Risk factors for undescended testis

Journal of Pediatric Urology (2012) 8, 59e66

Risk factors for undescended testis Marijn M. Brouwers a, Leonie M. de Bruijne a, Robert P.E. de Gier b, Gerhard A. Zielhuis a, Wouter F.J. Feitz b, Nel Roeleveld a,* a b

Radboud University Nijmegen Medical Centre, Department of Epidemiology, Biostatistics, and HTA, The Netherlands Radboud University Nijmegen Medical Centre, Paediatric Urology Centre, The Netherlands

Received 25 July 2010; accepted 5 November 2010 Available online 27 November 2010

KEYWORDS Cryptorchidism; Endocrine disruptors; Inguinal hernia; Risk factors; Undescended testis

Abstract Objective: To contribute to the understanding of the etiology of undescended testis (UDT), by exploring a wide range of potential risk factors in a case-referent study. Patients and methods: Cases and referents were recruited at five hospitals and included 200 boys with surgically corrected UDT and 629 boys with persistent middle ear effusion. Risk factor data were obtained by postal questionnaires to both parents. Clinical data were collected from medical files. Adjusted odds ratios (OR) with 95% confidence intervals (CI) were estimated using logistic regression. Results: The main findings include associations between UDT and familial occurrence of the disorder: OR 3.1 (95%CI 1.9e4.9), low birth weight: 2.2 (1.1e4.3), twinning: 2.2 (0.9e5.4), gestational preeclampsia: 1.9 (0.8e4.4), use of oral contraceptives after conception: 3.6 (1.0e12.5), in vitro fertilization/intracytoplasmic sperm injection treatment: 2.2 (0.8e6.0), paternal subfertility: 1.8 (0.8e4.1), and maternal occupational exposure to cosmetics: 3.0 (0.9e10.0). Subgroup analyses indicated differences in ORs for several factors between cases with (n Z 92) and without (n Z 103) inguinal hernia or hydrocele. Conclusion: The findings point towards a role for genetic predisposition, placental insufficiency, and possibly exposure to specific endocrine disrupting substances in the etiology of UDT. Further research should take into account potential etiologic differences between subgroups of cases with UDT. ª 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Introduction Undescended testis (UDT) is a common birth defect in boys, with a prevalence of around 1.2% according to a recent

Dutch investigation [1]. Because the inguinoscrotal phase of testicular descent normally takes place in the last trimester of pregnancy [2], UDT is especially common in boys who are born preterm [3]. In these cases, the testes often descend spontaneously before or shortly after the expected time of

* Corresponding author. Radboud University Nijmegen Medical Centre, Department of Epidemiology, Biostatistics, and HTA (133), PO Box 9101 6500 HB, Nijmegen, The Netherlands. Tel.: þ31 24 3618856; fax: þ31 24 3613505. E-mail address: [email protected] (N. Roeleveld). 1477-5131/$36 ª 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jpurol.2010.11.001

60 delivery. Spontaneous postnatal descent may also occur in at-term infants, resulting in substantial differences in the prevalence of UDT between age groups [3]. UDT has been hypothesized to partly share its etiology with other disorders of male reproductive health, including hypospadias, reduced semen quality and testicular cancer, collectively referred to as the testicular dysgenesis syndrome (TDS). TDS and its component disorders may result from prenatal exposure to endocrine disruptors (EDs), possibly combined with a genetic susceptibility [4]. A role of EDs in the etiology of UDT finds some support from reports of increased risks after occupational exposures to pesticides [5e7] and diesel exhaust fumes [8], and increased levels of several persistent endocrine disrupting chemicals in maternal breast milk and placenta tissue [9e12]. The association between UDT and low birth weight is well established, which can be interpreted as placental insufficiency in providing the fetus with both nutrients and gonadotropins [3,13e15]. In addition, several studies have demonstrated familial clustering of UDT [15e17]. Knowledge of other risk factors for UDT is restricted to indications for associations with specific obstetric conditions [8,18], pregnancy complications [8,18,19], intra-uterine insemination [20], increased maternal age [21], and parental life style factors [8,18,20,22e24], with sometimes contradictory study results. Our aim was to contribute to the understanding of the etiology of UDT, including the role of maternal and paternal exposure to EDs, by exploring a comprehensive set of risk factors in a case-referent study.

Methods Recruitment of cases and referents Cases and referents were recruited at one university medical centre and four general hospitals in the eastern part of The Netherlands, of which two hospitals provided either cases or referents, and three hospitals provided subjects for both study populations. Cases were recruited at departments of urology and pediatric surgery, and were boys who underwent an orchidopexy or orchidectomy, which is indicative of a persistent manifestation of UDT. Referents were boys who received ear ventilation tubes for a middle ear infection at the departments of otorhinolaryngology. This is a very common surgical procedure, so we expected these referents to be a good representation of the general population. By including children who had early childhood surgery as referents, we also aimed to minimize recall bias. The study population was restricted to boys who were born between 1996 and 2004 and residents from the catchment area of the participating hospitals. These criteria were met by 376 cases and 2911 referents, identified from the hospitals’ information systems. From these, we randomly sampled 1125 referents based on the geographical distribution of the cases. Personal data of all subjects were screened in order to identify twins and triplets (from whom one sibling was randomly selected), other siblings (oldest sibling was selected), and double records. Through these procedures, we selected 368 cases and 1091 referents.

M.M. Brouwers et al. In February 2005, we invited the parents of these children to participate by filling out a set of postal questionnaires. Two reminders were sent to increase response rates. The questionnaires included items on socioeconomic characteristics, familial occurrence of illnesses and birth defects, and the medical history of their son. Referring to the time just before and during the index pregnancy, parents were questioned about health status, pregnancy details, life style, various exposures at work or during leisure time activities, and diet. These items included questions about private and occupational use of personal care products, and exposure to pesticides, solvents or other chemicals at work and during home improvements or hobbies, as potential sources of exposure to EDs. Occupational exposure to other potential reproductive toxicants (X-rays, cytostatics, anesthetics) was assessed as well. Regarding dietary intake, we focused on soy replacements for meat or dairy (as rich sources of phytoestrogens) and seafood (as a rich source of persistent endocrine disrupting pollutants). Parents were first asked to list their current intake of foodstuffs, to help them recall their intake at the time of the index pregnancy. To avoid selective non-response and information bias, parents were informed that the study was designed to investigate the association between life style, occupation, illnesses, and complications at time of pregnancy with children’s health in general, not focusing on the specific disorder under study. Approximately 150 parents were contacted by phone to complete their questionnaire information. From the medical files of the cases, we collected information on testis position at surgery and previous physical examinations, and on the presence of an inguinal hernia or hydrocele. The study was approved by the Regional Committee on Research Involving Human Subjects. Referents with UDT or a major birth defect as defined in the EUROCAT registry protocol [25] were excluded from the study population. Cases with scrotal or retractile testes during surgery, with iatrogenic UDT due to an earlier hernia surgery, and cases with syndromal UDT or chromosome abnormalities were excluded as well. In addition, we performed subanalyses to see whether exclusion of cases with other major birth defects affected the main study results. Regarding the risk factor data, adjustments were made to the reported occupational exposures, particularly concerning cosmetics, paints, adhesives, and thinners, as the data indicated that occupational exposure was sometimes confused with private use of these products. For instance, only parents who were employed in the cosmetics industry (e.g. beauticians and hairdressers) or in health care (e.g. nurses and geriatric helpers) were considered likely to work with cosmetics.

Statistical analyses The data were analyzed using SPSS version 14.0. In the primary analyses, crude odds ratios (ORs) with 95% confidence intervals (95%CIs) were calculated for all potential risk factors. Next, multivariable logistic regression analyses were performed for factors with crude ORs  1.5 and a lower bound of the 95%CI  0.7 as well as for weaker ORs with more precision. Potential confounders

Risk factors for undescended testis Table 1

61

Results

Clinical breakdown of cases with UDT. Cases (n Z 200)a

Unilateral or bilateral UDT Location of UDT

Secondary UDT UDT accompanied by inguinal hernia or hydrocele

Unilateral Bilateral Inguinal or high scrotal Intra-abdominal Ectopic Unclassifiableb Absent testisc No Yes No Yes

162 33 166 14 9 1 5 161 34 103 92

a Totals do not add up to 200 because the medical files of 5 cases were not accessible. b Location was unclassifiable due to severe inguinal hernia. c Absent testis most often indicates atrophy due to testicular torsion.

included parental educational level, ethnic origin, and other identified or previously reported risk factors for UDT, except variables that shared their causal pathway with the risk factor of interest (e.g. low birth weight was not included in the analysis of preeclampsia). Potential confounders that changed the crude ORs by at least 10% were included in the regression models simultaneously. For variables with at least 30 cases and referents, homogeneity of strata-specific ORs was checked using contingency tables to exclude effect measure modification, but none was found. All effect estimates were adjusted for year of birth to account for potential trends in exposure levels or probabilities over time. To obtain indications for the influence of misclassification in case of long recall intervals, stratified subanalyses were performed to identify differences in effect estimates between short (1e5 years) and long (6e9 years) time periods between delivery and data collection.

Table 2

Descriptive statistics of cases and referents.

Mean age at time of data collection (SD) Educational level of parents (%) Mother

Father

Ethnic background of parents (%) Mother Father

Completed questionnaires were received from the parents of 230 cases and 741 referents, which corresponds to a response rate of 66% and 70%, respectively, taking into account 19 cases and 37 referents with unknown address changes. For 2 cases and 7 referents, essential questionnaire data were missing. An additional 105 referents were excluded because parents reported UDT (n Z 12) or major birth defects (n Z 93). These included 37 boys with orofacial clefts and 7 boys with Down syndrome, which are associated with persistent middle ear infections. We also excluded 3 cases with syndromes, 4 cases with iatrogenic non-scrotal testes, and 21 cases with scrotal or retractile testes during surgery, in which one testis had often atrophied due to testicular torsion. As a result, the primary analyses included 200 cases and 629 referents. UDT was most often unilateral and located within or just below the inguinal canal (Table 1). Ectopic and intraabdominal testes were found in 9 and 14 cases, respectively. Secondary ascending testes were assumed in 34 cases, for whom a urologist or other health care professional observed the testes within the scrotum at an earlier examination. In 92 cases, UDT was accompanied by inguinal hernia or hydrocele, for which subgroup analyses were performed. Mean age at time of data collection was around 5.5 years for both cases and referents (Table 2). Parents of cases and referents reported similar educational levels, but the parents of cases were more often of non-European ethnic origin, such as Turkish, Moroccan, Surinamese, Antillean, or Asian. Table 3 displays crude and adjusted associations between UDT and potential risk factors, which were generally little influenced by confounding. Occurrence of the disorder in 1st or 2nd degree relatives was associated with a three times increased risk of UDT: OR 3.1 (95%CI 1.9e5.0), but no associations were found with familial occurrence of hypospadias or testicular cancer. An increased occurrence of UDT in children with low birth weight was also reflected in our data: OR 2.2 (95%CI 1.1e4.3), and similar associations were found with being a twin or triplet: OR 2.2 (95%CI 0.9e5.5) and with preeclampsia: OR 1.9 (95%CI 0.8e4.5). We found no associations with gestational diabetes or other pregnancy

Referents (n Z 629)

Cases (n Z 200)

5.6 (2.1)

5.4 (2.0)

Low Intermediate High Low Intermediate High

153 306 164 164 227 190

(24.6) (49.1) (26.3) (28.2) (39.1) (32.7)

54 96 49 62 62 66

(27.1) (48.2) (24.6) (32.6) (32.6) (34.7)

European Caucasian Other European Caucasian Other

609 13 568 15

(97.9) (2.1) (97.4) (2.6)

184 15 174 17

(92.5) (7.5) (91.1) (8.9)

62 Table 3

M.M. Brouwers et al. Effect estimates of potential risk factors for UDT. Referents (n Z 629)

Familial history (1st or 2nd degree) UDT Hypospadias Testicular cancer Gestational and obstetric characteristics Low birth weight (<2500 g) Twin or triplet Preeclampsia Gestational diabetes Increased maternal age (>35 years) Mother is probably DES-daughter Factors associated with subfertility TTP > 6 months, excluding fertility treatments Subfertility of mother Subfertility of father Pregnancy achieved with IVF/ICSI Obesity (BMI > 30 kg/m2) Mother Father Prescriptive drug use Mother Any drug Antiemetic drugs Antibiotics Oral contraceptives: after conception Father Any drug Maternal intake of folic acid or other vitamin supplements Life style Mother Smoking during pregnancy Alcoholic beverages during pregnancy Father Smoking Private use of cosmetics Mother Body lotion Any product Father Body lotion Any product Occupational exposures (at least 8 h per week) Mother Cosmetics Domestic cleaning agents Industrial cleaning agents Paints, inks, adhesives, or thinners Exhaust fumes Pesticides (also less than 8 h per week) Dusts (any) Father Domestic cleaning agents Industrial cleaning agents Paints, inks, adhesives, or thinners Exhaust fumes Pesticides (also less than 8 h per week) Dusts (any) Diet Mother Vegetarian or vegan diet Soy meat replacements (at least once per week) Fish or seafood (at least three times per week)

Cases (n Z 200)

Crude OR (95%CI)

Adjusted ORa (95%CI) 3.1 (1.9e5.0) 0.8 (0.2e3.6) 2.1 (0.3e12.4)

45 8 3

39 2 2

3.1 (1.9e4.9) 0.7 (0.2e3.5) 2.0 (0.3e12.3)

43 20 15 10 99 16

23 12 9 4 28 2

2.4 1.9 1.9 1.3 0.9 0.4

98

30

0.9 (0.6e1.5)

0.9 (0.6e1.5)

61 17 10

15 10 7

0.8 (0.4e1.4) 1.8 (0.8e4.1) 2.2 (0.8e6.0)

0.8 (0.4e1.4) 1.8 (0.8e4.1) 2.2 (0.8e5.9)

30 25

11 4

1.1 (0.6e2.4) 0.5 (0.2e1.4)

1.1 (0.6e2.3) 0.5 (0.2e1.4)

340 23 64 7

104 14 11 6

0.9 2.0 0.5 2.7

0.9 2.0 0.5 3.5

42 459

23 145

1.7 (1.0e2.9) 1.0 (0.7e1.4)

1.8 (1.0e2.9) 1.0 (0.7e1.4)

109 94

39 27

1.1 (0.8e1.7) 0.9 (0.5e1.4)

1.1 (0.8e1.7) 0.9 (0.5e1.5)

204

62

0.9 (0.6e1.3)

0.9 (0.6e1.3)

302 603 38 503

89 192 19 168

0.9 0.9 1.6 1.3

(0.6e1.2) (0.3e2.2) (0.9e2.8) (0.7e2.2)

0.9 0.9 1.6 1.3

(0.6e1.2) (0.3e2.2) (0.9e2.8) (0.7e2.2)

9 39 7 6 6 12 7 7 12 27 41 30 52

6 19 1 2 4 4 7 6 6 8 15 9 19

2.1 1.5 0.4 1.0 2.1 1.0 3.2 2.6 1.6 0.9 1.1 0.9 1.2

(0.7e5.9) (0.9e2.7) (0.1e3.6) (0.2e5.2) (0.6e7.5) (0.3e3.2) (1.1e9.2) (0.9e8.0) (0.6e4.2) (0.4e2.0) (0.6e2.1) (0.4e2.0) (0.7e2.0)

3.0 1.5 0.4 1.0 2.1 1.0 5.1 1.0 1.6 0.9 1.1 0.9 1.2

(0.9e10.0)d (0.9e2.7) (0.1e3.6) (0.2e5.1) (0.6e7.4) (0.3e3.3) (1.6e16.4)d (0.2e5.0)f (0.6e4.2) (0.4e2.0) (0.6e2.1) (0.4e2.0) (0.7e2.0)

12 18 21

2 7 8

(1.3e4.7)b (0.9e4.0) (0.8e4.4) (0.4e4.0) (0.6e1.4) (0.1e1.8)

(0.7e1.3) (1.0e3.9) (0.3e1.0) (0.9e8.2)

0.5 (0.1e2.3) 1.2 (0.5e3.0) 1.2 (0.5e2.8)

2.2 2.2 1.9 1.3 0.9 0.4

(1.1e4.3)c (0.9e5.5)d (0.8e4.5) (0.4e4.0) (0.6e1.4) (0.1e1.8)

(0.7e1.3) (1.0e3.9) (0.3e1.0) (1.0e12.3)e

0.5 (0.1e2.4) 1.2 (0.5e3.0) 1.2 (0.5e2.7)

(continued on next page)

Risk factors for undescended testis

63

Table 3 (continued ) Referents (n Z 629) Father

Vegetarian or vegan diet Soy meat replacements (at least once per week) Fish or seafood (at least three times per week)

4 17 6

Cases (n Z 200) 2 3 4

Crude OR (95%CI)

Adjusted ORa (95%CI)

1.5 (0.3e8.5) 0.5 (0.2e1.9) 2.1 (0.6e7.7)

1.5 (0.3e8.6) 0.5 (0.2e1.8) 2.1 (0.6e7.7)

BMI Z body mass index, TTP Z time to pregnancy. NB Maternal exposure referred to 3 months prior to and/or during pregnancy, while paternal exposure referred to 3 months prior to pregnancy only. Proportions of missing values were less than 5% per variable, except for familial occurrence of UDT (16%) and hypospadias (9%), paternal prescriptive drug use (14%) and BMI (7%), maternal alcohol consumption (6%), and paternal intake of soy meat replacements (6%) and seafood (6%). a All associations were adjusted for year of birth. b Adjusted for gestational age. c Adjusted for year of birth, gestational age, and ethnic background of mother. d Adjusted for year of birth and familial occurrence of cryptorchidism. e Adjusted for year of birth, low birth weight, and gestational age. f Adjusted for year of birth, ethnic background of father, and familial occurrence of UDT.

complications, mother being a DES (diethylstilbestrol)daughter, and increased maternal age. UDT appeared to occur more often after in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment: OR 2.2 (95%CI 0.8e5.9), which seemed to correspond with the findings for paternal subfertility: OR 1.8 (95%CI 0.8e4.1); whereas subfertility of the mother was not associated with UDT. Mothers of cases more often reported the use of oral contraceptives: OR 3.5 (95%CI 1.0e12.3) and antiemetic drugs: OR 2.0 (95%CI 1.0e3.9) during pregnancy, while an inverse association was found with the use of antibiotics: OR 0.5 (95%CI 0.3e1.0). For fathers, use of any prescriptive drug in the 3 months prior to pregnancy appeared to be associated with UDT: OR 1.8 (95%CI 1.0e2.9), but the population size did not allow us to identify associations with specific drugs. Fathers of cases also slightly more often reported daily use of body lotion: OR 1.6 (95%CI 0.9e2.8). We identified no associations with any other personal care products (e.g. deodorant, make-up, perfume, or sunscreens), different combinations of these products, parental life style factors, obesity, or use of folic acid or other vitamin supplements. Regarding occupational exposures, a three times increased risk of UDT was found if mothers worked with cosmetics: OR 3.0 (95%CI 0.9e10.0). Mothers of cases also more often reported exposure to domestic cleaning agents: OR 1.5 (95%CI 0.9e2.7) and dusts: OR 5.1 (95%CI 1.6e16.4), although the latter finding was difficult to interpret due to the diversity in dusts that were listed (e.g. dust from hay, grain, hairdressing products, and textile manufacturing). Our data did not reflect an increased risk of UDT if parents worked with pesticides, nor for exposure to paints, adhesives, inks, thinners, industrial cleaning agents, metals, exhaust fumes, X-rays, anesthetics, or cytostatics, although the latter generally involved small numbers. The same was true for exposures during leisure time activities, including home improvements, gardening, and hobbies. No associations were detected either between UDT and a vegetarian or vegan diet, intake of soy meat replacements, and frequent consumption of fish or seafood by the parents in and around pregnancy. Exclusion of 24 cases with major birth defects or 34 cases with secondary ascending testes did not substantially affect any of the reported effect estimates, except that in the latter subanalyses the

adjusted OR for maternal occupational exposure to cosmetics was less strong: OR 2.1 (95%CI 0.5e8.5). For some risk factors, stronger ORs were calculated if the data referred to a more recent time period (1e5 vs 6e9 years ago), including being a twin or triplet: OR 4.9 (95%CI 1.2e20.6) vs OR 1.3 (95%CI 0.4e4.6), maternal use of antibiotics: OR 0.3 (95%CI 0.1e0.7) vs OR 1.1 (95%CI 0.4e2.7), and occupational exposure to cosmetics of the mother: OR 2.8 (95%CI 1.0e7.8) vs OR 0.3 (95%CI 0.1e1.5). An opposite pattern, however, was seen for several other factors, including familial occurrence of UDT: OR 2.2 (95%CI 1.1e4.6) vs OR 4.0 (95%CI 2.1e7.6), gestational preeclampsia: OR 0.4 (95%CI 0.1e3.1) vs OR 3.8 (95%CI 1.4e10.3), IVF/ICSI treatment: OR 1.6 (95%CI 0.5e5.5) vs OR 3.6 (95%CI 0.7e18.2), paternal subfertility: OR 1.3 (95% CI 0.4e4.5) vs OR 2.4 (95%CI 0.9e7.1), and maternal occupational exposure to household cleaning agents: OR 1.0 (95%CI 0.4e2.3) vs OR 2.4 (95%CI 1.1e5.3). Table 4 displays a breakdown for the co-occurrence of UDT with inguinal hernia or hydrocele based on multinomial logistic regression analyses, only showing effect estimates with substantial subgroup differences. In general, stronger effect estimates were calculated for the subgroup of UDT with inguinal hernia or hydrocele, whereas the estimates for UDT alone centered around one. An opposite pattern in subgroup-specific effect estimates was seen for maternal antiemetic drug use and occupational exposure to domestic cleaning agents, with stronger risk estimates in cases without inguinal hernia or hydrocele. The population size did not allow subgroup analyses that distinguished between unilateral and bilateral UDT or between specific locations of the UDT (inguinal, ectopic, or intra-abdominal). When the 33 bilateral cases were excluded, the effect estimates for maternal antiemetic drug use and occupational exposure to dusts were reduced to OR 1.5 (95%CI 0.7e3.3) and OR 3.5 (95%CI 0.9e3.4), respectively, whereas for being a twin or triplet: OR 2.8 (95%CI 1.1e6.7), gestational preeclampsia: OR 2.3 (95%CI 1.0e5.4), and IVF/ICSI treatment: OR 2.7 (95%CI 1.0e7.2), slightly stronger ORs were found. Exclusion of 23 cases with ectopic or intra-abdominal testes resulted in a somewhat weaker effect estimate for maternal dust exposure as well: OR 4.2 (95%CI 1.2e14.9).

64

M.M. Brouwers et al.

Table 4 Effect estimates of potential risk factors for the total case population and subgroups of cases with and without inguinal hernia or hydrocele.

Twin or triplet Gestational preeclampsia Pregnancy achieved with IVF/ICSI Subfertility of father Oral contraceptives after conception Mother used antiemetic drugs Father used any prescriptive drug Father used cosmetics (any product) Mother worked with domestic cleaning agents (at least 8 h per week)

All cases (n Z 200) Adj OR (95%CI)a

With hernia or hydrocele (n Z 92) Adj OR (95%CI)a

Without hernia or hydrocele (n Z 103) Adj OR (95%CI)a

2.3 (0.9e5.5)b 1.9 (0.8e4.5) 2.2 (0.8e5.9)

4.3 (1.6e11.2)b 2.4 (0.8e6.7) 2.7 (0.8e9.0)

0.9 (0.2e3.9)b 1.2 (0.4e4.3) 1.2 (0.3e5.6)

1.9 (0.4e1.4) 3.5 (1.0e12.3)c

2.4 (0.9e6.4) 5.9 (1.5e22.7)c

1.1 (0.3e3.7) 1.4 (0.2e12.1)c

2.0 (1.0e3.9) 1.8 (1.1e3.0)

0.9 (0.3e3.0) 2.1 (1.1e4.1)

3.1 (1.5e6.6) 1.4 (0.7e2.9)

1.3 (0.7e2.2)

1.0 (0.5e2.1)

1.5 (0.7e3.2)

1.5 (0.9e2.7)

1.0 (0.4e2.5)

1.9 (1.0e3.9)

NB The table is restricted to factors for which the ORs in the subgroups differed by at least 10% from the OR for the total case population. a All associations are adjusted for year of birth. b Adjusted for year of birth and familial occurrence of UDT. c Adjusted for year of birth, low birth weight, and gestational age.

Discussion This study demonstrated familial clustering of UDT and associations with low birth weight, being a twin or triplet, and gestational preeclampsia. Additionally, increased risks of UDT were found after IVF/ICSI treatment, if fathers reported subfertility, and if mothers used oral contraceptives during pregnancy. Associations with other prescriptive drugs used by the parents were found as well. Mothers of cases were more often occupationally exposed to domestic cleaning agents, dusts, and cosmetics, whereas slightly more case-fathers reported daily use of body lotion. We did not detect associations with other occupational or leisure time exposures, dietary intake of soy or seafood, pregnancy complications, life style factors, or familial occurrence of hypospadias and testicular cancer. Subanalyses revealed differences in the effect estimates of specific risk factors between cases with and without inguinal hernia or hydrocele, which, to our knowledge, have not been described previously. Before interpreting these results, we will discuss some methodological aspects of our study. Our case population was restricted to boys who underwent an orchidopexy or orchidectomy to represent persistent manifestations of UDT, as opposed to cases that are ascertained by means of a physical examination, after which the testis may spontaneously descend in up to 70% [3]. Additionally, physical examinations are prone to misdiagnosis, which is illustrated by the 21 cases with scrotal or retractile testes diagnosed during surgery that we excluded from the analyses. However, surgical cases also include cases with secondary ascending testes of which the pathogenesis is not clear [26,27], but subanalyses excluding these cases did not substantially change our findings. By using boys who received ear ventilation tubes as the referent population, we aimed to obtain a comparable group

of surgical patients of similar age and from the same geographical area. To our knowledge, persistent middle ear effusion is not associated with the risk factors that were studied in the present study [28]. This may not be true for the birth defects that were reported for some referents, who were therefore excluded from the analyses. The prevalence of birth defects among referents (14%) was much higher than expected (2e3%) [29], partly due to the increased occurrence of middle ear infections among children with orofacial clefts and Down syndrome. Subanalyses in which we also excluded cases with co-occurring birth defects did not lead to different findings. The parents of referents and cases were not informed about the aims of this study to prevent selective nonresponse and information bias, resulting in response rates of 70% for referents and 66% for cases. Therefore, an association between participation and exposure status seems unlikely. We cannot exclude random misclassification, however, as risk factor data were collected retrospectively with postal questionnaires. We assessed the reproducibility of a subset of items by means of telephone interviews with 72 mothers and 55 fathers a year later. Moderate to high agreement was found for questions on low birth weight (kappa 0.8), prolonged time to pregnancy (kappa 0.6), various pregnancy complications (kappas between 0.5 and 0.8), and maternal and paternal smoking (kappa 0.9 and 0.8, respectively). Poorer agreement was calculated for some questions on home improvements and occupational exposure at the time of the index pregnancy (kappas 0.0e0.7). The latter may explain why only a few associations with UDT were found. In the stratified subanalyses, we found some indications that specific effect estimates were affected by misclassification due to long recall intervals. Parents of cases more often reported the occurrence of UDT in 1st or 2nd degree relatives, which corresponds with previous reports [15e17]. Familial clustering may indicate

Risk factors for undescended testis a genetic component in the etiology of UDT, although family members may share environmental risk factors as well. Contrary to our expectation based on the TDS hypothesis, we did not identify familial clustering of hypospadias or testicular cancer in cases with UDT [4]. The association between UDT and paternal subfertility is in line with the TDS hypothesis. Placental insufficiency is considered to be a plausible underlying mechanism for the increased occurrence of UDT in children with low birth weight and may also explain the associations with preeclampsia and being a twin or triplet. For these factors, similar risk estimates were found for hypospadias [30], pointing towards a shared etiologic pathway for the two disorders, which has been suggested in previous literature as well [13e15]. The data also reflected similar risks of UDT and hypospadias in case of paternal subfertility and after IVF/ICSI treatment [30], which to our knowledge has not been described before. Interestingly, in vitro chorion formation during IVF/ICSI treatments has also been linked to placental insufficiency [31]. But other mechanisms through which IVF and ICSI may interfere with fetal development have been described as well, including disturbance of the fetal endocrine system by the administered ovulation stimulants [32] or progestins [33], and epigenetic defects induced by the in vitro procedures [34]. An approximately three times increased risk of UDT was found for mothers using oral contraceptives during pregnancy, although confidence intervals were broad due to low cell counts. Possibly, estrogens or progestins in oral contraceptives interfere with testicular descent, but the association may also reflect an increased risk of UDT for unplanned pregnancies in general. The findings for antiemetic drug use by the mother seem to correspond with the earlier reported increased risk of UDT in the case of severe gestational vomiting [8]. The biologically implausible associations with paternal prescriptive drug use and maternal use of antibiotics, however, cast doubts on the validity of the present data on prescriptive drugs. We speculate that mothers of boys with persistent middle ear effusion may have used antibiotics more often because of an increased familial predisposition to infections. Mothers of cases more often worked with cosmetics (e.g. hair sprays, hair dyes, nail polish, lotions, and ointments) during pregnancy, whereas fathers slightly more often reported daily use of body lotion. Most cosmetics, including body lotion, contain a mixture of phthalates, siloxanes, parabens, and UV screens, which have all been reported to have endocrine disrupting properties [35e38]. Domestic cleaning agents, which were also more often reported as used by mothers of cases, may also contain some level of endocrine disrupting chemicals, although we would expect more relevant exposures from the various industrial agents for which no associations with UDT were found. The strong association with exposure to dusts is also difficult to interpret. However, as indicated by the reproducibility study, the data on occupational exposures in general warrant extra caution about misclassification. In almost 50% of the cases, UDT was accompanied by inguinal hernia or hydrocele due to incomplete closure of the processus vaginalis. The processus vaginalis is thought to play an active role in testicular inguinoscrotal descent, but the etiological significance of an inguinal hernia or hydrocele in UDT is unclear [2,39,40]. The subgroup analyses revealed

65 that several risk factors (e.g. being a twin or triplet, gestational preeclampsia, IVF/ICSI, oral contraceptives during the pregnancy), appear to be associated with the combination of UDTand inguinal hernia or hydrocele, but not with UDTalone. This could indicate that these factors are predominantly associated with the risk of inguinal hernia or hydrocele, and do not interfere with testicular descent. Another explanation would be that the subgroup with UDT and co-occurring hernia or hydrocele represents a specific manifestation of the disorder, related to the timing or duration of risk factor interference with testicular descent. Distinguishing between bilateral and unilateral cases or between different locations of the UDT could be informative as well, especially because inguinal testes, intra-abdominal testes, and ectopic testes are thought to involve different causal mechanisms [2]. The population size did not allow assessment of the effect of risk factors for these subgroups separately, however. In conclusion, we found indications for several risk factors for UDT, which point towards a role of genetic predisposition, placental insufficiency, and possibly exposure to specific endocrine disrupting substances in its etiology. The differences in effect estimates between cases with and without inguinal hernia or hydrocele are difficult to explain, but seem interesting. We propose that further research should differentiate between subgroups of cases and include more extensive assessment of exposure to EDs, in order to obtain more insight into the etiology of UDT.

Conflict of interest statement There were no financial or other relations that may have led to a conflict of interest.

Acknowledgements We thank K. Graamans, P. Rieu, R. Corten, J. van Leeuwen, J. Hinderink, A. Meier, E. Theunissen, and all others who facilitated the recruitment of the study population at the Radboud University Nijmegen Medical Centre, the CanisiusWilhelmina Hospital in Nijmegen, The Alysis Health Care Group in Arnhem, The Regional Hospital Queen Beatrix in Winterswijk, and the VieCuri Medical Centre Northern Limburg. In addition, we thank M van de Leur and M. Loeffen for data entry and J. Groenewoud and E. Brummelkamp for data management assistance. We are very grateful to the parents for their willingness to participate. The study was funded by the Netherlands Organization for Scientific Research. This organization was not involved in the collection, analysis and interpretation of data, in the writing of the manuscript, and in the decision to submit the manuscript for publication.

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