Hypospadias occurs at around 8 to 14 weeks after conception. It is one of the most common structural malformations in humans, occurring in approximately 4 to 6 per 1000 male births.^4-6 Corticosteroids (ie, glucocorticoids) affect several mechanisms that are critical to urethral development, including sex steroid synthesis, placental function, and epithelial–mesenchymal cell interactions.^7-14 A recent experimental study demonstrated that prednisone significantly affected urethral seam closure in mice.¹⁵ Supraphysiologic doses of prednisone resulted in a more proximal urethral opening and thinner connective tissue around the urethral seam, and 25% of the male offspring had hypospadias. Experimental studies have also shown that administration of corticosteroids in utero results in reduced anogenital distance in male offspring, which is considered to be a measure of antiandrogenic exposure.^16-18 An epidemiologic study in Denmark found that prescriptions for inhalation or systemic glucocorticoids in the period from 90 days before conception through the first trimester of pregnancy were not associated with hypospadias (odds ratio [OR] = 1.1; 95% confidence interval [CI] = 0.4 to 2.7),¹⁹ A study in Sweden reported no association of hypospadias with anti-asthma medications, some of which were corticosteroids, in early pregnancy (OR 1.0, 95% CI 0.9, 1.3)²⁰, and a study in Hungary reported no increased risk associated with use of oral (OR 0.9, 95% CI 0.7, 1.3) or ointment (OR 0.4 95% CI 0.1, 1.2) corticosteroids at any time during pregnancy.²¹

The present study provides a detailed assessment of the association between corticosteroid use and hypospadias. Investigation of this hypothesis is important, given the biological plausibility and recent experimental evidence described above, along with limited previous epidemiologic studies. Specifically, using data from a recent large population-based, case-control study conducted in the United States, we explored whether women who reported corticosteroid use during pregnancy were more likely to deliver an infant with hypospadias compared with women who did not use corticosteroids during pregnancy.

This study included only cases of second- or third-degree hypospadias, that is, with the urethral opening at the penile shaft, scrotum, or perineum (modified British Pediatric Association codes 752.606, 752.607, 752.626, and 752.627). Medical record information (including operative reports when available) with anatomic descriptions or diagrams by pediatricians, urologists, geneticists, pathologists, or other health care providers was reviewed by a clinical geneticist at each study center who determined whether to include or exclude cases in the NBDPS database. Cases described as chordee alone, mild hypospadias (ie, first-degree, coronal, or glandular), hypospadias not otherwise specified, epispadias, or ambiguous genitalia without further description were excluded. Infants with recognized single gene disorders, female karyotypes, or chromosomal abnormalities also were excluded. Each case received a final review by a single clinical geneticist (R.O.) to ensure that cases from each study center met standard eligibility criteria. This geneticist also classified each case as isolated, if there was no concurrent major anomaly or only a minor anomaly (eg, sacral/pilonidal dimple), or multiple, if there was at least 1 unrelated accompanying major anomaly and in another organ system.²⁴

Maternal interviews were conducted using a standardized, computer-based telephone questionnaire in English or Spanish, no earlier than 6 weeks and no later than 24 months after the infant's estimated date of delivery (EDD). Final EDD was based on the mother's self-report; if this was not available, then EDD was estimated from information in the medical record (< 2% of subjects). Interviews were conducted with the mothers of 1165 cases (77% of eligibles) and 3000 controls. (The participation rate in the mothers of all controls was 75%; the rate in the mothers of male-only controls was not available.) The mean time from delivery to interview was 13.2 months in the mothers of cases and 8.9 months in the mothers of controls.

Exposure to corticosteroid medications was determined by asking the mothers whether they experienced various types of illnesses and injuries (eg, respiratory illness, infections) and what medications they used to treat them. The mothers were also asked to describe the specific illness or injury that they experienced. In a final section, the mothers reported the use of any other medications not reported in the preceding illness/injury-specific sections; indication was not recorded in this section. For each medication, information on start and stop dates of use and frequency of use was recorded. Those women who knew only either the start date or stop date of use, but not both, were asked about the duration of use. Medication exposure was assessed during the period extending from 4 weeks before conception through 18 weeks after conception. The date of conception was derived by subtracting 266 days from the EDD. A central coding facility assigned a drug code to each medication exposure, using the Slone Epidemiology Center Drug Dictionary. These codes were used to identify the primary components in each medication. Route of administration (eg, topical, systemic) was assigned based on exact wording from the mother's report or on the known formulation of the medication. Indication was assigned based on injury or illness reported in conjunction with the corticosteroid medication.

The following covariates were considered for inclusion in multivariate models given a possible association with the risk of hypospadias or reported corticosteroid use: maternal education (less than high school diploma, high school diploma, 1 to 3 years of college, 4 or more years of college), race/ethnicity (non-Hispanic white, non-Hispanic black, Hispanic, other), age (< 25, 25 to 34, ≥ 35 years at the time of conception), number of previous live births (0, 1, 2 or more), folic acid–containing supplement intake (any vs none from 1 month before through 3 months after conception), smoking (any vs none from 1 month before through 3 months after conception), body mass index (underweight, normal weight, overweight, or obese),²⁵ subfertility (ie, any fertility-related treatments or procedures), and study site. The subfertility variable was based on a positive response to any of the following 3 questions: (1) “Did you have any surgical procedures [to help you become pregnant]?”; (2) “In the 2 months before you became pregnant with [baby's name], did you take any medications to help you become pregnant?”; and (3) “Did you have any other procedures to help you become pregnant?”

We first examined the association between the risk of hypospadias and corticosteroid use versus no corticosteroid use during the periconceptional period (ie, 4 weeks before conception through 14 weeks after conception). We initially used this time window, which both precedes and includes the time of urethral and genital tubercle development, because some of the potential effects of corticosteroid medications extend beyond the actual period of use (eg, adrenal suppression). We then examined associations with more specific time windows of exposure, route of administration, and medication components. We also explored associations with corticosteroid use starting at 15 to 18 weeks after conception (i.e., after urethral closure would have been completed), to help identify possible reporting errors. ORs were estimated only if there were at least 2 exposed cases and 2 exposed controls in a particular comparison. To estimate relative risks, maximum likelihood estimates of ORs and their corresponding 95% CIs were calculated from logistic regression models using SAS version 9.1 (SAS Institute, Cary, North Carolina). Covariates were included in the models if their inclusion resulted in at least a 10% change in the OR for the association of any corticosteroid use with hypospadias; they were tested one at a time because of the limited number of exposed subjects.

We also examined whether the association of corticosteroid use varied depending on whether or not the mother took folic acid–containing supplements during the periconceptional period, using a cross-product term in the logistic model.Most of the women who took supplements used a prenatal formulation.²⁶ Experimental studies have shown that vitamin B₆ and folic acid, both of which are contained in prenatal vitamin formulations, are protective against the teratogenic effects of corticosteroids,^27,28 possibly because they suppress the activity of glucocorticoid receptors.^29,30

Analyses by route of administration and specific component found elevated ORs only for nasal spray/inhaled corticosteroids (Table I). As with the overall OR for any use, the ORs by route and component were reduced after adjustment for covariates, and all of the CIs included 1.

Analyses of more specific time windows of exposure found no elevated ORs for exposures occurring only during the 4 weeks before conception or the first 4 weeks after conception, after adjustment for potential confounders. The odds ratio for any exposure during weeks 5-14 after conception, which corresponds more closely to the actual time of genital tubercle and urethral development, was elevated (OR 1.8, 95% CI 1.1, 2.8), but closer to one after adjustment for covariates (OR 1.4, 95% CI 0.9, 2.4) (Table II). Separate analyses of exposures during weeks 5-8 versus weeks 9-14 yielded very similar results (data not shown). The OR for exposures starting in weeks 15 to 18 after conception (ie, after the completion of urethral development) was 1.0 (95% CI = 0.2 to 5.4), based on the exposures in 2 mothers of cases and 5 mothers of controls.

Separate analyses by indication were not conducted, because indication was not specified in a majority of the exposed cases (19/39) and controls (23/33), and also because the reported indications were diverse, especially within subgroups based on timing.

The association of any versus no corticosteroid use was examined separately in women who began taking vitamin supplements during the month before conception or the first month after conception (early intake), during the second or third month after conception (later intake), or not at all during this time period (no periconceptional intake). The results suggest an association between corticosteroid use and increased risk of hypospadias in those with later or no periconceptional supplement intake (OR = 3.0 and 2.9, respectively) but not in those with early supplement intake (OR = 1.1) (Table III). The P value for the interaction term included in the logistic regression model was .091. The stratum-specific ORs were similar after adjustment for the covariates.

Our findings suggest that the use of nasal spray/inhaled corticosteroids may be associated with a moderately increased risk of hypospadias, but that systemic corticosteroids are not associated with increased risk. One possible explanation for this difference is the longer duration of use for nasal spray/inhaled medications; However, the half-life and absorption of these medications tend to be less than systemic medications. Frequency of exposure tended to be daily for both medications and thus is an unlikely explanation for the findings.

Although we found no strong association between corticosteroid use and hypospadias overall, we did find a possible association between corticosteroid and increased risk of hypospadias in the mothers who did not take folic acid—containing supplements around the time of conception. In other words, early supplement intake may be protective against potential negative effects of corticosteroid intake. However, it is unclear why supplement intake during the second or third month of pregnancy, which would cover the time of urethral development, was not also protective. As noted earlier, experimental studies have demonstrated that vitamin B₆ and folic acid, which are contained in prenatal vitamin preparations, are protective against the teratogenic effects of corticosteroids,^27,28 and that these effects may result from the suppressed activity of glucocorticoid receptors. ^29,30 A limitation of our stratified analysis is we analyzed corticosteroid exposure only overall, that is, without regard to specific time windows or route of administration. We did not pursue further classification of corticosteroid use based on timing, because of the relatively small numbers of exposed women and variable patterns of use during the designated time period. We categorized supplement use based on timing because maternal characteristics have been shown to vary among women who start taking supplements at different times relative to pregnancy.²⁶

Strengths of our study include its population-based design, large size, geographic diversity, and ability to examine corticosteroid medication use based on specific time periods related to urethral development. It was also advantaged by its thorough case ascertainment from medical records, precise case definition, and differentiation of isolated and multiple cases. But even in this large study, certain comparisons were limited by small sample size, including comparisons based on type of medication and comparisons of ORs within the stratum of vitamin supplement use. Other limitations include the inability to examine dose of medications and underlying maternal conditions or indication. Recall bias (i.e., differential accuracy of reporting by case and control mothers) is possible, although the popular belief is that mothers of cases would tend to more accurately report exposures than mothers of controls, leading to spuriously strong results. The longer recall period among cases than controls could also contribute to recall bias. Given that about 25% of study subjects were not interviewed, selection bias is a potential concern, and the generalizability of our findings to the larger study population is uncertain. For example, it is possible that non-participation could have selectively removed corticosteroid-exposed cases and therefore incurred some bias in our results, but we cannot determine whether this occurred. In addition, given that our study was limited to moderate/severe cases, an association with milder forms of hypospadias (the majority of cases) is possible, but this was not examined.