Phthalates are ubiquitous man-made chemicals that emerged in our modern life with the increase of plastic use in households [1]. Di-(2-ethylhexyl) phthalate (DEHP) is one of the most abundant and studied compounds. It is used mainly as plasticizers in wallpaper, carpet, flooring and adhesives industries [2,3,4,5], medical devices, clothes, food containers and many automotive, household and building products [5]. It is also used as a fragrance carrier in personal care products and cosmetics [6, 7]. Phthalates are not integrated in the products since they do not form covalent bonds. Therefore, they can leak into the environment and food during their use [8]. Exposure to phthalates may happen by ingestion, dermal absorption, or inhalation [1].

Phthalate metabolites have biologic activity and show antiandrogenic, antiestrogenic or anti-thyroid activity [1, 9]. These chemicals are thus called endocrine disruptors. Chronic exposure to phthalates has been linked with many adverse health outcomes. Exposure to higher levels of phthalates has been associated with menstruation disturbances, ovulation abnormalities, and a higher risk of endometriosis [10, 11]. In couples attempting to conceive, exposure to phthalates has been linked to longer times to conception and possible diagnosis of infertility later-on (inability to conceive for 1 year) [12]. Studies have also associated exposure to phthalates with decreased egg and sperm quality [13, 14], and increased risk of miscarriage and pregnancy loss [15,16,17]. Phthalates have been detected in amniotic fluid, and exposure to high levels of phthalates during pregnancy may lead to poor obstetric outcomes like preterm delivery [18].

Phthalates are rapidly metabolized and almost fully eliminated in the urine. The amount of phthalate metabolites found in human urine characterizes a measurement of exposure to the original parent compound that has occurred in the last 24 h [19, 20]. Advances in human biomonitoring have led to further possibilities in evaluating exposures to phthalates, because biomonitoring methods now use the secondary oxidized metabolites as biomarkers to exposure. These metabolites are not susceptible to external contamination and possess longer elimination half-lives, making them more appropriate to assess the average exposure [21].

To our knowledge, no studies have yet screened the prevalence and quantified the magnitude of exposure to phthalates in the Jordanian population. Moreover, most previous studies were limited by small sample sizes. Additionally, with the increase in incidence of abnormal reproduction outcomes in our community, no studies have been done to see the potential role of phthalates as the main culprit in these outcomes. This study will be the first study to screen for phthalates levels in the Jordanian population and try to potentially link it with fertility outcomes. Biomonitoring will elucidate our knowledge on the role of phthalates in negative reproductive outcomes. We aim to increase awareness to environmental exposure to phthalates, point attention to the importance of implementation of public health interventions to control and minimize the effects of phthalate exposure, and provide a base for further studies and future research to aid in the formation of policies and guidelines for the manufacturing and use of phthalates.


Subjects and sample collection

Study participants were recruited from visitors of clinics in King Abdullah University Hospital (KAUH) in the period between September 2019 and March 2020. Ethical approval for this study was in compliance with the Institutional Review Board (IRB) Guidelines at Jordan University of Science and Technology (IRB# 9/119/2018). All subjects were females who have been married for 12 months or more and visited either the Assisted Conception Center or obstetrics and gynecology clinics. For the purpose of population screening, subjects were classified into four groups: cases, controls, previous infertility diagnosis, and others. Those who have previous or current fertility problems, have not been able to conceive, and have been trying to get pregnant for 12 months or more were considered cases. This is in line with the definition of infertility in the Glossary of definitions for fertility and infertility care as “a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse” [22]. Cases with male fertility problems (n = 22) and no female infertility problems were excluded. Subjects who have had no fertility problems either previously or currently and have children were considered controls. Pregnant women with no fertility problems were eligible to be enrolled as controls. The previous group was those who were previously struggling with inability to conceive and had fertility problems but have undergone treatment, conceived, and are not complaining of any problems in the present. Those with none of the previous characteristics applied to them were classified as others such as women who did not conceive and/or did not want to conceive for 12 months or more. Others were included for comparisons to other biomonitoring studies, but were not included to assess relationships between fertility and phthalate concentrations. The flowchart for subjects included as cases or controls was summarized in Supplementary Fig. 1. All controls and cases were used in the case-control analyses.

Written informed consent was obtained from each participant. Approval from the Institutional Review Board 9/119/2018 of JUST/KAUH was obtained before the study was started.


Information was obtained by direct interviews regarding sociodemographic characteristics, medical and surgical history, reproductive history, and a variety of potential sources of exposure to phthalates. The reproductive and surgical history of subjects were confirmed from patient medical files. The questionnaire contained questions about sources of phthalates exposure in the past 48 h. We assessed the use of plastic containers used to package or store food (tupperwares, cups, pre-packaged food and beverages), consumption of foods and beverages packaged in plastic, chewing gum, use of gloves, personal care items, cosmetics, and nail care. We also assessed if the subjects had done renovations in their homes within the last two years and if PVC was used in the renovations. Monthly income was grouped into middle-or-high income (500 JD or more) and low income (less than 500 JD). Based on occupation there were two groups; currently employed and not employed. Education was categorized as high school or less, college education, or graduate level. Body mass index categories used were less than 18.5 kg/m2 (underweight), 18.5–24.9 kg/m2 (normal), 25–29.9 kg/m2 (overweight), more than 30 kg/m2 (obese). Pre-pregnancy weight was taken for first trimester control. Smoking status of both wife and husband was assessed and packs per year was calculated. Household smoking is a general variable that represents either the wife or husband or both smoking.

Sample collection

Participants provided one spot urine sample in sterile urine sample containers while visiting the clinic. We stored all the samples at −80 °C after collection until preparation for analysis. Samples with large volume were divided into more than one sample container (n = 16, 5.90%). More than one sample on different visit dates were obtained from some patients (n = 16, 5.90%).

Sample preparation

All samples were thawed before analysis, then assigned 1 ml of each sample to a numbered glass tube. From most of the samples, duplicates were made.

The urine samples were analyzed in the Pharmaceutical Research Center—Jordan University of Science and Technology (PRC-JUST). The urine specimens were tested for two DEHP metabolites (MEHHP, MEOHP). The total amount of each metabolite was quantified using high-performance liquid chromatography with isotope dilution coupled with our group’s tandem mass spectrometry (HPLCMSMS). Information has been provided elsewhere about this analytical method [23]. In short, an aliquot 1.0 mL of urine samples were sealed with Teflon-lined screw caps and gently mixed. The samples underwent subsequent incubation at 37 °C for 90 min with b-glucuronidase to hydrolyze the metabolites. Next, using the Sigma SupelTM SELECT SAX cartridge, the target compounds were collected. The target fraction was eluted with an ethyl acetate content of 5.0 mL. Eluates were concentrated to near dryness using nitrogen and then reconstituted for instrumental analysis in 50 percent acetonitrile solution. Chromatographic separation was performed on the Acquity UPLC BEH C18 column (100 mm × 2.1 mm × 1.7 mm) with 0.1% ammonium water solution and 0.1% ammonium water solution containing methanol as a mobile step in gradient elution.

Exposure assessment

The methodology described by refs. [24, 25] was used to assess exposure to DEHP. The measured concentrations of phthalate metabolites (MEHHP, MEOHP) in urine were used to evaluate the amount of exposure to the parent compound DEHP by using the equation:

$${{{{{\mathrm{Estimated}}}}}}\,{{{{{\mathrm{Parent}}}}}}\,{{{{{\mathrm{Phthalate}}}}}}\,{{{{{\mathrm{Concentration}}}}}} = \frac{{Metabolite\,Concentration}}{{Exceretion\,Fraction}}$$

Statistical analysis

All urinary DEHP metabolite concentrations were log transformed. The analysis was performed using SAS 9.4 Cary NC, USA. Maximum of replicate concentrations were used. Summary stats (means, minimum and maximum, medians, interquartile ranges) were calculated for total DEHP and its metabolites in urine (MEHHP, MEOHP). Parametric T-test was used to assess the presence of significant differences in the means of cases and controls for each metabolite. Non-parametric Wilcoxon test was used to assess the presence of significant differences in the medians of cases and controls for each metabolite. Differences in sociodemographic characteristics between cases and controls were assessed using chi square tests. Chi square tests were also used after logistic regression analysis and categorizing DEHP metabolite concentrations into lower (0–25%), middle (25–75%), and higher (>75%) using quartiles. Pearson and Spearman correlation coefficients matrixes were used to assess linear relationships between metabolite concentrations. Multivariate logistic regression for candidate covariates, identified in literature such as maternal age, BMI, and smoking status, were used as confounders for each metabolite. The DEHP median concentration obtained in this study was compared to the medians from various biomonitoring studies. p values were set at 0.05. Figures were drawn in GraphPad Prism 5.


The study included a total of 325 women who visited clinics in KAUH. There were 191 cases (73% had primary infertility and 26.5% had secondary infertility) and 95 controls. There were 22 women with previous fertility problems who underwent treatment and 16 labeled as others who were not considered either a case or a control. Some of the controls (n = 37) were pregnant women accounting for 39% of all controls. All couples with female fertility problems known and unknown diagnoses were taken into account as cases. Those with a diagnosis of polycystic ovarian syndrome were around 28% (n = 60). A percentage of about 12% (n = 26) of the cases had fallopian tube issues. Nearly 11% (n = 23) had secondary infertility. Secondary infertility is defined as the inability to become pregnant or carry a pregnancy to full term after at least one previous successful conception [22]. Table 1 shows the sociodemographic and reproductive history characteristics for all the participants, cases and controls separately. None of the differences between cases and controls regarding sociodemographic characteristics were significant. The subjects had a good representation across the age ranges with the lowest percentage being 7.5% in the range 18–24. Cases had the most participants in the range 30–34 (23.8%) and the least percentage in the range 18–24 (9.4%). The age range with the highest percentage for controls was 35–39 (32.6%). The lowest percentage for controls was in the range of 18–24 (3.5%). The majority of participants fell in either the normal or overweight BMI category. More than half of the total sample (56.8%), 56.3% of cases, and 57.7% of controls had a college education.

Most of them also had an occupation and were employed. Most of the sample (73% overall, 75% cases, 69% controls) had low income. Participants were asked about both the husband-and-wife smoking status. A percentage of 50% of husbands and 5% of wives were smokers. About two thirds of households had a smoker (64.3%). Percentages of smokers for cases were higher than for controls but was only significant in smoking husbands (p = 0.04). A higher percentage of cases have been married for less than 5 years (41.7%) compared to controls who had a higher percentage who have been married for more than 10 years (40.7%) (p = 0.029). More than half of the controls had more than two children (57.3%) while most of the cases had no children (60.7%) (p < 0.001). Almost 40% of both cases and controls reported a history of abortions (p = 0.933). Out of the controls, 60.7% were planning to have no more than two children while 54.6% of cases reported planning for three to five children (p = 0.001).

Potential sources of exposure to DEHP and their frequencies are shown in Supplementary Table 1. Participants were asked about various sources of exposure to DEHP such as the frequency of using plastic objects, some personal care products, and cosmetics and if they had used them within the previous two days. Those who reported never heating plastic containers or plates in the microwave were 75.2% of cases compared to 91.2% of controls (p = 0.017). A percentage of 87.3% and 96.2% of the cases and controls respectively had not heated anything plastic in the microwave in the past two days (p = 0.029). Around 24 and 39% of cases and controls respectively have never used skin make up (p = 0.045). A percentage of 22.6% of cases and 39.2% of controls have never used eye make-up (p = 0.026). Those who had not used eye make up in the past two days were 42.6% of cases and 57.7% of controls (p = 0.029).

Furthermore, 64.7% of cases and 79.5% of the controls have not used sunscreen in the past two days (p = 0.021). As for use of nail polish, 65.4% of cases and 83.3% of the controls have reported never using nail polish (p = 0.033).

Figure 1 shows the histogram distribution of the urinary concentrations of Total DEHP, MEHHP, and MEOHP at the diagonal panels. Correlation scatter plots and values are summarized in Fig. 1 between total DEHP, MEOHP, and MEHHP urine concentrations. A significant moderate positive correlation (r = 0.49, p < 0.0001) was identified between MEHHP and MEOHP. The finding suggests that women who have high MEOHP levels were also more likely to have high MEHHP levels. As expected, DEHP had a strong significant positive correlation with the two metabolites. Shown in Table 2 is the summary statistics of the two DEHP metabolites MEOHP, MEHHP, and total DEHP for cases, PCOS cases, and controls. It is clearly apparent that cases, and PCOS cases had higher means and medians than controls. Figure 2 illustrates the comparisons between cases and controls for the three phthalates. The urinary concentrations of MEOHP and total DEHP were significantly higher in cases than controls (p = 0.002 for both). Although the cases had higher MEHHP concentrations than controls, it did not reach statistical significance (p = 0.33). Although, pregnant controls had lower concentrations of phthalates than non-pregnant controls the difference was not significant (p = 0.12) for MEOHP. MEOHP was significantly higher in cases compared to controls (p = 0.0017). On the other hand, pregnant controls were significantly lower (p = 0.017) than both cases and non-pregnant controls for MEHHP. The urinary concentrations of total DEHP and DEHP metabolites MEHHP, MEOHP were categorized as quartiles and summarized in Supplementary Table 2. The upper quartile for total DEHP and MEOHP has a higher percentage of cases, and the lower quartile has a higher percentage of controls. These differences in distribution were significant with p-values of p = 0.026 for DEHP and p = 0.04 for MEOHP.

Unadjusted odds ratios (Table 3) suggested an increased risk of infertility with greater concentrations of MEOHP (p = 0.0027). The odds of having infertility among high MEOHP were 1.71 times higher among case-patients than controls. After adjusting for BMI, Age, Age at-first pregnancy, and smoking the adjusted odds ratio of being an infertile case in the high MEOHP exposure was 1.66 (95% CI: 1.14–2.40) the odds of being a control (p = 0.0075). After adjusting for BMI, Age, Age at-first pregnancy, and smoking the adjusted odds ratio of being an infertile case in the high MEEHP exposure was not significant and it was 1.09 (95% CI: 0.70–1.72) the odds of being a control (p = 0.71).

Table 1 Sociodemographic and reproductive history characteristics of participants in the study.
Fig. 1: Correlation matrix for DEHP and its two metabolites (MEHHP, MEOHP) in urine on a ten logarithmic scale.
figure 1

The diagonal figures represent the histogram distribution for each compound. The upper right boxes contain the Pearson r value for correlation. The lower left boxes show the scatter plots for each pair. p value less than 0.05 is considered significant. “*”: p value less than 0.05, “**”: p value less than 0.01, “***”: p value less than 0.001.

Table 2 Summary statistics for the urinary phthalate metabolites and total DEHP, di(2-ethylhexyl) phthalate, (in µg/L or ppb) MEHHP, mono (2-ethyl-5-hydroxyhexyl) phthalate; MEOHP, mono (2-ethyl-5-oxohexyl) phthalate, Actual metabolite concentrations were used. The concentrations were shown for all cases and PCOS alone, as well as in all controls then in pregnant vs non-pregnant.
Fig. 2: Case-control comparisons for DEHP and its two metabolite (MEHHP, MEOHP) concentrations in urine.
figure 2

The graph shows the side-by-side boxplots for the three compounds. The middle line represents the median while the upper and lower box borders represents first and third quartiles. Whiskers represent 1.5*IQR or largest value. P-values from Wilcoxon tests. p values of less than 0.05 were considered significant. The ** represents values less than 0.01.

Table 3 Multivariate Logistic Regression for fertility cases compared to controls. Comparisons for DEHP metabolites: MEHP mono (2-ethyl-5-hydroxyhexyl) phthalate; and MEOHP, mono (2-ethyl-5-oxohexyl) phthalate, concentrations (log 10 scales of ppb) between cases (n = 184) and controls (n = 95). *p values less than 0.05 were considered significant.

While both the crude and adjusted odds ratios for MEHHP were not significant (Table 3).

Table 4 shows the median of total DEHP and DEHP metabolites (MEHHP, MEOHP) in our study as well as from other biomonitoring studies. Our results are higher than those reported in other studies except for MEHHP which is in line with other studies.

Table 4 Urinary MEHP, MEHHP, MEOHP, and total DEHP median concentrations (µg/L) in biomonitoring studies.


Our study measured for DEHP exposure among North Jordanian women and investigated the association between DEHP levels and reproductive and fertility outcomes. To the best of our knowledge, this is the first study to measure the exposure to phthalates in Jordan. We chose DEHP as it is the most widely used phthalate. It is also the most extensively screened for in literature. We chose MEHHP, MEOHP as the DEHP metabolites to measure. We chose KAUH as it is the largest tertiary hospital in the north of Jordan with specialized clinics. It covers the entire northern region.

Our study demonstrates high exposure to DEHP among women of reproductive age in the Northern part of Jordan. Many studies suggest diet to be the primary source of exposure to DEHP [8, 26, 27]. In our study, it was shown that heating food with plastic packaging or containers in the microwave may play an essential role in ingestion of DEHP. Previous studies proved that plastic leaching from containers, especially with high temperatures is the main source of DEHP exposure [28, 29]. Food is commonly packaged and stored in plastic wrapping or containers and many people may find it easier and faster to heat the food as is in the microwave without transferring it to something not plastic such as glass plates. People may also be unaware of the effects of storing and heating food in plastic. Another possible source of exposure to phthalates are cosmetics [6]. Since women are the major users of cosmetics, this suggests that it is a source of high exposure for them. Our study showed a significant difference between cases and controls in terms of use of skin and eye make-up (p = 0.045, 0.029, respectively), sunscreen (p = 0.021), and nail polish (p = 0.033). This is in line with previous studies that stated the presence of DEHP in skin make-up products, sunscreen, and nail polish [30,31,32,33]. DEHP is a common ingredient in these products. As for eye make-up, DEHP is not commonly used in eye make-up. Interestingly, our findings indicate the presence of DEHP in commonly used products among Jordanian women. Our findings showed that median concentrations of MEOHP and total DEHP were found to be higher compared to biomonitoring studies, while MEHHP median was in line with other studies. Kuwait was reported to have the highest concentrations of total phthalate metabolites among other countries with biomonitoring studies for phthalates [34]. Other countries reported to have high concentrations of total phthalate metabolites are Saudi Arabia, India, and Korea [34]. Possible explanations for the high metabolite concentrations found in our study could be due to: first, the existence of legislations that ban the use of DEHP in the U.S., Europe, and other countries resulting in lower exposure of their people to it. Second, using highly sensitive method (HPLCMSMS) with new modern instruments may be another reason for screening high concentrations results compared to others. Third, no regulations or monitoring of DEHP use in products in Jordan exist nor are there any limits set. Cosmetic importing does not fall under any single authoritative regulation. It is treated like any other imported goods and the Jordan Food and Drug Administration (JFDA) does not have any clear rule in monitoring these products. Additionally, from our experience in interviewing our subjects, there is a lack of awareness about phthalates, what products may contain them, and their possible effects on health. An explanation for the high MEOHP median compared to other studies while the median MEHHP is in line with them may be the conversion of MEHHP to MEOHP by oxidation according to the metabolic pathway of DEHP [8]. Another reason for the high phthalate concentration is that our sample included women only, which are known to have higher urine metabolites than men due to their greater use of personal care products [17, 35, 36]. The positive significant correlation shown in our study between MEHHP and MEOHP can be explained by having the same parent compound and shared metabolic pathway following DEHP exposure [37].

In comparing cases and controls, we found that cases had higher concentrations of MEOHP and total DEHP than controls. This shows that DEHP exposure could be associated with infertility among Jordanian women. DEHP cannot be measured in the urine, it is broken down to primary metabolite MEHP, and secondary metabolites, MEHHP and MEOHP. Most studies use MEHP, MEHHP, and MEOHP metabolites with different outcomes [17, 38]. We chose only MEHHP, MEOHP as the DEHP metabolites to measure. MEHHP and MEOHP are secondary metabolites, which are excreted around three times more than primary metabolites, and are the most detected [39, 40]. Furthermore, MEHP, shows higher variability when compared to MEOHP and MEHHP [41]. There is a moderate significant correlation between all phthalate metabolites [42]. Indicating that if one is high, the others might be high as well.

Phthalate metabolites might have short biological half-lives in urine [41, 43]. In our study, we measured phthalates metabolites at least two to three times per person for at least of 80% of our women and averaged the concentrations as a proxy for the overall exposure. This may not fully characterize exposure variability. Albeit a study showed that changes in phthalates concentration in pregnant women were small (11% for DEHP) and 12% for MEHP [43]. Another study showed moderate significant intraclass correlations for MEHHP (0.43), and MEOHP (0.41) in 12 pregnant women in a single spot urine. Which could reflect longer-term exposures to the corresponding metabolites [44]. Moreover, we might not have the power to derive a temporal sequence of exposure preceding the outcome such as cohort studies, since the exposure was taken at the case ascertainment. We are under the crude assumption that people with high exposure (as shown in their urine) are having a lifestyle pattern that allows this exposure to be chronic. However, cases and controls were recruited and measured in the same way making this kind of study reasonable enough to compare the two groups. Future studies adapting a cohort approach might be needed in the future to confirm the findings. Pregnant women with no fertility problems were eligible to be enrolled as controls in our study. Pregnancy might be a period for increased susceptibility to the effects of phthalate exposure, an animal study in rats suggest that pregnancy-related changes in metabolism may result in longer biological residence times for some phthalates, potentially increasing the effect of a given exposure [45]. Very few human studies directly compared phthalate exposure between non-pregnant and pregnant women. Thus, we consider having controls, that are either pregnant or not, as an advantage to compare their levels. One study found that the urinary levels of DEHP metabolites in pregnant women were lower, particularly at the later stages of pregnancy, than those in non-pregnant women [46]. Another study compared pre-pregnancy concentrations of different endocrine disruptors with after-pregnancy concentrations. Moreover, they found that MEHP (the primary metabolite for DEHP that has the secondary metabolites we tested in our study: MEHHP, and MEOHP), had very close levels pre- and post-pregnancy levels. They concluded that absolute concentrations of phthalate metabolites were similar before and during pregnancy and the variability during pregnancy than before pregnancy was similar for ΣDEHP [43]. The pregnant controls in our study accounted for almost (~40%) of all controls. MEHHP concentrations in pregnant controls were significantly lower which is consistent with ref. [46], while MEOHP did not show significant difference between pregnant and non-pregnant controls which is consistent with ref. [43]. This indicates that pre-and post-pregnancy levels are metabolite dependent.

To the best of our knowledge, this is the first study to examine the association of infertility outcomes with phthalates concentrations in cases and controls. Other recent epidemiologic studies examined other endpoints, such as pregnancy loss in relation to urinary phthalate metabolites in women planning or attempting pregnancy [17], gestational weight gain [38]. Our study had a dual aim, to establish measuring levels in women in Jordan and to potentially see its relationship to fertility outcomes. Only (7.38%) of the sample exceeded the EPA RfD and (0.62%) exceeded the EFSA TDI. Since our study shows an association between exposure to phthalates and increased risk of fertility problems, this may suggest the need of reassessment of the existing guidance values. The cut points of these values may need to be lower to correctly reflect exposure and possible health outcomes.

Previous biomonitoring studies for urinary DEHP had samples of both men and women, women and their children, occupationally exposed individuals, couples undergoing fertility treatment, pregnant women, and/or either infertile or fertile women all summarized in Table 4. These studies were mostly cohorts. Unlike previous studies, our sample included both fertile and infertile women where a case-control comparison was conducted. The median total DEHP concentration for the whole sample in our study is 5322.7 with a significant difference between cases and controls. This concentration is much higher than other biomonitoring studies.

A study from Japan compared infertile women with endometriosis to infertile women without endometriosis, they showed median total DEHP concentrations of 89.3 µg/L and 72.7 µg/L for both groups, respectively [47]. A study done in France including pregnant women showed high exposure to DEHP with a median total DEHP concentration of 84.6 µg/L [48]. Another study from the Netherlands also included pregnant women and showed a median total DEHP concentration of 61.8 µg/L [49]. A Korean study included mothers and showed a median total DEHP concentration of 55.7 µg/L [50].

This study demonstrates high exposure of the Jordanian population to DEHP. It confirms the association between DEHP exposure and infertility. Furthermore, our study is the first done in Jordan to measure the phthalate exposure and one of the limited studies done in the area. More investigations and studies are needed to fully assess the specific sources of exposure to DEHP among Jordanians. Rules and legislations related to the use of DEHP in products are needed to decrease exposure to it and minimize the risk of potential health effects.

The strengths of our study are: having good sample size for a unique sample population that has not been studied before, collection of repeated urine samples from subjects over time, using highly sensitive techniques for screening, and the investigation of phthalate role in infertility. We are the first study to do screening and baseline levels of DEHP metabolite exposure in Jordan and one of the few limited studies in the surrounding area. Using HPLC/MS-MS as the analytical method to detect the DEHP metabolites provides high selectivity and sensitivity where which is becoming the method of choice for the detection of phthalate metabolites in biological samples [51, 52]. Our current study has several limitations: First, the lower number of controls compared to cases. Subjects considered cases were more willing to participate than those considered controls. Additionally, due to the Covid-19 pandemic we were unable to recruit additional subjects. However, this should not affect the study outcome. We selected the controls from the same referral region that applied for the cases. Some studies reported that increasing the number of cases rather than the number of controls is the best way to increase precision. The increase in precision from adding a case is greater than that from adding a control [53, 54]. Second, we did not adjust for urinary dilution as we could not obtain creatine levels or specific gravity values. Moreover, we assessed urinary DEHP metabolite levels through single-spot urine samples, which may not accurately reflect long term exposure since phthalates are metabolized and excreted rapidly within hours. However, Koch et al. reported that means and medians obtained from single spot sampling and 24-h collection are very comparable [54, 55]. Thus, we used MEOHP and MEHHP as indicators for DEHP exposure. However, measurements of MEHP level may be more important and more relevant to study the association between exposure to DEHP and adverse health outcomes [39].