9

Effects on Veterans’ Fertility and Reproductive Success

Chapter Overview

Based on new evidence and a review of prior studies, the committee for Update 2014 did not find any new significant associations between the relevant exposures and fertility or gestational outcomes. The current evidence supports the findings of earlier studies that

• None of the fertility or gestational outcomes had sufficient evidence of an association with the chemicals of interest.
• None of the fertility or gestational outcomes had limited or suggestive evidence of an association between the chemicals of interest.
• There is inadequate or insufficient evidence to determine whether there is an association between the chemicals of interest and endometriosis; decreased sperm counts or sperm quality, subfertility, or infertility; spontaneous abortion, stillbirth, neonatal death, or infant death; and low birth weight or preterm delivery.
• There is limited or suggestive evidence of no association between paternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and spontaneous abortion.

This chapter summarizes the scientific literature published since Veterans and Agent Orange: Update 2012,¹ hereafter referred to as Update 2012 (IOM,

________________

¹Despite loose usage of “Agent Orange” by many people, in numerous publications, and even in the title of this series, this committee uses “herbicides” to refer to the full range of herbicide exposures experienced in Vietnam, while “Agent Orange” is reserved for a specific one of the mixtures sprayed in Vietnam.

2014), on the association between exposure to herbicides and adverse effects on fertility and during gestation. (The analogous shortened names are used to refer to the updates for 1996, 1998, 2000, 2002, 2004, 2006, 2008, and 2010 [IOM, 1996, 1999, 2001, 2003, 2005, 2007, 2009, 2011] of the original report Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam [VAO; IOM, 1994].) The literature considered in this chapter includes studies of a broad spectrum of reproductive effects in Vietnam veterans and in other populations exposed occupationally or environmentally to the herbicides sprayed in Vietnam or to TCDD. Because some polychlorinated biphenyls (PCBs), some polychlorinated dibenzofurans (PCDFs), and some polychlorinated dibenzodioxins (PCDDs) other than TCDD have dioxin-like biologic activity, studies of populations exposed to PCBs or PCDFs were reviewed if their results were presented in terms of TCDD toxic equivalents (TEQs). Although all studies reporting TEQs based on PCBs were reviewed, those studies that reported TEQs based only on mono-ortho PCBs (which are PCBs 105, 114, 118, 123, 156, 157, 167, and 189) were given very limited consideration because mono-ortho PCBs typically contribute less than 10 percent to total TEQs, based on the World Health Organization’s (WHO’s) revised toxicity equivalency factors (TEFs) of 2005 (La Rocca et al., 2008; van den Berg et al., 2006).

The adverse outcomes evaluated in this chapter include impaired fertility (in which declines in sperm quality may be involved), endometriosis, increased fetal loss (spontaneous abortion and stillbirth), neonatal and infant mortality, and the adverse gestational outcomes of low birth weight and preterm delivery. In this update, consideration of the possibility of adverse health outcomes at any time during the lives of all progeny of Vietnam veterans has been moved to a separate chapter: Chapter 10, “Effects on Veterans Descendants.”

Because the vast majority of Vietnam veterans are men, the primary focus of the VAO series has been on potential adverse effects of herbicide exposure on men, and the etiologic importance of the exposed party’s sex does not play the same dominant role in nonreproductive outcomes that it does in reproductive outcomes. However, about 8,000 women served in Vietnam (H. Kang, US Department of Veterans Affairs, personal communication, December 14, 2000), so findings relevant to female reproductive health, such as those concerning endometriosis, are also included in the present chapter. Whenever the information was available, an attempt has been made to evaluate the effects of exposure on adult men and women separately.

The categories of association and the approach to categorizing the health outcomes are discussed in Chapters 1 and 2. To reduce repetition throughout the report, Chapter 6 characterizes study populations and presents design information related to new publications that report findings on multiple health outcomes or that revisit study populations considered in earlier updates.

BIOLOGIC PLAUSIBILITY OF EFFECTS ON
FERTILITY AND REPRODUCTION

This chapter opens with a general discussion of the plausibility of the various suggested adverse reproductive effects of TCDD and the four herbicides used in Vietnam. There have been few reproductive studies of the four herbicides in question, particularly picloram and cacodylic acid, and those studies generally have shown toxicity only at very high doses, so the preponderance of the following discussion concerns TCDD, which other than in controlled experimental circumstances, usually occurs in a mixture of dioxins (dioxin congeners in addition to TCDD).

TCDD is stored in fat tissue and has a long biologic half-life, so internal exposure at generally constant concentrations may continue after an episodic, high-level exposure to an external sources ceases. If a person had a high exposure, then high amounts of dioxins may still be stored in fat tissue and be mobilized, particularly at times of weight loss. That would not be expected to be the case for nonlipophilic chemicals, such as cacodylic acid.

Dioxin exposure has the potential to disrupt male reproductive function by altering gene expression that is pertinent to spermatogenesis and by altering steroidogenesis (Wong and Cheng, 2011) and to disrupt female reproductive function by altering gene expression relevant to ovarian follicle growth and maturation, uterine function, placental development, and fetal morphogenesis and growth.

A father’s direct contribution to a pregnancy is limited to the contents of the sperm that fertilizes an egg; those contents had long been thought to consist of greatly condensed, transcriptionally inert deoxyribonucleic acid (DNA) constituting half the paternal genome (a haploid set of chromosomes). Consequently, it was believed that paternally derived damage to the embryo or offspring could only result from changes in sperm DNA, and dioxins have not been shown to mutate DNA sequence. However, as discussed in Chapter 4, TCDD can have epigenetic effects that modify expression of a cell’s genetic material that persist in the daughter cells following cell division, whether the division involves an individual’s own somatic tissues or production of his (or her) gametes. This provides an alternative pathway to creating permanent (heritable) changes in gene expression without altering the DNA sequence. Epigenetic changes include chemical modifications made to DNA (usually involving methylation) or to other cellular components such as histones and RNAs (Jirtle and Skinner, 2007). As a sperm matures, most of its histones are replaced by protamines, which renders it transcriptionally quiescent and permits extensive DNA compaction. The core histones that are retained in human sperm carry epigenetic modifications to maintain open nucleosomes, which permits transcription of genes that are important during embryo development (Casas and Vavouri, 2014). Sperm also carry a considerable collection of ribonucleic acid (RNA) fragments (Kramer and Krawetz, 1997; Krawetz et al., 2011) including ribosomal RNAs (rRNAs), messenger

RNAs (mRNAs), and small noncoding RNAs (miRNAs and piRNAs) (Casas and Vavouri, 2014; Lane et al., 2014). Small RNAs have been found to play critical roles in fertilization (Amanai et al., 2006), early embryonic development (Hamatani, 2012; Suh and Blelloch, 2011), and epigenetic modifications (Gapp et al., 2014; Kawano et al., 2012). Therefore, male infertility or fetal loss associated with exposure to the chemicals of interest (COIs) might be mediated by epigenetic modifications to components of sperm other than their DNA (Krawetz, 2005).

A mother’s contribution to a pregnancy is obviously more extensive, and damage to an embryo or offspring can result from epigenetic changes in the egg DNA or from the direct effects of exposure on placenta formation and the fetus during gestation. The mobilization of dioxin during pregnancy may be increased because the body is drawing on fat stores to supply nutrients to the developing fetus. TCDD has been measured in human circulating maternal blood, cord blood, and placenta. Thus, dioxin in the mother’s bloodstream could cross the placenta and expose the developing embryo and fetus. Data indicate that dioxin can accumulate in placental tissue, but the amount of TCDD that can transfer to the fetus appears to be very limited—TCDD’s transfer index was the lowest of 13 environmental toxicants evaluated in perfusion studies of human placentas (Mose et al., 2012).

On the basis of laboratory animal studies, it is known that TCDD can affect reproduction, so a connection between TCDD exposure and human reproductive and gestational effects is biologically plausible. However, making definitive conclusions based on animal studies about the potential for TCDD to cause reproductive and gestational toxicity in humans is complicated by differences in sensitivity and susceptibility among different species including strain-specific differences; by the lack of strong evidence of organ-specific effects across species; by differences in the route, dose, duration, and timing of exposure in experimental protocols and real-world exposure; and by substantial differences between laboratory animals and humans in the toxicokinetics of TCDD. Experiments with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) indicate that these chemicals have subcellular effects that could constitute a biologically plausible mechanism for reproductive and gestational effects. However, the preponderance of evidence from animal studies indicates that these chemicals do not have reproductive effects. There is insufficient information on picloram and cacodylic acid to assess the biologic plausibility of their potential reproductive or gestational effects.

The sections on the biologic plausibility of the specific outcomes considered in this chapter present more detailed toxicologic findings that are of particular relevance to the outcomes discussed.

ENDOMETRIOSIS

Endometriosis (International Classification of Diseases, 9th revision [ICD-9], code 617) affects 5.5 million women in the United States and Canada at any

given time (NICHD, 2007). The endometrium, the tissue that lines the inside of the uterus, is built up and shed each month during menstruation. In endometriosis, endometrial cells are found outside the uterus—usually in other parts of the reproductive system, in the abdomen, or on surfaces near the reproductive organs. The ectopic tissue develops into growths or lesions that continue to respond to hormonal changes in the body and break down and bleed each month in concert with the menstrual cycle. Unlike blood released during normal shedding of the endometrium, blood released from endometrial lesions has no way to leave the body and results in inflammation and internal bleeding. The degeneration of blood and tissue can cause scarring, pain, infertility, adhesions, and intestinal problems.

There are several theories of the etiology of endometriosis, including one that posits a genetic contribution, but the cause remains unknown. Estrogen dependence and immune modulation are established features of endometriosis but do not adequately explain its cause. It has been proposed that endometrium is distributed through the body via blood or the lymphatic system; that menstrual tissue backs up into the fallopian tubes, implants in the abdomen, and grows; and that all women experience some form of tissue backup during menstruation but only those who have immune-system or hormonal problems experience the tissue growth associated with endometriosis. Despite numerous symptoms that can indicate endometriosis, diagnosis is possible only through laparoscopy or a more invasive surgical technique. Several treatments for endometriosis are available, but there is no cure.

Conclusions from VAO and Previous Updates

Endometriosis was first reviewed in this series of reports in Update 2002, which identified two relevant environmental studies. Additional studies considered in later updates have not changed the conclusion that the evidence is inadequate or insufficient to support an association with herbicide exposure. Table 9-1 provides a summary of relevant studies that have been reviewed.

Update of the Epidemiologic Literature

No Vietnam-veteran, occupational, or case-control studies of exposure to the COIs and endometriosis have been published since Update 2012.

Environmental Studies

Upson et al. (2013) measured persistent organic pollutants in a subset of women enrolled in the Women’s Risk of Endometriosis Study. An increased risk of endometriosis was observed for exposure to β-hexachlorocylohexane (HCH), which is a component of the insecticide lindane, and mirex, but the authors did not measure the COIs.

TABLE 9-1 Selected Epidemiologic Studies—Endometriosis

NOTE: BMI, body mass index; CALUX, chemical activated luciferase gene expression; CI, confidence interval; dl, dioxin-like; DLC, dioxin-like compound; GA, Georgia; GC/MS, gas chromatography/mass spectrometry; OR, odds ratio; PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzo-p-dioxin; PCDF, polychlorinated dibenzofuran; RR, relative risk or risk ratio; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEQ, (total) toxic equivalent.

Biologic Plausibility

Laboratory studies that used animal models and examined gene-expression changes associated with human endometriosis provide evidence of the biologic plausibility of a link between TCDD exposure and endometriosis. Genetic polymorphisms in the aryl hydrocarbon receptor (AHR) signaling complex have recently been associated with a susceptibility to advanced endometriosis in humans (Li Y et al., 2013; Wu et al., 2012), although another recently published study found no association in Japanese women (Matsuzaka et al., 2012). The first suggestion that TCDD exposure may be linked to endometriosis came as a secondary finding of a study that exposed female rhesus monkeys (Macaca mulatta) chronically to low concentrations of dietary TCDD for 4 years (Bowman et al., 1989). Ten and 13 years after the exposure ended, the investigators documented an increased incidence of endometriosis in the monkeys that correlated with the TCDD exposure concentration (Rier et al., 1993, 2001). The sample was too small to,

yield a definitive conclusion that TCDD was a causal agent of endometriosis, but this study led to additional studies of the ability of TCDD to promote the growth of pre-existing endometriotic lesions (Bruner-Tran et al., 1999; Johnson et al. 1997; Yang et al., 2000).

There are a number of mechanisms by which TCDD may promote endometrial lesions, which provide additional support of the biologic plausibility of a link between TCDD and endometriosis. Human endometrial tissue and cultured human endometrial epithelial cells both express the AHR; its dimerization partner, the aryl hydrocarbon nuclear translocator (Khorram et al., 2002); and three AHR target genes—CYP1A1, 1A2, and 1B1 (Bulun et al., 2000; Willing et al., 2011). These findings suggest that endometrial tissue is responsive to TCDD. MN Singh et al. (2008) showed that CYP1A1 expression is greater in ectopic endometrial tissue than in eutopic uterine tissue in the absence of TCDD exposure, which suggests that CYP1A1 may play a role in the etiology of the disease or that AHR and its signaling pathway have been activated by an endogenous ligand other than TCDD, such as bilirubin (Seubert et al., 2002). Other mechanisms by which TCDD may promote endometriosis include altering the ratio of progesterone receptor A to progesterone receptor B and blocking the ability of progesterone to suppress matrix metalloproteinase expression—actions that promote endometrial-tissue invasion and that are observed in women who have endometriosis (Igarashi et al., 2005).

TCDD also induces changes in gene expression that mirror those observed in endometrial lesions. In addition to the induction of CYP1A1 noted above, TCDD can induce expression of histamine-releasing factor, which is increased in endometrial lesions and accelerates their growth (Oikawa et al., 2002, 2003). TCDD disrupts cannabinoid signaling in endometrial stromal cells by inhibiting progesterone-induced expression of cannabinoid receptor type 1 (CB1-R), which is also observed in women with endometriosis (Resuehr et al., 2012). TCDD also stimulates the expression of RANTES (regulated on activation, normal Tcell–expressed, and secreted protein) in endometrial stromal cells, and RANTES concentration and bioactivity are increased in women who have endometriosis (Zhao et al., 2002). The two CC-motif chemokines (chemotactic cytokines), RANTES and macrophage-inflammatory protein 1α (MIP-1α), have been identified as potential contributors to the pathogenesis and progression of endometriosis. Previous studies have shown that the combination of 17β-estradiol and TCDD increases the secretion of RANTES and MIP-1α in endometrial stromal cells (Yu et al., 2008), and a more recent study showed that the same combination suppresses the expression of tetraspanin CD82, a tumor-metastasis suppressor, and thus promotes the invasion of endometrial stromal cells (Li MQ, 2011). Those results support the idea that TCDD in combination with estradiol may contribute to the development of endometriosis by increasing the invasiveness of endometrial cells. Despite that evidence, chronic exposure of rats to TCDD, PCB 153, dioxin-like PCB 118 or PCB 126, or 2,3,4,7,8-PeCDF (the furan congener,

with the highest TEF), either individually or in various combinations, fails to alter endometrial histology in a consistent manner (Yoshizawa et al., 2009). The differences between rodent and human endometrium could account for the lack of observed effects in rats.

In summary, experimental studies, particularly ones that used human eutopic and ectopic endometrial tissue, provide evidence of the biologic plausibility of a link between TCDD exposure and endometriosis.

Synthesis

The studies linking dioxin exposure with endometriosis are few and inconsistent. Although animal studies support the biologic plausibility of an association, contemporary human exposures may be too low to show an association consistently.

Conclusion

On the basis of the evidence reviewed here, in VAO, and in the previous VAO updates, the committee concludes that there is inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and human endometriosis.

FERTILITY

Male reproductive function is under the control of a variety of components whose proper coordination is important for normal fertility. Several of these components and some health outcomes related to male fertility, including reproductive hormones and sperm characteristics, can be studied as indicators of fertility. The reproductive neuroendocrine axis involves the central nervous system, the anterior pituitary gland, and the testis. The hypothalamus integrates neural inputs from the central and peripheral nervous systems and regulates the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Both are secreted into the circulation in episodic bursts by the anterior pituitary gland and are necessary for normal spermatogenesis. In the testis, LH interacts with receptors on Leydig cells, where it stimulates increased testosterone synthesis. FSH and the testosterone from the Leydig cells interact with Sertoli cells in the seminiferous tubule epithelium to regulate spermatogenesis. A more detailed review of the male reproductive hormones can be found elsewhere (Strauss and Barbieri, 2013). Several agents, such as lead and dibromochloropropane, affect the neuroendocrine system and spermatogenesis (for reviews, see Schrader and Marlow, 2014; Sengupta, 2013). Reviews on the effects of various environmental toxicants, including TCDD, on testicular steroidogenesis and spermatogenesis provide insights into potential underlying mechanisms, including reducing

testosterone production in Leydig cells and inhibiting the formation of cAMP (Mathur and D’Cruz, 2011; Svechnikov et al., 2010).

Studies of the relationship between chemicals and fertility are less common in women than in men. Some chemicals may disrupt the female hormonal balance necessary for proper functioning. Normal menstrual-cycle functioning is also important in the risk of hormonally related diseases, such as osteopenia, breast cancer, and cardiovascular disease. Chemicals can have multiple effects on the female system, including modulation of hormone concentrations that result in menstrual-cycle or ovarian-cycle irregularities, changes in menarche and menopause, and impairment of fertility (Bretveld et al., 2006a,b).

Conclusions from VAO and Previous Updates

The committee responsible for the original VAO report (IOM, 1994) concluded that there was inadequate or insufficient evidence of an association between exposure to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid and alterations in sperm characteristics or infertility. Additional information available to the committees responsible for Update 1996, Update 1998, Update 2000, Update 2002, Update 2004, Update 2006, Update 2008, Update 2010, and Update 2012 did not change the conclusion that exposure to the COIs had not been found to be associated with impaired fertility in either men or women. Reviews of the relevant studies are presented in the earlier reports. Tables 9-2 and 9-3 summarize the studies related to male and female fertility, respectively.

Update of the Epidemiologic Literature

Male Fertility

Ferguson et al. (2012) reported on a number of male fertility markers in a study of 358 men seeking infertility treatment with their partners. Of the four PCB congeners reported on individually, only the common, mono-ortho PCB 118 has dioxin-like activity, but results were reported for a group of PCBs (95/66, 74, 77/110, 105/141, 118, 156, 167, 128, 138, 170) characterized by Wolff et al. (1997) as having antiestrogenic and dioxin-like activity (although only the four congeners bolded in the preceding list are recognized by WHO as having dioxin-like activity). After adjusting for age, BMI, and serum lipids, inverse relationships were observed for PCB 118 with steroid hormone binding globulin (SHBG) (β = −0.13, p < 0.01) and with total testosterone (β = −22.1, p = 0.08). A similar, but weaker pattern was seen for the group including dioxin-like PCBs with SHBG (β = −0.08, p = 0.08) and with total testosterone (β = −25.9, p = 0.09); it is not clear what impact on fertility might arise from these at-best marginally significant changes in a PCB grouping of questionable relevance with respect to dioxin-like activity.

TABLE 9-2 Selected Epidemiologic Studies—Male Fertility (Altered Hormone Concentrations, Decreased Sperm Counts or Quality, Subfertility, or Infertility) (Shaded entry is new to this update)

NOTE: 2,4-D, 2,4-dichlorophenoxyacetic acid; AFHS, Air Force Health Study; AHR, aryl hydrocarbon receptor; CALUX, assay for determination of dioxin-like activity; CDC, Centers for Disease Control and Prevention; CI, confidence interval; COI, chemicals of interest; dl, dioxin-like; DLC, dioxin-like chemical; DNA, deoxyribonucleic acid; FSH, follicle-stimulating hormone; ICD, International Classification of Diseases; IU, international unit; LH, luteinizing hormone; ln, natural logarithm; nr, not reported; PAH, polycyclic aromatic hydrocarbon; PCB, polychlorinated biphenyl; POP, persistent organic pollutants; SEA, Southeast Asia; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEF, toxicity equivalency factor; TEQ, (total) toxic equivalent; VOC, volatile organic compound.

^aGiven when available; results other than estimated risk explained individually.

^bFor this study, relative risk has been replaced with standardized fertility ratio, for which value less than 1.0 indicates adverse effect.

^cFor this study, relative risk has been replaced with fecundability ratio, for which value less than 1.0 indicates adverse effect.

^dTable 1 in reference reverses these figures—control, 82.9%; exposed, 37.1%—but text (“The percentages of asthenospermia, mobility, necrosperma and teratospermia were greater in the exposed group than in controls…”) suggests that this is a typographical error.

TABLE 9-3 Selected Epidemiologic Studies—Female Fertility (Altered Hormone Concentrations, Subfertility, or Infertility)

NOTE: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; BMI, body mass index; CATI, computer-assisted telephone interviewing; CI, confidence interval; HR, hazard ratio; IARC, International Agency for Research on Cancer; IQR, inter-quartile range; OR, odds ratio; PCB, polychlorinated biphenyl; ppt, parts per trillion; TCDD, 2,3,7,8–tetrachlorodibenzo- p-dioxin; TCDF, tetrachlorodibenzofuran; TEQ, (total) toxic equivalent.

^aGiven when available; results other than estimated risk explained individually.

Female Fertility

No Vietnam-veteran, occupational, environmental, or case-control studies of exposure to the COIs and female fertility have been published since Update 2012.

Biologic Plausibility

Although a recent study reported that doses of 2,4-D greater than 50 mg/kg/day produce acute testicular toxicity in male rats (Joshi et al., 2012), there is little evidence that lower doses of either 2,4-D or 2,4,5-T (when free of TCDD contamination) given chronically have substantial effects on reproductive organs or fertility (Charles et al., 2001; Munro et al., 1992). The no-observed-adverse-effect level [NOAEL] for 2,4-D is recognized as 15 mg/kg/day (Gervais et al., 2008). In contrast, many diverse laboratory studies have provided evidence that TCDD can affect reproductive-organ function and reduce fertility in both males and females.

The administration of TCDD to male animals elicits reproductive toxicity by affecting testicular, epididymal, prostate, and seminal vesicle weight and function and by decreasing the rate of sperm production (Foster et al., 2010; Rider et al., 2010; Schneider et al., 2014). The mechanisms underlying those effects are not known, but the primary hypotheses are that they are mediated through the dysregulation of testicular steroidogenesis, altered Sertoli cell function, and increased oxidative stress. The exposure of cultured testicular Leydig cells to 25 nM TCDD markedly alters gene expression (Naville et al., 2011), and the exposure of cultured Sertoli cells to 5 nM TCDD decreases viability and increases markers

of oxidative stress (Aly and Khafagy, 2011). The exposure of adult rats or mice to TCDD (2–7 μg/kg/week for 45–60 days) reduces testicular and reproductive function, and these effects can be attenuated by co-treatment with various antioxidants (Beytur et al., 2012; Ciftci et al., 2012; Sönmez et al., 2011; Yin et al., 2012). The results of those studies are supported by the transgenic mouse model that harbors a constitutively active AHR in which testicular and ventral prostate weights and sperm number are reduced (Brunnberg et al., 2011).

Many studies have examined the effects of TCDD on the female reproductive system. Two primary mechanisms that probably contribute to abnormal follicle development and decreased numbers of ova after TCDD exposure are the “cross-talk” of the AHR with the estrogen receptor and the dysregulation of the hypothalamic–pituitary–gonadal axis (Pocar et al., 2005; Safe and Wormke, 2003). Oocytes are directly responsive to TCDD, so TCDD’s effects on hormone concentrations, hormone-receptor signaling, and ovarian responsiveness to hormones all probably contribute to TCDD-induced female reproductive toxicity. The data of Jung et al. (2010) in rats show that a single gavage treatment of 32 μg/kg TCDD reduces the proliferation of granulosa cells and thus attenuates cell-cycle progression and potentially contributes to the reduction in ovulation rates observed in other studies. In contrast, Karman et al. (2012) found that 1 nM TCDD exposure in vitro did not reduce the rates of growth of murine antral follicles, but did reduce the secretion of progesterone and estradiol by the follicles. The concentrations of those hormones could be restored by the addition of the precursor pregnenolone, which suggests that TCDD acts upstream of pregnenolone formation. This would be consistent with previous observations in zebrafish that 10, 40, and 100 ppb TCDD in food depressed estradiol biosynthesis (Heiden et al., 2008).

The effects on early embryo development and the effects on placenta formation attributable to dioxin are well documented (Chen et al., 2010; Ishimura et al., 2009; Tsang et al., 2012). Petroff et al. (2011) used a rat in vitro fertilization model to demonstrate that 100 nM TCDD perturbs chromatin and cytoskeletal remodeling at the earliest stages of embryo development, but these changes failed to result in any apparent morphologic changes at later stages of development. The long-term potential effects of these early changes on pregnancy outcome are unknown. It has previously been shown that TCDD may have direct effects on human trophoblast formation at 0.2–2.0 nM in vitro and thus may have the capacity to influence the developing fetus (Chen et al., 2010). That idea is supported by a study showing the ability of 5 nM TCDD to activate the AHR signaling pathway in both rat and human placental trophoblasts (Stejskalova et al., 2011). Finally, a study has demonstrated that TCDD at 0.1, 1.0, and 10.0 nM reduces in a dose-dependent fashion the ability of trophoblastic spheroids (which constitute an embryo surrogate) to attach to endometrial epithelial cells (Tsang et al., 2012). The more recent literature continues to support the biologic plausibility of TCDD having effects on male and female reproduction.

Sex Ratio

Although it would not constitute an adverse health outcome in an individual veteran, perturbations in the sex ratio of children born to an exposed population would suggest that the exposure had an impact on the reproductive process. As shown in Table 9-4, studies of the sex ratios observed among children born to people exposed during the 1976 Seveso accident (Mocarelli et al., 1996, 2000) suggested that paternal exposure to dioxin results in a lower sex ratio (i.e., a smaller-than-expected proportion of male infants at birth), particularly when the father was exposed early in his life (sex ratio [SR] = 0.382). However, a consideration of all 481 singleton births in 1994–2005 to women who resided in Zones A and B and were less than 28 years old at the time of the Seveso accident (ages 18–46 years at the beginning of period when births were identified) generated crude sex ratios showing that male births slightly exceeded female births in Zones A and B (SR = 0.516) and that the increase (SR = 0.571) was more pronounced for the 56 births in Zone A (Baccarelli et al., 2008).

A similar depression in the sex ratio concentrated in fathers who were under 20 years old at the time of the Yucheng poisoning with oil contaminated with PCBs, PCDFs, and PCDDs was reported by del Rio Gomez et al. (2002). On the other hand, Yoshimura et al. (2001) found a nonsignificant increase in the sex ratio (SR = 0.574) of children born in the 4 years following the similar 1967 Yusho poisoning by rice oil contaminated with PCBs and PCDFs (but not TCDD) when at least one parent was exposed. Following up on the Yusho cohort, however, Tsukimori et al. (2012b) noted modest nonsignificant decreases in the sex ratio when either the mother (SR = 0.450) or the father (SR = 0.465) was less than 20 years old at the time of the poisoning. In considering the second generation of Yusho offspring, Tsukimori et al. (2012b) found no effect on the sex ratio in the grandchildren of the exposed men, but the daughters of exposed women showed a tendency toward decreased sex ratios, especially if the grandmother had been young when exposed (results not tabled).

Chao et al. (2007) mentioned that they did not find an association between the sex ratio of offspring and the TEQ concentrations of dioxins, furans, or PCBs in the placentas from 119 Taiwanese women. Hertz-Picciotto et al. (2008) found evidence of an effect on sex ratio in an analysis of the serum concentrations of nine PCB congeners (of which the two dioxin-like congeners were the mono-ortho PCBs 105 and 118) in blood gathered during the 1960s from 399 pregnant women in the San Francisco Bay area. The adjusted odds of male birth were significantly decreased when the 90th percentile of the total concentration of all nine PCBs was compared with the 10th percentile (OR = 0.45, 95% CI 0.26–0.80, p = 0.007). The proportion of male births was significantly reduced for only two of the PCBs when analyzed separately: Dioxin-like, mono-ortho PCB 105 and non–dioxin-like PCB 170 (p = 0.02 for each).

TABLE 9-4 Selected Epidemiologic Studies—Sex Ratio^a

NOTE: 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; AFHS, Air Force Health Study; dl, dioxin-like; IARC, International Agency for Research on Cancer; NIOSH, National Institute for Occupational Safety and Health; ns, not significant; nr, not reported; OFFHS, Ontario Farm Family Health Study; OR, odds ratio; PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzodioxin; PCDF, polychlorinated dibenzofurans; ppt, parts per trillion; SEA, Southeast Asia; SR, sex ratio; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDF, tetrachlorodibenzofuran; TEQ, (total) toxic equivalent.

^aVAO reports before Update 1998 did not address association between perturbations in sex ratio of offspring and exposure to chemicals of interest.

^bGiven when available.

Reductions in the expected number of male offspring have also been reported in cohorts of men who were occupationally exposed to dioxin (Moshammer and Neuberger, 2000; Ryan et al., 2002), but other such cohorts did not manifest this relationship (Heacock et al., 1998; Savitz et al., 1997; Schnorr et al., 2001). In the single report relevant to this outcome in Vietnam veterans, however, the sex ratio was increased in the Ranch Hand group that had the highest serum dioxin concentrations (Michalek et al., 1998a), but no formal analysis of this outcome was reported.

A population-level finding of a paternally mediated effect would be a strong indicator that dioxin exposure can interfere with the male reproductive process. James (2006) has interpreted the perturbation of sex ratios by dioxins and other agents as being an indicator of parental endocrine disruption. If James’s hypothesis were demonstrated to be true, then it would be concordant with a reduction

in testosterone in exposed men. Another pathway to an altered sex ratio might involve male embryos’ experiencing more lethality from induction of mutations due to their unmatched X chromosome. A genotoxic mechanism has not been expected to apply to TCDD, but sex-specific adverse consequences of the modified imprinting of gametes might be a possible mechanism leading to the observation of altered sex ratios at birth. To date, however, results regarding the proportion of sons among the children of fathers exposed to dioxin-like chemicals do not present a clear pattern of reduction.

No experimental animal studies have specifically examined the effects of TCDD on the sex ratios of offspring, nor have any alterations in sex ratio been reported in animal studies that examined the developmental effects of TCDD on offspring.

Synthesis

Reproduction is a sensitive toxic endpoint of TCDD and dioxin-like compounds (DLCs) in rodents, and the fetal rodent is more sensitive than the adult rodent to the adverse effects of TCDD. The sensitivity of these endpoints in humans is less apparent. There is little evidence that exposure to dioxin is associated with a reduction in sperm quality or a reduction in fertility. However, the committee for Update 2008 noted that the evidence that TCDD exposure reduces serum testosterone in men is consistent across several epidemiologic studies with an appropriate consideration of confounders, including one of Vietnam veterans that found a dose–response relationship. The biologic plausibility of such a relationship is supported by concomitant increases observed in gonadotropins and the results of animal studies. Human populations showing evidence of reduced testosterone with exposure to DLCs include a general population sample (Dhooge et al., 2006), occupationally exposed people (Egeland et al., 1994), and Vietnam veterans in the Air Force Health Study (AFHS) (Gupta et al., 2006b). The evidence that DLCs may modify the sex ratio lends credence to the hypothesis that these chemicals have an effect on male reproductive functioning.

Despite the relative consistency of the finding of a reduction in testosterone, the testosterone concentrations observed even in the highest-exposure groups studied are well within the normal range. The small reduction in testosterone is not expected to have adverse clinical consequences. There is evidence of compensatory physiologic mechanisms. The occupational study by Egeland et al. (1994) found increased gonadotropins in addition to reduced testosterone. Gonadotropins stimulate the production of testosterone in men.

Eskenazi et al. (2010) published the only study to date that has examined dioxin exposure in women with respect to time-to-pregnancy (number of contraceptive-free months before pregnancy) and infertility (more than 12 contraceptive-free months to pregnancy). Dose–response relationships between TCDD serum levels in women who were less than 40 years of age at the time of

the Seveso accident and both time to pregnancy and infertility were observed, which is consistent with published observations in the rat model. Epidemiologic studies have not provided sufficient data to interpret the effects of dioxin specifically on menstrual-cycle function in humans.

Conclusions

On the basis of its evaluation of the evidence reviewed here and in previous VAO reports, the present committee concludes that there is inadequate or insufficient evidence of an association between exposure to the COIs and decreased sperm counts or sperm quality, subfertility, or infertility.

SPONTANEOUS ABORTION, STILLBIRTH,
NEONATAL DEATH, AND INFANT DEATH

Spontaneous abortion is the expulsion of a nonviable fetus, generally before 20 weeks of gestation, that is not induced by physical or pharmacologic means. The background risk of recognized spontaneous abortion is generally 11 to 22 percent (Avalos et al., 2012, but it is established that many more pregnancies terminate before women become aware of them (Wilcox, 2010). Such terminations are known as subclinical pregnancy losses and generally are not included in studies of spontaneous abortion. Estimates of the risk of recognized spontaneous abortion vary with the design and method of analysis. Studies have included cohorts of women asked retrospectively about pregnancy history, cohorts of pregnant women (usually those receiving prenatal care), and cohorts of women who are monitored for future pregnancies. The value of retrospective reports can be limited by differential recall of details (e.g., exposure history) specific to pregnancies that occurred long before the interview. Studies that enroll women who appear for prenatal care require the use of life tables and specialized statistical techniques to account for differences in the times at which women seek medical care during pregnancy. The enrollment of women before pregnancy provides the theoretically most valid estimate of risk, but it can attract non-representative study groups because the study protocols are demanding for the women.

Countries and US states have different legal definitions of the age of fetal viability and apply these terms differently, but typically “stillbirth” or “late fetal death” refers to the delivery at or after 20 weeks of gestation of a fetus that shows no signs of life, including a fetus that weighs more than 400 g regardless of gestational age (Lamont et al., 2015); “neonatal death” refers to the death of a liveborn infant within 28 days of birth (Whitworth et al., 2015); and “postnatal death” refers to a death that occurs before the first birthday (Andrews et al., 2008).

The causes of stillbirth and early neonatal death overlap considerably, so they are commonly analyzed together in a category referred to as “perinatal mortality” (Andrews et al., 2008). Stillbirths make up less than 1 percent of all births (CDC, 2000). The most common causes of mortality during the neonatal period are low birth weight (< 2.5 kg at birth), preterm delivery, congenital malformations, pregnancy or delivery complications, and placenta or cord conditions. The most common causes of postnatal death of infants is SIDs (sudden infant death syndrome) (Andrews et al., 2008).

Conclusions from VAO and Previous Updates

The committee responsible for the original VAO report concluded that there was inadequate or insufficient evidence of an association between exposure to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid and spontaneous abortion or perinatal death. Additional information available to the committees responsible for Update 1996, Update 1998, and Update 2000 did not change that conclusion.

The committee responsible for Update 2002, however, found that there was enough evidence available concerning paternal exposure specifically to TCDD to conclude that there was “limited or suggestive evidence” of no association between that paternal exposure to TCDD and the risk of spontaneous abortion. That conclusion was based primarily on the National Institute for Occupational Safety and Health study (Schnorr et al., 2001), which investigated a large number of pregnancies fathered by workers whose serum TCDD concentrations were extrapolated back to the time of conception; no association was observed up to the highest exposure group (1,120 ppt or higher). Indications of a positive association were seen in studies of Vietnam veterans (CDC, 1989c; Field and Kerr, 1988; Stellman SD et al., 1988b), but the committee for Update 2002 asserted that they might be due to an exposure to phenoxy herbicides rather than to TCDD and concluded that there was insufficient information to determine whether there is an association between maternal exposure to TCDD and the risk of spontaneous abortion or between maternal or paternal exposure to 2,4-D, 2,4,5-T, picloram, or cacodylic acid and the risk of spontaneous abortion.

The additional information (none of which concerned paternal exposure) reviewed by the committees responsible for Update 2004, Update 2006, Update 2008, Update 2010, and Update 2012 did not change these conclusions.

The relevant studies concerning perinatal death are reviewed in the earlier reports, and Table 9-5 summarizes the findings of studies concerning spontaneous abortion.

TABLE 9-5 Selected Epidemiologic Studies—Spontaneous Abortion^a (Shaded entry is new to this update)

NOTE: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; CDC, Centers for Disease Control and Prevention; CI, confidence interval; COI, chemical of interest; IARC, International Agency for Research on Cancer; NIOSH, National Institute for Occupational Safety and Health; nr, not reported; ns, not significant (usually refers to p < 0.05); PeCDF, 2,3,4,7,8-pentachlo-rodibenzofuran; PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzo-p-dioxin; PCDF, polychlorinated dibenzofuran; ppt, parts per trillion; SEA, Southeast Asia; SWHS, Seveso Women’s Health Study; TCDD, 2,3,7,8–tetrachlorodibenzo-p-dioxin; TEF, toxic equivalency factor; TEQ, (total) toxic equivalent; VA, US Department of Veterans Affairs.

^aUnless otherwise indicated, results are for paternal exposure.

^bGiven when available; results other than estimated risk explained individually.

^aSpontaneous abortion rate per 100 full-term pregnancies for 1991–1997.

Update of the Epidemiologic Literature

No Vietnam-veteran, occupational, or case-control studies of exposure to the COIs and spontaneous abortion or perinatal death have been published since Update 2012.

Environmental Studies

Wesselink et al. (2014) reported the results of a 30-year updated analysis of pregnancy outcomes in the Seveso Women’s Health Study (described in Chapter 6). Overall, the lack of association between TCDD and spontaneous abortion, fetal growth, and gestational length observed in the 20-year follow-up (Eskenazi et al., 2003a) was confirmed in this updated analysis. No effect of note was observed between a 10-fold increase in TCDD levels at the time of the accident and the risk of spontaneous abortion (OR = 0.78, 95% CI 0.59–1.02, n = 160) or when only the first births after the explosion were considered (OR = 0.81, 95% CI 0.55–1.18, n = 75).

Biologic Plausibility

Laboratory animal studies have demonstrated that TCDD exposure during pregnancy can alter the concentrations of circulating steroid hormones and disrupt placental development and function and thus contribute to a reduction in the

survival of implanted embryos and to fetal death (Huang et al., 2011; Ishimura et al., 2009; Wang J et al., 2011; Wu Y et al., 2013, 2014). There is no evidence of a relationship between paternal or maternal exposure to TCDD and spontaneous abortion. Exposure to 2,4-D or 2,4,5-T causes fetal toxicity and death after maternal exposure in experimental animals. However, that effect occurs only at high doses and in the presence of maternal toxicity. No fetal toxicity or death has been reported to occur after paternal exposure to 2,4-D.

Laboratory studies of maternal TCDD exposure during pregnancy have demonstrated the induction of fetal death; neonatal death, however, is only rarely observed and is usually the result of cleft palate, which leads to an inability to nurse. Studies addressing the potential for perinatal death as a result of paternal exposure to TCDD or herbicides are inadequate to support conclusions.

Synthesis

A single study concerning the COIs and spontaneous abortion, stillbirth, neonatal death, or infant death has been published since Update 2012, but it did not provide supporting evidence of an association with the COIs and these outcomes. Furthermore, toxicologic studies do not provide clear evidence for the biologic plausibility of an association.

Conclusions

On the basis of the evidence reviewed to date, the committee concludes that there is limited or suggestive evidence that paternal exposure to TCDD is not associated with risk of spontaneous abortion and that insufficient information is available to determine whether there is an association between maternal exposure to TCDD or either maternal or paternal exposure to 2,4-D, 2,4,5-T, picloram, or cacodylic acid and the risk of spontaneous abortion. The committee concludes that there is inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and stillbirth, neonatal death, or infant death.

BIRTH WEIGHT AND PRETERM DELIVERY

Birth weight and the length of the gestation period can have important effects on neonatal morbidity and mortality and on health over the life span. Typically, low birth weight (LBW) is defined as a birth weight under 2,500 g (UNICEF, 2004). In the absence of congenital malformations or chromosomal anomalies, LBW is the consequence of either preterm delivery (PTD) or intrauterine growth-restriction (IUGR). PTSD is delivery at less than 259 days or 37 weeks gestation from the date of the first day of the last menstrual period (Jones and Lopez, 2013), and IUGR is birth weight that is lower than average according to local or national fetal-growth graphs (Romo et al., 2009). LBW occurs in about

7 percent of live births. When no distinction is made between IUGR and PTD, the factors most strongly associated with LBW are maternal tobacco use during pregnancy, multiple births, and race or ethnicity. Other potential risk factors are low socioeconomic status, malnutrition, maternal weight, birth order, maternal complications during pregnancy (such as severe pre-eclampsia or intrauterine infection) and obstetric history, job stress, and cocaine or caffeine use during pregnancy (Alexander and Slay, 2002; Alexander et al., 2003; Ergaz et al., 2005; Jones and Lopez, 2013; Peltier, 2003). Established risk factors for PTD include race (black), extremes of maternal age, low socioeconomic status, previous LBW or PTD, multiple gestations, tobacco use, and low maternal prepregnancy weight or poor pregnancy weight gain (Rubens et al., 2014).

The importance and interpretation of associations with birth weight are often unclear and a subject of controversy among researchers (Barker et al., 2012; Wilcox, 2010). Across populations, the frequency distribution of birth weight is Gaussian, with an extended lower tail, or “residual distribution,” that includes preterm and LBW infants. The predominant, normal distribution corresponds largely to term births. In general, shifts in the predominant distribution do not tend to correspond to notable shifts in infant mortality (Wilcox, 2001). A number of factors may result in shifts in the predominant distribution; altitude, race or ethnicity, and maternal smoking are among the better studied, producing a larger (or smaller) percentage of LBW babies. However, populations that have a larger percentage of LBW infants do not always have higher infant mortality (Wilcox, 2001, 2010). While birth weight is tracked internationally as a public health indicator to identify opportunities for intervention and to understand country-specific infant mortality (UNICEF, 2004), strategies to increase birth weight have not been effective in reducing mortality.

Conclusions from VAO and Previous Updates

The committee responsible for VAO concluded that there was inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and LBW or PTD.

Additional information available to the committees responsible for Update 1996, Update 1998, Update 2000, Update 2002, Update 2004, Update 2006, Update 2008, Update 2010, and Update 2012 did not change that conclusion. Reviews of the relevant studies are presented in the earlier reports. The most relevant findings on birth weight after paternal and maternal exposure to the COIs are summarized in Tables 9-6 and 9-7, respectively.

Update of the Epidemiologic Literature

No Vietnam-veteran, occupational, or case-control studies of exposure to the COIs and LBW or PTD have been published since Update 2012.

TABLE 9-6 Selected Epidemiologic Studies—Birth Weight Following Paternal Exposure

NOTE: AFHS, Air Force Health Study; AO, Agent Orange; CDC, Centers for Disease Control and Prevention; IUGR, intrauterine growth restriction; LBW, low birth weight; OR, odds ratio; RR, relative risk; SEA, Southeast Asia; SGA, small for gestational age; TCDD, 2,3,7,8-trichlorodibenzo-p-dioxin; VA, US Department of Veterans Affairs.

Environmental Studies

Since Update 2012, several studies have examined potential relationships between the COIs and birth weight. Papadopoulou et al. (2013a) analyzed infant weight, length, and head circumference at birth using estimated maternal dietary intake of dioxins during pregnancy. They considered the entire Norwegian Mother and Child (MoBa) cohort enrolled from 2002 to 2008, of which the births occurring in 2007–2008 form a subcohort of the NewGeneris cohort discussed below. The estimated dietary intakes of dioxin-like activity for the 50,651 eligible mothers were partitioned into quartiles inversely associated with birth weight (−62.1 g, 95% CI −73.8 to −50.5). A similar association was observed when the sexes were looked at separately; boys (−68.9 g, 95% CI −85.2 to −52.2) and girls (−55.2 g, 95% CI −71.7 to −38.6). Two other measures of infant growth (length and head circumference at birth) showed similar patterns.

Papadopoulou et al. (2014) used the CALUX assay to measure dioxin-like activity in maternal blood samples collected at delivery from the 604 mothers in the NewGeneris cohort. Maternal dietary intake of dioxins was estimated using data from food frequency questionnaires and was correlated with the TEQ concentration measured in maternal blood. For mothers in the highest category of dietary intake of dioxin-like compounds, an inverse relationship with birth weight (−121 g, 95% CI −232 to −10) was observed after adjustment for maternal education, energy intake, age, prepregnancy BMI, parity, smoking, and country of

TABLE 9-7 Selected Epidemiologic Studies—Birth Weight Following Maternal Exposure (Shaded entries are new information for this update)

NOTE: BMI, body mass index; BW, birth weight; CI, confidence interval; I-TEQ, International (total) toxic equivalent; LBW, low birth weight; OR, odds ratio; PCB, polychlorinated biphenyls; PCDD, polychlorinated dibenzo-p-dioxin; PCDF, polychlorinated dibenzofurans; SAB, spontaneous abortion; TCDD, 2,3,7,8-trichlorodibenzo-p-dioxin; TEQ, (total) toxic equivalent; VA, US Department of Veterans Affairs.

cohort, plus infant’s gestational age and gender. When the sexes were examined separately, the inverse relationship was maintained, but it only achieved statistical significance for boys (−170 g, 95% CI −332 to −8). Similarly, when the highest dietary intake group was compared with the lowest, a small, inverse relationship with gestational age was observed (−1.4 days, 95% CI −3.8–1.0 days). The estimate of gestational exposure employed in these two studies (Papadopoulou et al., 2013a; 2014) is a more indirect and presumably less precise metric than the CALUX results used in the next article.

Vafeiadi et al. (2014) examined dioxin-like activity measured by CALUX in maternal and cord blood samples and birth outcomes in 967 mother–child pairs enrolled in four European cohorts. When the highest tertile of TEQs values measured in cord blood was compared with the lowest exposure category, inverse associations were observed for birth weight (−82 g, 95% CI −216–53; p-trend = 0.225) and gestational age (−0.4 weeks, 95% CI −0.8 to −0.1; p-trend = 0.029). In analyses performed on the two sexes independently, nonsignificant inverse relationships with dioxin-like activity in cord blood and birth weight were observed in both boys (−124 g, 95% CI −391–144) and girls (−57 g, 95% CI −300–185). No indication of association was observed for dioxin-like activity measured in maternal plasma and birth outcomes.

Wesselink et al. (2014) reported the results of an updated analysis of pregnancy outcomes in the Seveso Women’s Health Study (described in Chapter 6). Overall, the lack of association between TCDD and spontaneous abortion, fetal growth, and gestational length observed in the first 20-year follow-up (Eskenazi et al., 2003a) was confirmed in this updated analysis. A small inverse association (−22.8 g, 95% CI −80.1–34.6) between a 10-fold increase in serum TCDD levels estimated at pregnancy and birth weight was observed, with the strongest reduction observed for the first births after the explosion (−47.7g, 95% CI −107.3–11.9).

Biologic Plausibility

The available evidence from experimental animal studies indicates that TCDD exposure during pregnancy can reduce body weight at birth, but only at high doses. A recent study in human placental explants suggests that TCDD exposure may enhance placental inflammation and may influence pre-term births associated with infection (Peltier et al., 2013). Laboratory studies of the potential male-mediated developmental toxicity of TCDD and herbicides as a result of exposure of adult male animals are inadequate to support conclusions. TCDD and herbicides are known to cross the placenta, which leads to the direct exposure of the fetus. Data from studies of experimental animals also suggest that the preimplantation embryo and developing fetus are sensitive to the toxic effects of 2,4-D and TCDD after maternal exposure.

Synthesis

Two analyses from European birth cohorts observed a small decrease in birth weight in relation to maternal dietary intake of DLCs (Papadopoulou et al., 2013, 2014). A small decrement in gestational age (1.4 days) was also observed when comparing the highest to lowest dietary intake categories. A similar reduction in birth weight was observed in an analysis of mother–infant pairs enrolled in four European birth cohorts, with an inverse association with birth weight observed

when comparing cord blood measurements of the highest to lowest categories. In all three analyses, the reduction in birth weight was less than 200 grams and not likely to be of clinical relevance. In a final study, an update of the Seveso cohort observed no association between TCDD and fetal growth, confirming an earlier analysis of this cohort.

There are a number of challenges in conducting these types of epidemiologic studies in a rigorous way. First, the prenatal and immediate postpartum period is not a stable pharmacokinetic state, because it involves substantial changes in body volume and fat mobilization. Biomarker measures during pregnancy may be substantially affected by weight change during pregnancy. Moreover, the extrapolation of a more recent biomarker measure back many years to a more relevant period is complicated by intervening pregnancy and breastfeeding events, which result in a substantial uncertainty in the index exposure level. Overall, although the committee notes that the animal literature does support an effect of TCDD exposure at high doses on birth weight, the epidemiologic literature is insufficiently robust to allow a final determination.

Conclusions

On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that there is inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and low birth weight or preterm delivery.

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