In this large case–control study, we found 2- to 3-fold increased risk of hypospadias among children of mothers exposed to hair spray and phthalates in the workplace during pregnancy and a 36% reduction in risk associated with folate supplementation during the first 3 months of pregnancy. These findings were robust to control for potential confounders. In contrast with two smaller studies (Akre et al. 2008
; North and Golding 2000
), we did not find an association between vegetarianism/veganism and hypospadias.
This is the first study to report a significant association between maternal occupational exposure to hair sprays, some of which may contain phthalates, and risk of hypospadias. A previous study reported that from 1980 to 1989, women hairdressers had slightly reduced risk of giving birth to a boy with hypospadias, whereas during 1992–1996, risk was significantly increased (OR = 1.50; 95% CI, 1.02–2.09) (Vrijheid et al. 2003
). The risk was reduced after adjustment for parental social class (OR = 1.18; 95% CI, 0.80–1.64), suggesting possible confounding (Vrijheid et al. 2003
). In contrast, our study showed that adjustment for household income (or maternal educational level) as a proxy for social class did not materially affect the risk estimates.
We are unaware of studies that have reported urinary phthalate metabolites in hairdressers or women applying hair sprays, so it is unclear to what extent their exposure may have been elevated at the time of the study. Phthalates, predominantly diethyl phthalate (DEP) and dibutyl phthalate (DBP), were present in many cosmetics including deodorants, fragrances, and nail and hair products (Hubinger and Havery 2006
; Koo and Lee 2004
). However, since 2005, certain phthalates including DBP have been prohibited for use in cosmetic products in Europe. The phthalates or their metabolites, for example, monoethyl phthalate and mono-n
-butyl phthalate, are associated with androgen-lowering activities and abnormal Leydig cell function and have been linked to a decrease in anogenital distance in male infants (Main et al. 2006
; Swan et al. 2005
); androgen lowering is associated with reproductive tract malformations including hypospadias (Lottrup et al. 2006
; Mylchreest et al. 2000
). Inhalation contributes significantly to the uptake of these phthalates (Adibi et al. 2003
), which may explain some species differences for DEP between human and animal studies, where oral administration has mainly been used (Lottrup et al. 2006
; Mylchreest et al. 2000
). A number of other substances included in hair sprays may have toxic effects if inhaled. They include polyvinyl alcohol, polyvinylpyrolidone, hydrofluoro-carbon, and propylene glycol, although for these substances the concern is acute effects on the cardiorespiratory system, skin, and eyes rather than on the reproductive system (U.S. National Library of Medicine 2008
This is also the first study to show a protective effect of folate supplementation on risk of hypospadias. A recent Dutch case–control study found no association of maternal folic acid supplements and hypospadias (Brouwers et al. 2007
), nor was there an effect in a trial of folic acid/multivitamin supplementation based on small numbers of cases (Czeizel 1996
). However, in a Hungarian case–control study, use of dihydrofolate reductase inhibitors (folic acid antagonists) in pregnancy was associated with a (nonsignificant) 20% excess risk of hypospadias (Czeizel et al. 2001
Although we cannot exclude recall bias in our study, we believe it is unlikely to explain our findings, given that folate has not previously been associated with hypospadias, and, to explain the association reported here, the size of any such bias would need to be large. In the United Kingdom, 400 μg folate supplementation is recommended in the first trimester of pregnancy for the prevention of neural tube defects (Department of Health 2000
). Although we did not collect quantitative data on dietary intake of folate, the National Diet and Nutrition Survey estimated that mean intake of folate from foods ranged from 229 μg/day for women at 19–24 years of age to 255 μg/day at 35–49 years of age (National Diet and Nutrition Survey 2003
). Thus, folate supplementation is likely to have more than doubled the daily folate intake. Like the neural tube, the urethra is a midline structure. Biochemical, genetic, and epidemiologic observations suggest that folic acid may prevent neural tube defects by stimulating cellular methylation reactions, although this methylation hypothesis requires further exploration (Blom et al. 2006
). Folic acid may also protect against other congenital anomalies such as orofacial clefts, cardiac, and urinary tract defects (Hernandez-Diaz et al. 2000
). In addition, recall bias for hair spray is unlikely, because only one previous study reported a possible association (Vrijheid et al. 2003
). Recall bias is not an issue for phthalate exposure, because it was assessed by job exposure matrix. However, some exposure misclassification is possible with the job exposure matrix because of uncertainty in expert assessment (Van Tongeren et al. 2002
Major strengths of our study include the large sample size, wide population-based coverage, and extensive interviewer-based questionnaire. Most previous studies of hypospadias have relied on routinely collected registry data with limited information on potential risk factors and confounders, and varying levels of quality control and completeness (Aho et al. 2003
; Irgens 2000
; Kallen 1988
; Kallen et al. 1986
; Kristensen et al. 1997
; Vrijheid et al. 2003
). We ascertained slightly more cases than the hospital registries and many more cases than the national congenital anomalies system (Nelson et al. 2007
). Our study also has limitations. One potential weakness is the low proportion of control women who replied to our invitation to participate. Because of constraints imposed by the ethics committee, we were not able to contact the women in the control group directly after they were randomly selected from the birth registry. Instead, we had to follow a convoluted procedure requiring both health authorities and the mothers’ GPs to forward our invitation pack, with the result that an estimated 72% of nonresponders never received the participant invitations. Although the controls appeared to be of slightly higher social class than the cases, adjusting for socioeconomic status (i.e., income or education) made no difference to our findings.
Furthermore, we investigated more formally the potential for selection bias by socioeconomic status (SES) in selection of cases and controls using a weighting procedure analogous to poststratification for adjustment of item nonresponse in the survey literature (Park et al. 2004
). In brief, this involves reweighting the estimates of probabilities of exposure conditional on case/control status according to the distribution of SES in the target (i.e., unbiased population). The idea is that if, for example, individuals with low SES are underrepresented in the study, these estimates can be up-weighted using the SES distribution of the target population. We found no evidence of selection bias mediated by SES in the study (Geneletti et al. 2008
A particular difficulty affecting research on hypospadias is the wide variation in case definition, from mild displacement of the urethral orifice to severe anomalies requiring major corrective surgery (Baskin et al. 2001
). We relied on clinical judgment of surgeons who operate on cases, and thus excluded milder cases who were not referred for surgical correction. We also excluded cases where hypospadias was part of a wider syndrome. Thus our study is likely to have included a greater proportion of more severe, isolated cases than some previous studies.
In conclusion, this is the first study to report increased risks of hypospadias associated with exposure to phthalates and hair sprays and a protective effect of folate supplementation. Previous association of vegetarianism with risk of hypospadias was not confirmed. Measurements of exposure to phthalates and/or biomonitoring may help to understand possible pathways of exposure and toxicology and provide quantitative estimates. Our findings with respect to folate use may have important implications for public health and prevention.