace exposures; they were then followed prospectively through pregnancy (18). Control patients had an ongoing pregnancy, whereas the cases had miscarried. Serum levels of DDT and DDT metabolites were measured. Patients who miscarried were found to have higher levels of DDE in their serum (22 vs. 12 ng/g) than controls (P¼ .025) (18). Another prospective study by Venners et al. followed Chinese textile workers prospectively with serum hCG levels (19). In this study DDT levels were drawn before conception. Venners et al. found that there was a relative odds of pregnancy loss of 1.17 (95% CI, 1.05–1.29) associated with a 10 ng/g increase in serum total DDT (19). Overall, these studies consistently show an increase in spontaneous abortion in patients with higher serum levels of DDT or DDT metabolites (Table 1) (11, 18, 20).
BISPHENOL A (BPA) BPA [2,2-bis(4-hydroxyphyenyl) propane] was first synthesized in 1891 as a synthetic estrogen for the pharmaceutical industry. It was taken out of production once a much more potent estrogen was developed, diethylstilbestrol. Then, in the 1950s, its production resumed as it was found to be useful as an epoxy resin and the basis for polycarbonate plastics. It is now one of the highest-volume chemicals produced worldwide. BPA is an estrogenic compound used in the manufacture of polycarbonate plastic and epoxy resins. The use of BPA is so widespread that it is estimated that 100 tons of this chemical are released into the atmosphere annually (24, 34). While BPA is used to manufacture plastics frequently used for food storage, studies have shown that VOL. - NO. - / - 2016
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TABLE 1 Endocrine disrupting chemicals and their reproductive outcomes. Compound
Mechanism
Oocyte effect
Endometrial effect
DDT
Estrogen receptor agonist, androgen receptor antagonist
Decrease in mature oocytes (zebrafish), increased granulosa aromatase activity (human)
Decreased proliferation and increased apoptosis
BPA
Estrogen agonist
Meiotic errors, epigenetic changes, early apoptosis
Altered gene expression of HOXA10, Era, ERb, B3integrin, and ITGB3
PCBs and dioxin
Mixed estrogen agonist/antagonist, androgen receptor agonist
Inhibit meiotic spindle assembly, prevent normal oocyte maturation
Altered expression of estrogen responsive genes
Phthalates
P receptor agonist in silico, interacts with PPAR with decrease in aromatase activity
Anovulation, abnormal granulosa steroidogenesis
Abnormal expression of Era, PR, and E-cadherin
Miscarriage
Reference
OR, 1.4 (1.1–1.6) per 60 mg/L increase (prospective, n ¼ 1,717); OR, 1.13 (1.02–1.26) (case control study); OR, 1.17 (1.05–1.29) prospective (n ¼ 372) Sporadic: OR, 1.97; recurrent: OR, 3.33 in highest quartile (n ¼ 115); highest concentration: recurrent spontaneous abortion OR, 9.34 (3.06–28.44; n ¼ 264) OR, 1.6–2.52 depending on type of PCB; Agent Orange relative risk, 0.99 (0.85– 1.16) Increased miscarriage OR, 2.87 (1.09– 7.57)
(18–23)
(24–27)
(28–30)
(31–33)
Krieg. Environmental toxins and miscarriage. Fertil Steril 2016.
this compound can leach into the food and, accordingly, be ingested. BPA is also used in thermal receipt paper and some dental sealants. Given that BPA is used in so many frequently used products it is not surprising that detectable levels of this compound are found in 90% of urine samples in a representative U.S. population that was tested (24, 34). The ubiquitous nature of BPA exposure is particularly concerning as it is a known EDC. Indeed, in the 1970s it was found to be a reproductive toxin by the national toxicity program. In 2008 Canada placed a limited ban on BPA, classifying it as a toxic substance. While the Food and Drug Administration declared that BPA is safe at current estimated levels of human exposure, retailers began pulling baby bottles and children's food containers that contained BPA in 2008. Meanwhile, the impact of continuous lowdose exposures remains in debate (34). BPA has been linked to reproductive disorders such as polycystic ovarian syndrome, endometriosis, infertility, and thyroid disease (24, 34, 35). Detectable concentrations of BPA have also been identified in reproductive tissues such as follicular fluid and amniotic fluid, suggesting that exposure can occur during the time of implantation and establishment of early pregnancy (24, 35). Alteration of expression of genes associated with implantation has been associated with BPA as BPA is able to weakly bind the estrogen receptor (24, 36). As a result of its estrogen agonist properties, an increase in expression of estrogen-regulated
genes is found with BPA. This includes genes such as HOXA10, B3integrin, and ITGB3. These genes are known to be important for normal implantation. BPA has also been shown to impact endocrine signaling by downregulating expression of ERa and PR. Endometrial angiogenesis is also impacted by BPA by downregulating vascular endothelial growth factor expression (24, 36). These gene expression data, taken together, suggest that BPA has the potential to disrupt implantation, with the potential downstream effects of miscarriage, antenatal complications, and even implications in adult health and disease. While many have questioned the impact of BPA upon fertility, development, and adult disease, the focus of this review will be upon miscarriage. It is known that mice exposed to BPA have a lower number of implantation sites (37). In fact, when mice are exposed to BPA during pregnancy, their offspring have abnormal endometrial stromal signaling pathways. BPA has also been shown to result in thinner endometrial lining in human studies, further supporting a negative impact on the deciduae (35). BPA has been associated with an increase in aneuploidy oocytes in murine studies, which could also be a cause of miscarriage. BPA has been shown in several animal models to affect oocyte meiosis, leading to aneuploidy (38). A similar result has been found in human oocytes during culture (39). This increase in oocyte aneuploidy can certainly lead to miscarriage. In fact, a recent study by Lathi and coworkers demonstrated a statistically
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ENVIRONMENTAL EXPOSURES: PREGNANCY, PLACENTA, AND MISCARRIAGE significant increase in both euploid and aneuploid losses in patients in the fourth quartile of urinary BPA concentration (25). The relative risk of loss for sporadic miscarriage was 1.97 (95% CI, 1.08–3.59) and for recurrent loss was 3.33 (95% CI, 1.04–10.71) in the highest quartile of serum BPA concentration (25). Similarly, another group has demonstrated an increase in idiopathic miscarriage in patients with higher urinary BPA levels, but the sample size was small (n ¼ 45) and there was no fetal karyotyping performed (40). Shen and coworkers found that a higher urinary BPA level is associated with recurrent miscarriage with an OR of 3.91 (95% CI, 1.23–12.45) in the 0.4–0.93 mg/g Cr group and an OR of 9.34 (95% CI, 3.06–28.44) in the .93 mg/g Cr group adjusting for confounding factors; however, these samples were not collected during pregnancy for the miscarriage group (26). Taken together, BPA has been shown in laboratory models to increase the risk of miscarriage at the level of both the endometrium and the oocyte. These molecular changes as a result of BPA exposure do appear to confer an increased risk of miscarriage in patients with higher BPA accumulation. These preliminary data suggest a critical need for larger epidemiological studies to confirm an association between BPA exposure and miscarriage.
PCBs Dioxin-related toxins including polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans are ubiquitous contaminants of industrial combustion processes. PCBs were first introduced in the late 1920s, but by the early 1970s many countries began banning or restricting their use. Dioxins are perhaps one of the most infamous members of this group, having a well-accepted role in carcinogenesis. These compounds are highly lipophilic, resulting in accumulation and concentration through the food chain. Unfortunately, because of their resistance to degradation, these compounds still persist in the environment, despite their use being banned (28, 41). These compounds can act not only as estrogen agonists or antagonists but also can have androgenic properties. PCBs have been shown to inhibit meiotic spindle assembly in vitro (42). These chemicals also hamper maturation of oocytes in vitro. Both of these effects have the potential to cause miscarriage. The endometrium also appears to be impacted by PCBs. In vitro endometrial cultures show an alteration in estrogen-responsive genes upon exposure to PCBs (29). Given that the PCBs including dioxin have the potential to alter both normal oogenesis and endometrial function, how is pregnancy impacted? Dioxin exposure in pregnancy has been associated with fetal loss and low birth weight in rodents and monkeys. PCB exposure has also been associated with a developmental syndrome involving hydronephrosis, cleft palate, and thymic atrophy in mice (43). Monkeys exposed to PCBs have an increased number of miscarriages and higher rates of fetal demise (44). Human studies generally show similar findings. One example of PCB toxicity is Yusho oil disease. In 1968 in western Japan, rice oil was contaminated with PCBs and other dioxin-
related compounds. As a result over 1,900 people presented with clinical symptoms including acne-like eruptions, pigmentation changes, eye discharge, and paresthesias. More than 500 people died as a result. The levels of PCBs and dioxins remained elevated for a prolonged period of time in these individuals. In a study done by Tsukimori et al., a statistically significant increase in miscarriage was noted, with OR ranging from 1.6 (95% CI, 1.1–2.33) to 2.52 (95% CI, 0.92–6.87) depending on the type of PCB measured (28). This study included interviews with 214 women (512 pregnancies) regarding pregnancy outcomes over 36 years; these patients had also had blood levels of PCBs measured. Other groups have shown an increase in low birth weight and growth restriction in this population. The Yusho oil incident is not isolated; similarly the Yu-sheng accident and Vietnamese exposure to Agent Orange have shown an increase in spontaneous abortion (30, 45). After an explosion at a chemical factory in Seveso, Italy, that involved dioxin, no increase in miscarriage after evaluating pregnancy outcomes in 510 women was observed (41). This could be explained by different concentrations of dioxin, PCBs, and dibenzofurans in these exposures and the timing of exposure. Another dioxin-related compound is Agent Orange, a dioxin-contaminated defoliant used during the Vietnam War. Agent Orange is a mixture of the herbicides 2.4-D [(2,4-dichlorophenoxy)acetic acid] and 2,4,5-T [(2,4,5-trichlorophenoxy)acetic acid]. During the Vietnam War, Agent Orange was contaminated with 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). A systematic review by Ngo et al. showed that Agent Orange appeared to be associated with an increased risk of birth defects (46). Later epidemiological studies demonstrated an increase in spina bifida after paternal exposure to Agent Orange. There are conflicting data regarding Agent Orange and risk of spontaneous abortion. While many studies have been limited by small sample size or inadequate exposure, the data are inconsistent. Most studies have evaluated paternal exposure; however, studies of exposed Vietnamese women have shown an increase in miscarriage and premature deliveries (47). Larger studies have found a trend toward increased risk of spontaneous abortion in wives of veterans with low exposure but not at high exposure levels (48). A larger crosssectional study of 281 workers exposed to TCDD during production of Agent Orange, showed primary outcomes of increase in stillbirth, spontaneous abortion, and sex ratio. In this study no increase was seen in any of these outcomes (45). However, this study was vulnerable to recall bias, and it occurred over a time period when early pregnancy tests were not available (1950–1960s), so early losses would not be captured. While PCBs represent a large class of chemicals, it appears from largely retrospective studies that they are associated with an increase miscarriage and other adverse reproductive outcomes. Further longitudinal studies are needed to further understand the reproductive repercussions of PCB use not only on pregnancy complication but also on the effect on offspring. VOL. - NO. - / - 2016
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PHTHALATE AND PHTHALATE METABOLITES Phthalates are frequently used in plastic manufacturing and are used to describe multiple phthalate monoesters. This group of plasticizers is used to make plastic more flexible and is found in many personal care products as well as medical supplies. In fact, exposures during medical interventions have been found to be up to 150 times the baseline exposure risks per day as phthalates are used in medical supplies such as IV tubing and bags. Globally, more than 18 billion pounds of phthalates are used annually. Phthalates have been associated with developmental abnormalities of the male reproductive system, miscarriage, endometriosis, and low sperm counts. The mechanism by which phthalates cause adverse reproductive outcomes includes P-like effects; in fact, di-(2ethyl)phthalate (DEHP) has been modeled in silico to interact with the P receptor. Other studies have shown in vitro that some forms of phthalates activate the PPARs and ultimately decrease aromatase activity (31, 32, 49). These possible P-like effects and decrease in aromatase activity have been theorized to contribute to adverse reproductive outcomes. In rat models, multiple studies have shown ovarian toxicity stemming from DEHP exposure. While rodent studies may be suggestive of human health risks, they allow the observation of titrated effects. Administration of DEHP to female rats has shown a decrease in serum E2 levels and an increase in anovulation (50). Histologically, these rats showed cystic follicles with abnormal granulosa cells and smaller follicle size. In human studies of patients undergoing IVF, a lower number of mature oocytes are found in patients with higher phthalate concentrations (51). These patients are also found to have lower implantation rates. In the mouse endometrium, titrated phthalate dosages are found to cause abnormal gene expression of ERa, PR, and E-cadherin. These gene products are important for normal implantation, and, concordantly, these mice had a lower number of implantation sites (52). In rodents that did become pregnant, a decrease in the number of litters, litter size, and proportion of live-born offspring was observed after DEHP exposure (53). Another form of phthalate, di-n-butyl phthalate, was found to confer an increase in midpregnancy miscarriage in rats. Chronically high exposure levels associated with manufacture have been linked to miscarriage and lower fertility rates in humans (54). Similarly, in humans, elevated urinary concentration of the phthalate monoethylhexyl phthalate around the time of conception was associated with an increase in miscarriage, with an OR of 2.87 (95% CI, 1.09, 7.57) in the highest quartile of exposure (54). Mu and coworkers also found an increase in miscarriage rates after measuring eight different urinary phthalate metabolites, dividing patients into four different quartiles of exposure. Participants falling into the first and fourth quartile of exposure had an increase in miscarriage (33). Exposure to phthalates has other effects in pregnancy. Elevated concentrations of phthalates have also been associated with adverse antenatal complications such as preeclampsia and gestational anemias (55). In utero exposure to DEHP in particular is associated with abnormal reproductive development (56, 57). These developmental abnormalities include
early breast development, shortened anogenital distance, and cryptorchidism, among others (33). Many of these effects are thought to be secondary to aromatase inhibition or other hormonal mimicry. As EDCs that have widespread use, phthalates appear to have an impact not only on miscarriage rates but also on offspring. Further studies with larger sample sizes are needed to delineate these effects.
CONCLUSIONS Environmental contaminants have been suggested to play a role in spontaneous miscarriage. The focus of this review was limited to endocrine disruptors. This group of chemicals, which are able to interact with hormone receptors, is known to impact wildlife, laboratory animals, and humans. Some of the effects on wildlife are profound, including marked decreases in population, inability to reproduce, physical changes in the reproductive organs, birth defects, and miscarriage. These exposures may occur in the workplace, from ingesting contaminated food, while receiving medical treatment (i.e., IV tubing), or from the pesticides/herbicides used in agriculture or for pest control. Many of these exposures are cumulative and as a result can be bioconcentrated in the food chain. The reproductive outcomes impacted by endocrine disruptors are many, but the focus of this review paper is on miscarriage. Many studies have shown a dose-dependent increase in miscarriage with these endocrine disrupters (Table 1). There are some considerations when interpreting these data. Many of these studies rely on small sample sizes and often rely on recall of pregnancy history. These are obvious confounders in interpreting the data. That being said, many of these compounds are extremely stable, allowing researchers to measure serum or urinary levels reliably. These studies suggest that women of reproductive age should exercise caution in exposure to these endocrine disruptors. Unfortunately, these compounds are ubiquitous in the environment and are often difficult to avoid. One concern with the ubiquitous nature of these chemicals is that many of these studies could be confounded by the presence of multiple chemicals. Ideally, multiple endocrine disruptors would be assayed for, permitting an assessment of the actual compound(s) causing an effect. Also there is the potential that these EDCs could behave in a synergistic manner, exacerbating a preexisting effect. These studies show the need and importance of more prospective studies of adequate sample size and design so that we can understand the full extent of the impact of these hormone-like compounds on the establishment and maintenance of pregnancy.
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