Hypospadias and Maternal Intake of Phytoestrogens

Abstract

Experimental data indicate that gestational exposures to estrogenic compounds impact risk of hypospadias. We examined whether risk of hypospadias (i.e., a congenital malformation in which the opening of the penile urethra occurs on the ventral side of the penis) was associated with maternal intake of phytoestrogens, given their potential impact on estrogen metabolism. The analysis included data on mothers of 1,250 hypospadias cases and 3,118 controls who delivered their infants from 1997 to 2005 and participated in the National Birth Defects Prevention Study, a multistate, population-based, case-control study. After adjustment for several covariates, high intakes of daidzein, genistein, glycetin, secoisolariciresinol, total isoflavones, total lignans, and total phytoestrogens were associated with reduced risks; odds ratios comparing intakes ≥90th percentile with intakes between the 11th and 89th percentiles ranged from 0.6 to 0.8. For example, the odds ratio for total phytoestrogen intake was 0.7 (95% confidence interval: 0.5, 1.0). This study represents the first large-scale analysis of phytoestrogen intake and hypospadias. The observed associations merit investigation in additional populations before firm conclusions can be reached.

The birth defect hypospadias occurs when the urethral opening is on the ventral side of the penis. It is a relatively common congenital malformation, affecting 4–6 per 1,000 male births (1). Experimental data indicate that maternal exposures to estrogenic compounds may impact risk of hypospadias by interfering with the production or action of fetal androgens, which are critical to normal urethral closure (2–5).

North and Golding (6) reported a 4-fold increased risk of hypospadias among boys born to vegetarian women. One proposed explanation for the increased risk was higher intake of phytoestrogens among vegetarians, given their estrogenic properties. Subsequent studies have not confirmed an association with a vegetarian diet (7–11), with 1 exception (12). One study specifically estimated phytoestrogen intake and did not find an association with hypospadias, but it included only 56 cases (7).

Phytoestrogens are plant constituents named for their estrogenic properties. The main types are isoflavones, lignans, and coumestans. Lignans have been shown to interfere with conversion of testosterone to dihydrotestosterone (13), which is critical to normal urethral closure, and the isoflavone genistein has been shown to induce hypospadias in mice (14). Genistein has also been associated with reduced anogenital distance in male rats (15). High intake of isoflavones increases 2-hydroxylation of estradiol (16), which results in estrogen derivatives that are mostly non- or weakly estrogenic (17). Thus, isoflavones could protect against hypospadias. Given their varied effects on the levels and activity of sex hormones such as estrogen and testosterone (13, 18), phytoestrogens have been likened to natural selective estrogen receptor modulators (19). As such, it is reasonable to hypothesize that they are associated with hypospadias, but it is not straightforward to predict the direction of such an association.

The objective of this analysis was to investigate whether the congenital malformation hypospadias was associated with intake of phytoestrogens among a large group of women participating in the National Birth Defects Prevention Study (NBDPS).

MATERIALS AND METHODS

Study design

The NBDPS is a large population-based, multicenter, case-control study of selected structural congenital malformations. Recruitment and data collection began with deliveries that had estimated due dates in October 1997 and are ongoing; the current analysis includes deliveries through the end of 2005 (the year through which the phytoestrogen data are available). Detailed study methods and descriptions of the surveillance systems in the 10 NBDPS study centers have been published (20). In brief, 7 included liveborn, stillborn (fetal deaths at >20 weeks’ gestation), and prenatally diagnosed and electively terminated cases (Arkansas, California, Georgia, Iowa, North Carolina, Texas, Utah); 1 included only liveborn and stillborn cases (Massachusetts); 1 included only liveborn cases (New Jersey); and 1 included liveborn cases from 1997 to 1999 and added stillborn cases in 2000 (New York).

Case review and classification

The NBDPS includes second- and third-degree hypospadias; that is, the urethra opened at the penile shaft, scrotum, or perineum. Medical record information (including operative reports) including anatomical descriptions or diagrams was reviewed by a clinical geneticist at each study center who decided about inclusion or exclusion. Cases described as chordee alone, mild (i.e., first degree, coronal, or glanular), hypospadias not otherwise specified, epispadias, or having ambiguous genitalia without further description were excluded. Infants with recognized single-gene disorders or chromosomal abnormalities were excluded. Each case received a final review by 1 clinical geneticist (R.S.O.) to ensure that cases from each study center met eligibility criteria. This geneticist also classified each case as isolated if there was no concurrent major anomaly or only minor anomalies (e.g., sacral/pilonidal dimple) or as nonisolated if there was at least 1 accompanying major anomaly (21).

Control selection

Each participating center randomly selected approximately 150 liveborn controls without birth defects per study year from birth certificates (Arkansas 2000–current, Georgia 2001–current, Iowa, Massachusetts, North Carolina, New Jersey, Utah) or birth hospitals (Arkansas 1997–1999, California, Georgia 1997–2000, New York, Texas) to represent the populations from which cases were derived. The current analysis was restricted to mothers of male controls.

Maternal interviews

Maternal interviews were conducted by using a standardized, computer-based questionnaire, nearly exclusively by telephone, in English or Spanish, from 6 weeks to 24 months after the infant's estimated due date. Exposures to many factors were assessed, relative to the woman's estimated date of conception, which was derived by subtracting 266 days from the woman's expected due date, which was primarily self-reported. Interviews were conducted with mothers of 72% of cases (n = 1,355) and 67% of male controls (n = 3,432). The median time from actual date of delivery to interview was 12.9 months for cases and 8.8 months for controls.

Food frequency questionnaire

Mothers reported their average intakes of foods using a shortened version of the food frequency questionnaire developed by Willett et al. (22) for the Nurses’ Health Study that included 58 food items. Participants reported how often, on average, they consumed food items in the year before they became pregnant. Fifteen response categories were possible ranging from once per month to 6 or more per day. Foods eaten less than once a month were recorded as “never or none.” Intakes of breakfast cereals, sodas, food supplements, and caffeinated tea and coffee were assessed by separate, more detailed questions, which covered intake during the 3 months before pregnancy. Because few women (mothers of 10% of cases and 10% of controls) consumed food supplements (which included items such as powdered drink supplements) and nutrient data were not available for many of these products, food supplements were not included in nutrient calculations. The US Department of Agriculture nutrient database, version 20, was the source of nutrient values (23).

Phytoestrogen intake

Phytoestrogen values published by Kuhnle et al. (24–27) were used to estimate maternal phytoestrogen intake. These values are recent, derive from a single laboratory using state-of-the-art techniques (28), are available for a relatively consistent set of specific phytoestrogens across all food items, and were available for most of the foods in this study's food frequency questionnaire (if not, we selected proxies). Phytoestrogen values were assigned to each food item in the food frequency questionnaire, with details available in the report by Carmichael et al. (29). We assessed total intake of specific isoflavones and lignans as well as their totals, intake of coumestans, and total phytoestrogen intake.

Analyses

Mothers with <500 or >5,000 kcal of energy intake and mothers with >1 food item missing (i.e., not queried) from the food frequency questionnaire (54 cases, 105 controls) were excluded, leaving 1,301 cases and 3,327 male controls available for analyses. A total of 1,250 cases and 3,118 controls had complete data on all covariates. Logistic regression analyses were conducted to estimate odds ratios and 95% confidence intervals reflecting the association of hypospadias with phytoestrogen intake. We compared intake ≥90th percentile and intake ≤10th percentile, relative to intakes between the 11th and 89th percentiles, using cutoffs determined from the distribution among control mothers. Bivariate analyses were followed by analyses adjusted only for energy intake (kilocalories, as a continuous variable), to adjust for the overall caloric density of the diet. We adjusted analyses for the following covariates, which were selected a priori on the basis of potential associations with hypospadias and/or nutritional status: maternal race/ethnicity (non-Hispanic white, Hispanic, African American, other); education (less than, equal to, or greater than high school); age (years); number of previous livebirths; body mass index (weight (kg)/height (m)²); intake of folic acid–containing supplements during the month before or the first 3 months of pregnancy; and study center.

RESULTS

Mothers of cases were more likely to be non-Hispanic white, have higher education, be older and nulliparous, and take folic acid–containing supplements, relative to control mothers (Table 1).

Table 1.

Descriptive Characteristics of Mothers of 1,250 Infants With Hypospadias and 3,118 Male Controls, National Birth Defects Prevention Study, 1997–2005

	Cases		Controls
	No.	%a	No.	%a
Maternal race/ethnicity
Non-Hispanic white	909	73	194	62
Non-Hispanic black	152	12	364	12
Hispanic	105	8	609	20
Other	84	7	204	7
Maternal education
<High school	98	8	454	15
High school	235	19	786	25
>High school	917	73	1,878	60
Maternal age, years
<25	284	23	1,029	33
25–34	698	56	1,643	53
≥35	268	21	446	14
No. of previous livebirths
0	685	55	1,272	41
1	371	30	1,050	34
≥2	194	16	796	26
Maternal prepregnancy BMIb
Underweight (<18.5)	61	5	189	6
Normal weight (18.5–<25.0)	678	54	1,736	56
Overweight (25–<30.0)	295	24	702	23
Obese (≥30.0)	216	17	491	16
Folic acid-containing supplement intakec
Yes	1,169	94	2,749	88
No	81	6	369	12

Abbreviation: BMI, body mass index.

^a Numbers may not add to 100% because of rounding.

^b BMI: weight (kg)/height (m)².

^c During the month before or the first 3 months of pregnancy.

For each phytoestrogen, intake ≥90th percentile was associated with reduced risk of hypospadias in unadjusted models, with odds ratios ranging from 0.4 to 0.7 and 95% confidence intervals excluding 1.0 with the exception of matairesinol (odds ratio = 0.9) (Table 2). After adjustment for energy intake, high intakes of biochanin A, formonoetin, and coumestrol were not associated with reduced risks. After adjustment for additional covariates, high intakes of daidzein, genistein, glycetin, secoisolariciresinol, total isoflavones, total lignans, and total phytoestrogens were associated with reduced risks, although the odds ratios tended to be slightly closer to 1 than before adjustment (ranging from 0.6 to 0.8), and some of the confidence intervals included 1.0.

Table 2.

Association of Hypospadias With Intake of Phytoestrogens Among Women in the National Birth Defects Prevention Study, 1997–2005

Phytoestrogens, µg/daya	Unadjusted Odds Ratio	95% CI	Energy-adjusted Odds Ratio	95% CI	Covariate-adjusted Odds Ratiob	95% CI
Daidzein
≤4.9	1.1	0.9, 1.4	1.0	0.8, 1.2	1.0	0.8, 1.3
5.0–24.1	1.0	Referent	1.0	Referent	1.0	Referent
≥24.2	0.4	0.3, 0.6	0.6	0.4, 0.8	0.7	0.5, 1.0
Genistein
≤10.7	1.3	1.1, 1.6	1.1	0.9, 1.4	1.1	0.9, 1.4
10.8–41.7	1.0	Referent	1.0	Referent	1.0	Referent
≥41.8	0.4	0.3, 0.6	0.6	0.4, 0.8	0.6	0.4, 0.9
Biochanin A
≤6.7	1.0	0.8, 1.3	0.8	0.7, 1.0	1.0	0.8, 1.2
6.8–26.0	1.0	Referent	1.0	Referent	1.0	Referent
≥26.1	0.7	0.5, 0.9	1.1	0.8, 1.4	0.9	0.7, 1.3
Formonoetin
≤2.5	1.0	0.8, 1.2	0.8	0.7, 1.0	1.0	0.8, 1.3
2.6–9.6	1.0	Referent	1.0	Referent	1.0	Referent
≥9.7	0.7	0.6, 0.9	1.1	0.9, 1.5	0.9	0.7, 1.2
Glycetin
≤3.7	1.2	1.0, 1.5	1.0	0.8, 1.3	1.0	0.8, 1.3
3.8–18.7	1.0	Referent	1.0	Referent	1.0	Referent
≥18.8	0.4	0.3, 0.5	0.5	0.3, 0.6	0.7	0.4, 1.0
Matairesinol
≤9.1	1.1	0.9, 1.3	0.8	0.7, 1.1	1.0	0.7, 1.2
9.2–39.5	1.0	Referent	1.0	Referent	1.0	Referent
≥39.6	0.9	0.7, 1.1	1.2	0.9, 1.5	1.0	0.8, 1.3
Secoisolariciresinol
≤40.4	1.0	0.8, 1.2	0.9	0.7, 1.1	1.0	0.8, 1.3
40.5–225.0	1.0	Referent	1.0	Referent	1.0	Referent
≥225.1	0.5	0.4, 0.6	0.6	0.5, 0.8	0.6	0.4, 0.9
Coumestrol
≤3.6	1.2	1.0, 1.4	1.0	0.8, 1.2	1.1	0.9, 1.4
3.7–17.3	1.0	Referent	1.0	Referent	1.0	Referent
≥17.4	0.7	0.5, 0.9	0.9	0.7, 1.2	1.0	0.7, 1.3
Total isoflavones
≤25.7	1.1	0.9, 1.4	1.0	0.8, 1.2	1.0	0.8, 1.3
25.8–103.4	1.0	Referent	1.0	Referent	1.0	Referent
≥103.5	0.4	0.3, 0.6	0.6	0.4, 0.8	0.7	0.5, 1.0
Total lignans
≤50.8	0.9	0.7, 1.1	0.8	0.6, 1.0	0.9	0.7, 1.2
50.9–258.6	1.0	Referent	1.0	Referent	1.0	Referent
≥258.7	0.5	0.4, 0.7	0.7	0.6, 1.0	0.8	0.6, 1.1
Total phytoestrogens
≤86.6	1.0	0.8, 1.3	0.9	0.7, 1.1	1.0	0.8, 1.2
86.7–376.3	1.0	Referent	1.0	Referent	1.0	Referent
≥376.4	0.4	0.3, 0.6	0.6	0.4, 0.8	0.7	0.5, 1.0

Abbreviation: CI, confidence interval.

^a Nutrient values reflect intakes ≤10th percentile and ≥90th percentile, with intakes between the 11th and 89th percentiles as reference. These categories were determined from nutrient intake levels among control mothers.

^b Adjusted for maternal age (years), parity (ordinal), race/ethnicity, education, body mass index (weight (kg)/height (m)²), study center, folic acid–containing supplement intake, and energy intake (kcal).

DISCUSSION

This study found that higher intake of some phytoestrogens was associated with reduced risks of delivering infants with hypospadias (second and third degree), even after adjustment for several covariates. This finding applied to overall intake of phytoestrogens as well as intake of specific phytoestrogens. Lower intake, however, did not tend to be associated with increased risk.

The study that first reported an association between hypospadias and a maternal vegetarian diet also reported increased risk associated with intake of soy products and legumes, but estimates were imprecise and not adjusted for any covariates (6). Another small study reported that legume intake was not associated with hypospadias (9). A third small study administered a phytoestrogen-specific food questionnaire and quantified intake of lignans and isoflavones; phytoestrogen intake was not associated with hypospadias (7). Several factors may contribute to the differences in the findings of the current study relative to the previous one, including in the previous study, the use of a different food frequency questionnaire, the use of a different database to estimate phytoestrogen content of the queried foods, the examination of tertiles rather than more extreme ends of the distribution, and chance.

Our previous investigations of hypospadias with nutritional factors, also using NBDPS data, have not found an association with vegetarian diet, frequency of consumption of animal products, general diet quality, or intake of vitamin supplements (11, 30). The mechanism by which phytoestrogens may protect against hypospadias is not clear. Phytoestrogens may protect against various other health outcomes, such as certain cancers, diabetes, and cardiovascular disease (13, 31–35). Their endocrine-related activities as well as their antiproliferative and antioxidant properties may contribute to these associations (31, 36). Further, several recent experimental studies in rodents have suggested either teratogenic or protective effects of phytoestrogens with respect to fetal development (14, 37–40). Nonetheless, our findings warrant replication.

A critical strength of this study is its ability to estimate phytoestrogen intake from a variety of foods, rather than relying only on food groups that are high in phytoestrogens as proxies for intake, as has occurred in previous studies. We developed a database that reflects the phytoestrogen content of foods that were queried for this study (29), using recent values that were generated by using state-of-the-art measurement techniques, for multiple specific phytoestrogens (26). Our study is also strengthened by its large sample size, population-based control selection, and careful case review. A limitation is incomplete or absent collection of data on intake of certain items that contribute to phytoestrogen intake, such as coffee and tea (NBDPS assessed only caffeinated versions), alfalfa sprouts, flax seed, food supplements, and specific soy-containing products, as well as lack of information on bioavailability or absorption. These limitations could have led to misclassification, which we expect to be nondifferential. Foods with modest phytoestrogen content but high frequency of intake tended to be primary contributors to phytoestrogen intake in this study population (29), which may stem from lack of information on high-phytoestrogen foods like soy products. The current study cannot determine whether more extreme phytoestrogen intake, for example, among women consuming large quantities of soy products, is associated with hypospadias, either positively or negatively. This subset of women, although important, is likely to be small. On the basis of recent NBDPS data collection, which involved a modified questionnaire (but insufficient hypospadias cases for a separate analysis), only 3%–4% of control mothers consumed items such as soy milk or tofu at least once per week (29). In addition, we could not validate our phytoestrogen estimates against a “gold standard,” such as serum values. However, previous studies of phytoestrogen intake derived from food frequency questionnaires have demonstrated good validity when compared with serum or urine values (41, 42). The study relied on women's recall of dietary intake 1 year before pregnancy. The quality of self-reported information and the predictive value of such information specifically for gestational intake may be subject to error. If this error is unrelated to case status, it would tend to bias effects toward 1.0. We should also note that the NBDPS excluded mild (first-degree) cases by design, so the generalizability of our findings to less severe cases is uncertain.

This study represents the first large-scale analysis of phytoestrogen intake and hypospadias (second and third degree). The observed protective associations merit investigation in additional study populations before firm conclusions can be reached.