Phthalate metabolite levels and menopausal hot flashes in midlife women.

Phthalates are a large class of ubiquitous synthetic chemicals, which are used as plasticizers and stabilizers in a myriad of consumer products, including shower curtains, children’s toys, cosmetics, and personal care products such as perfumes, nail polishes, deodorants, and lotions [ 1 – 3 ]. Phthalates are also used in pesticides, wood finishes, adhesives, solvents, lubricants, and in medical devices including tubing, blood bags, surgical gloves, and dialysis equipment [ 1 ]. The wide range of products in which phthalates are incorporated results in a global production and use of phthalates that is greater than 18 billion pounds per year [ 1 ]. The chemical structures of phthalates consist of esters of ortho-phthalic acid and are named based on the alcohol that generates the varying lengths of the alkyl chain in a linear or branched format. Phthalate esters with long alkyl chains (more than 6 carbons) have higher molecular weights and are likely to undergo chemical modifications for renal excretion. At least six different parent phthalates are used in consumer products, including diethyl phthalate (DEP; short alkyl chain), di(2-ethylhexyl) phthalate (DEHP; long alkyl chain), dibutyl phthalate (DBP; short alkyl chain), diisobutyl phthalate (DiBP; short alkyl chain), diisononyl phthalate (DiNP; long alkyl chain), and butyl benzyl phthalate (BBzP; short alkyl chain) [ 1 , 3 ]. The parent compounds can be converted mainly by the gastrointestinal tract or liver to various metabolites that can be more toxic than the parent compound [ 1 , 3 – 5 ]. The ubiquity of phthalates and phthalate metabolites is further evidenced by their detection in nearly all tested human urine samples [ 6 – 10 ]. Interestingly, the measured levels of urinary phthalate metabolites are higher in women compared to men [ 1 , 11 ]. This finding is possibly due to a greater use of personal care products by women compared to men. Previous epidemiological studies indicate that phthalate metabolites can reach the reproductive tissues and adversely affect reproductive function [ 8 , 12 , 13 ]. For example, phthalate metabolites have been associated with an increased risk of endometriosis [ 10 ] and earlier age at menopause [ 14 ]. Further, animal studies indicate that exposure to phthalates inhibits ovarian synthesis of sex steroid hormones that are required for normal reproductive function [ 3 , 4 , 15 – 17 ], and epidemiological studies indicate that phthalate exposure is associated with reduced sex steroid hormone levels [ 8 , 12 , 18 , 19 ]. It is not clear, however, whether and how phthalates impact reproductive function as women age and enter the menopausal transition. During the menopausal transition, a woman’s reproductive capacity declines, her hormone milieu changes, and her risk of hot flashes increases [ 20 , 21 ]. Hot flashes are transient periods of intense heat in the upper parts of the body and are often followed by flushing of the skin, profuse sweating, chills, palpitations, and anxiety [ 22 ]. Despite the high prevalence of hot flashes among women undergoing the menopausal transition, little is known about the etiology or the risk factors for hot flashes. However, the predominant hypothesis is that drastic changes in estrogen levels lead to the onset of menopausal hot flashes [ 20 , 22 ] and that low estradiol levels are associated with an increased risk of any, frequent, and severe hot flashes [ 20 , 23 – 25 ]. With evidence from animal studies that phthalates reduce estradiol levels [ 3 , 4 , 15 , 16 ], evidence from epidemiological studies that low estradiol levels are associated with an increased risk of hot flashes [ 20 , 23 , 25 ], and information that women commonly use personal care products containing phthalates [ 1 , 2 ], we tested the hypothesis that higher urinary levels of phthalate metabolites, including those combinations of phthalate metabolites present in personal care products, are associated with an increased risk of midlife hot flashes.

Baseline characteristics for women with and without hot flashes are presented in Table 1 . Women who ever experienced hot flashes were more likely to be of older age ( p = 0.0005), to report less than some college education ( p = 0.01), and to be perimenopausal ( p <0.0001) than those who did not experience hot flashes. Further, women who ever experienced hot flashes had lower levels of estradiol ( p <0.0001) and progesterone ( p =0.001) compared to women who never experienced hot flashes. The majority of the phthalate metabolites detected in our study were above the limit of quantification (LOQ) in most tested samples ( Table 2 ). Further, the metabolite levels in our study sample were similar to those published from the National Health and Nutrition Examination Survey 2011–2012 [ 18 ]. Sum PCP was significantly and positively associated with ever experiencing hot flashes ( Table 3 ). Further, the associations between both sum DEHP and sum AA with ever experiencing hot flashes were of borderline statistical significance ( Table 3 ). In addition, some individual phthalate metabolite levels in the mixtures were significantly and positively associated with ever experiencing hot flashes. Specifically, MECPP (OR =1.37; 95% CI=1.03, 1.81), MEHHP (OR=1.40; 95% CI=1.03, 1.91), and MEP levels (OR=1.39; 95% CI=1.07, 1.82) were significantly associated with ever experiencing hot flashes. Sum PCP was significantly associated with experiencing hot flashes in the past 30 days ( Table 4 ). Only one individual phthalate metabolite in the mixture, MEP, was significantly associated with having hot flashes in the past 30 days (OR=1.36; 95% CI=1.03, 1.79); however, the associations between both MECPP (OR=1.27; 95% CI=0.95, 1.70) and MEHPP (OR=1.30; 95% CI =0.94, 1.79) and having hot flashes in the past 30 days were of borderline statistical significance. Although none of the phthalate mixtures were significantly associated with moderate/severe hot flashes, the associations between sum PCP, sum DEHP, and sum AA with this outcome were all of borderline statistical significance ( Table 5 ). MECPP (OR=1.45; 95% CI=1.06, 1.99) and MEHPP levels in the mixtures (OR=1.41; 95% CI=1.01, 1.97) were significantly associated with moderate/severe hot flashes; and the association between MEP (OR=1.26; 95% CI=0.95, 1.68) and moderate/severe hot flashes was of borderline statistical significance. Sum PCP was significantly associated with more frequent (daily) hot flashes ( Table 6 ). Only one individual phthalate metabolite in the mixture, MEP, was significantly associated with more frequent hot flashes (OR=1.42; 95% CI=1.16, 1.91), but the associations between both MECPP (OR=1.37; 95% CI=0.99, 1.92) and MEHPP (OR=1.40; 95% CI=0.98, 2.01) and more frequent hot flashes were of borderline statistical significance.

In the current study, we examined the associations between phthalate metabolite levels and menopausal hot flashes in generally healthy midlife women. Our data suggest that some, but not all, phthalate metabolite mixtures and individual metabolites may be associated with menopausal hot flashes. Interestingly, the PCP summary variable, which estimates phthalate exposure from women’s personal care products, was associated or borderline associated with most of the hot flashes outcomes (ever experiencing hot flashes, experiencing hot flashes in the past 30 days, and more frequent hot flashes). These findings add to the current literature that correlates exposure to phthalates and their metabolites with aberrant reproductive function [ 1 , 3 , 38 ]. Additionally, studies in mice indicate that phthalate exposure may result in early reproductive senescence. Specifically, in adult mice, oral exposure to DEHP accelerates primordial follicle recruitment [ 16 ] and this may accelerate reproductive aging [ 49 ]. Moreover, in utero MEHP exposure in mice accelerates folliculogenesis and causes premature reproductive senescence in the female offspring [ 50 ]. These studies are important in the light of epidemiological studies such as that by Gibson-Helm et al. [ 51 ] indicating that women diagnosed with premature ovarian failure or medically induced ovarian failure report hot flashes more frequently than women prior to their menopausal transition. Strikingly, Grindler et al. reported significant associations between higher levels of DEHP metabolites (i.e., MEHHP and MEOHP) and early age at menopause [ 14 ]. Similarily, Hart et al . reported a significant association between higher exposure levels of MEP during pregnancy and lower anti-Müllerian hormone levels in the adolescent daughters [ 52 ]. Interestingly, lower anti-Müllerian hormone levels were reported as a marker for reduced ovarian reserve in adolescent girls [ 53 ] and reduced ovarian reserve can result in early age at menopause [ 54 ]. Further, previous studies indicate that early age at menopause is associated with hot flashes severity [ 55 ] and our study suggests that MEHHP is associated with hot flashes severity. Hence, it is possible that exposure to some of the phthalates increases the risk for early menopause and thus, the likelihood to experience menopausal hot flashes and in a more severe form. The underlying mechanism by which phthalate metabolites are associated with hot flashes is unknown. However, it likely involves hormone levels. In our current study and in other epidemiological studies, low levels of estradiol and progesterone are associated with hot flashes [ 20 , 23 , 24 , 56 ]. Further, several experimental studies indicate that phthalate exposures decrease both estradiol and progesterone levels [ 3 , 4 , 15 – 17 ]. Thus, future studies should examine whether the observed associations between urinary phthalate metabolites and hot flashes are mediated by low estradiol and/or progesterone levels. In our current study, not all phthalate metabolites were associated with hot flashes. It is possible that some of the phthalate metabolites were not associated with hot flashes because they do not play a role in the etiology of hot flashes. It is also possible that certain aspects of the study limited our ability to detect real associations between certain phthalate metabolites and hot flashes. For example, our study examined single urine samples of 195 women and this only provides a snap shot of phthalate exposure. However, all samples were collected under similar conditions around the same time of the day and the levels that were measured in our study are similar to the levels published by other epidemiological studies [ 18 ]. We collected blood samples without considering the day of the menstrual cycle because women undergoing the menopausal transition have extremely variable cycles; however, we used the average of the four blood draws per participant to account for potential variation. Second, there may be additional factors that were not assessed in the current study that may mask some of the potential associations between phthalate metabolites and hot flashes. Such factors may include differences in the metabolic capacity of the women, environmental sources of phthalate exposures such as house dust, medical issues such as consumption of medications that are coated with phthalates, exposure to additional endocrine disrupting chemicals, or even a potential combined effect of several metabolites/chemicals that were out of the scope of our current study.

Our study shows that a phthalate mixture and some individual phthalate metabolite levels are associated with hot flashes in midlife women. This observation is consistent with studies indicating that some, but not all phthalate metabolites, are associated with selected reproductive outcomes. For example, Meeker et al. showed that some, but not all, phthalate metabolites are associated with decreased testosterone levels [ 18 ]. Even though not all phthalate mixtures and individual metabolites were associated with hot flashes, our study provides important information on which phthalate metabolites should be of concern and to which we should consider limiting human exposure. In our study, most of the metabolites that were positively associated with hot flashes were derived from the parent phthalate DEP. Thus, it could be that exposure to DEP poses a greater health risk in women than other parent compounds. However, future studies are needed to determine whether this is the case and to determine the potential mechanisms by which phthalates may increase the risk of hot flashes.

All participants gave written informed consent according to procedures approved by the University of Illinois and Johns Hopkins University Institutional Review Boards (file number: 06741). Women (primarily Caucasian and African American) residing in Baltimore city (Maryland, USA) and its surrounding counties were enrolled in the Midlife Women’s Health Study from 2007–2015. Specifically, women with and without natural hot flashes between the ages of 45 and 54 years were invited to participate in a study of women’s midlife health by mail. To be eligible for the study, women must have had their ovaries and uterus, must have been late premenopausal, early or late perimenopausal, and not be pregnant. Women were excluded from the study if they were taking hormone therapy, herbal agents, or other natural agents for treatment of menopausal symptoms, taking oral contraceptives, being treated for any cancer, or postmenopausal. Menopausal status was defined as follows: pre-menopausal women were those who experienced their last menstrual period within the past 3 months and reported 11 or more periods within the past year. Perimenopausal women were those who experienced: 1) their last menstrual period within the past year, but not within the past 3 months or 2) their last menstrual period within the past 3 months and experienced 10 or fewer periods within the past year. Postmenopausal women were those women who had not experienced a menstrual period within the past year. Eligible women were invited to the clinic site at Johns Hopkins University. At the clinic visit, women had their weights and heights recorded to calculate their body mass index (BMI). Women also donated spot urine samples for measurements of phthalate metabolites and they donated blood samples for measurements of sex steroid hormone levels. Further, women were asked to complete a detailed questionnaire that included questions regarding their hot flashes history along with additional demographic, medical and reproductive history, and lifestyle information. Hot flashes status was determined based on women’s answers on the questionnaires (see below). Overall, in the current study, a sample of 195 participants (96 with hot flashes and 99 without hot flashes) was evaluated for urine phthalate metabolite levels. Additional details related to the study design, recruitment of study participants, and hormone levels measurements are described in Gallicchio et al . [ 23 ]. On the study questionnaires, hot flashes were defined for participants as “a sudden feeling of heat in the face, neck, or upper part of the chest. Hot flashes are often accompanied by reddening or flushing of the skin followed by sweating and chills.” At baseline, a detailed hot flash history was obtained through a series of questions on the survey that asked for information on the following: whether the woman had ever experienced hot flashes, whether she had a hot flash in the past 30 days, the usual severity of hot flashes, the frequency of hot flashes, and the length of time that the woman experienced hot flashes. Women who responded no to ever experiencing hot flashes were prompted to skip the more detailed hot flash questions and were categorized as “never experiencing hot flashes”. The selected hot flashes questions have been used to collect data on hot flashes in the Midlife Health Studies for over 10 years [ 23 – 27 ]. In terms of severity, each woman was asked to describe her hot flashes as: mild (sensation of heat without sweating), moderate (sensation of heat with sweating), or severe (sensation of heat with sweating that disrupts usual activity). In terms of frequency of hot flashes, each woman was asked to describe her hot flashes as occurring: every hour, every 2–5 hours, every 6–11 hours, every 12–23 hours, 1–2 days per week, 5–6 days per week, 2–3 days per month, 1 day per month, less than 1 day per month, or never. For analysis of the data, the following hot flash variables were examined as dependent variables (outcomes): ever experienced hot flashes (yes versus no); experienced any hot flashes in the past 30 days (yes versus no); moderate or severe hot flashes (yes versus no); and daily hot flashes (yes versus no). Urine samples were analyzed blinded by isotope dilution high-performance liquid chromatography negative-ion electrospray ionization-tandem mass spectrometry (HPLC-MS/MS) at the Environmental Health Laboratory & Trace Organics Analysis Center, School of Public Health at the University of Washington as previously described [ 10 , 28 ]. The following metabolites were measured: mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP), monobutyl phthalate (MBP), monoethyl phthalate (MEP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-benzyl phthalate (MBzP), mono-isobutyl phthalate (MiBP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and mono-2-ethylhexyl phthalate (MEHP). These phthalate metabolites were selected because they are the major urinary metabolites of common phthalate parent compounds DEP, DEHP, DBP, DiBP, and BBzP [ 6 , 8 , 9 , 29 ]. Further, the selected metabolites or their parent compounds have been associated with adverse reproductive outcomes in animal models and epidemiological studies [ 1 , 3 – 5 , 12 , 16 – 18 , 30 – 32 ]. All values (ng/mL) were normalized to the specific gravity value of the sample to account for any potential hydration differences between the participants and volume of the donated sample as described in other studies [ 33 – 35 ]. In cases in which values were lower than the limit of quantitation (LOQ) of the assay, a value of LOQ/square root 2 was assigned to the sample [ 36 ]. Humans are exposed to mixtures of phthalates that can contain different parent compounds or metabolites. These parent compounds and metabolites often have different toxicities, depending on dose, tissue, type, and species [ 1 , 3 , 12 , 37 , 38 ]. Thus, it is important to estimate exposure to relevant phthalate mixtures. To do so, we used calculations that have been performed in other studies on phthalate exposures in humans [ 32 , 39 – 42 ]. For the estimation of the phthalates present in women’s personal care products (PCP), we summed the metabolite molar concentrations (normalized to their respective specific gravity values) of MBP (MW 222 µg/µmol) and MEP (MW 194 µg/µmol). This provides an estimate of phthalate exposure from pharmaceutical agents, shampoos, conditioners, perfumes, nail polishes, and other personal care products [ 39 , 41 , 43 ]. For the calculation of the sum of DEHP metabolites (sum DEHP) per sample, we added the metabolite concentrations (normalized to their respective specific gravity values) of MECPP (MW 308 µg/µmol), MEHHP (MW 294 µg/µmol), MEOHP (MW 292 µg/µmol), and MEHP (MW 278 µg/µmol) per sample. This provides an estimate of phthalate exposure from DEHP-containing products such as polyvinylchloride plastics, building products, and medical devices [ 39 , 41 , 44 – 46 ]. Finally, we calculated the sum phthalate metabolites based on their anti-androgenic activity (sum AA) by adding the DEHP metabolite concentrations (normalized to their respective specific gravity values) of MBP, MBzP, and MiBP. This provides an estimate of exposure to phthalates with known anti-androgenic activity in experimental and/or epidemiological studies [ 3 , 8 , 47 ]. After conducting analyses using the various mixture formulas described above, we examined the associations between individual phthalate metabolites and the hot flashes outcomes. This was done to determine which phthalate metabolite in the mixture may be responsible for an observed association between a summary measure and a hot flashes outcome. Phthalate concentration data were log-transformed as data for these variables were not normally distributed. Differences in demographic, reproductive, and health habit characteristics by hot flash status (ever versus never) were examined using chi-square tests for categorical variables. Logistic regression was carried out to calculate odds ratios (ORs) and 95% confidence intervals (95% CI) for the associations between phthalate metabolite concentrations and the hot flash outcomes, adjusted for age, race, and education. Age, race, and education were included in the regression models as they have been shown to be significantly associated with hot flashes in either this study or the literature [ 48 ]. Menopausal status was not entered into the logistic regression models, as it was strongly associated with age (and, thus, collinear). All analyses were performed with SAS, version 9.2 (SAS Institute, Inc., Cary, North Carolina). A two-sided p -value of equal or less than 0.05 was considered statistically significant.