Hum. Reprod. Advance Access published online on May 24, 2008
Human Reproduction, doi:10.1093/humrep/den186
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Cryptorchidism at birth in Nice area (France) is associated with higher prenatal exposure to PCBs and DDE, as assessed by colostrum concentrations
1 Endocrinology Department, Hôpital l'Archet 1, CHU Nice, 151 route de Saint-Antoine, 06200 Nice, France 2 INSERM Unit 895, 06200 Nice, France 3 Pediatrics Department, CHU Nice, 06200 Nice, France 4 Conseil Général des Alpes Maritimes, 06200 Nice, France 5 INSERM Unit 822, 94276 le Kremlin Bicêtre, France 6 Biochemistry Laboratory, Hôpital Saint-Roch, CHU Nice, 06000 Nice, France 7 Obstetrics Department, Hôpital l'Archet 2, CHU Nice, 06200 Nice, France
8 Correspondence address. Tel: +33-4-92-03-55-19; Fax: +33-4-92-03-54-25; E-mail: brucker-davis.f{at}chu-nice.fr
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Abstract |
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BACKGROUND: Since fetal exposure to anti-androgenic and/or estrogenic compounds has adverse effect on animal reproduction, such exposure could be harmful to human fetus. Data are scarce on cryptorchidism and human exposure to endocrine disruptors.
METHODS: We performed a prospective case–control study to assess the incidence of cryptorchidism and fetal exposure to selected chemicals in the Nice area. One hundred and fifty-one cord bloods (67 cryptorchid, 84 tightly matched controls) and 125 colostrums (56 for cryptorchid and 69 for controls) were screened for xenobiotics, including anti-androgenic dichloro-diphenyl-trichloro-ethylene (DDE), polychlorinated biphenyls (PCBs), and dibutylphthalate (and metabolite monobutylphthalate, mBP).
RESULTS: Median concentrations in colostrum were higher, although not statistically significantly, in cryptorchid versus controls. Cryptorchid boys were more likely to be classified in the most contaminated groups in colostrum for DDE, 
PCBs and the composite score PCB + DDE. The same trend, but again not statistically significantly was observed for mBP. Odds ratio for cryptorchidism was increased for the highest score of PCB, with a trend only for DDE and PCB + DDE versus the lowest score of those components.
CONCLUSIONS: Our results support an association between congenital cryptorchidism and fetal exposure to PCBs and possibly DDE. Higher concentrations in milk could be a marker of higher exposure or for an impaired detoxification pattern in genetically predisposed individuals.
Key words: endocrine disruptors/fetal exposure/cryptorchidism/DDE/PCB
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Introduction |
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Since the 1990s, questions have been raised about a decline in male reproductive health (Skakkebaek, 1998
), including the increasing incidence of cryptorchidism. Controversy remains (Safe, 2004), because of the absence of accurate registries in many parts of the world (Toppari et al., 2001), including France, and the lack of standardization for the diagnosis of cryptorchidism, preventing meaningful geographical and temporal comparisons. Lifestyle changes and exposure to compounds with endocrine disrupting properties have been suggested as the cause of the proposed increase (Bay et al., 2006). Exposure to endocrine disruptors (ED) is unavoidable from conception throughout life, the prenatal period and infancy being the most vulnerable (Landrigan et al., 2003). Some consequences of prenatal exposure to EDs are immediately visible at birth (congenital malformations), whereas others may occur later in adulthood (like infertility and hormone-dependent cancers), or even afflict the next generation via epigenetic mechanisms (Skinner and Anway, 2005). On the basis of apparent epidemiological links and similar incidence trends, cryptorchidism has been described with hypospadias, poor sperm quality and testis cancer as part of the testicular dysgenesis syndrome (TDS) (Skakkebaek et al., 2001). It has been suggested that fetal exposure to compounds with anti-androgenic and/or estrogenic activity may contribute to TDS, at least in susceptible individuals (Bay et al., 2006; Toppari et al., 2006). However, though the potential pathways of disruption are well established (Sharpe, 2006), the evidence of fetal adverse effect of ED is still scarce in humans. So far, most of the solid evidence stems from wildlife observations and experimental studies (Facemire et al., 1995; Vos et al., 2000; Gray et al., 2001; Kavlock et al., 2002; Edwards et al., 2006), or from indirect circumstantial human observations (Garcia-Rodriguez et al., 1996; Hosie et al., 2000), with, however, few recent conclusive human studies (Swan et al., 2005; Damgaard et al., 2006; Main et al., 2007). The most studied chemicals may be the phthalates, with some consistent and coherent effects from benchmark to human studies (Kavlock et al., 2002; Fisher et al., 2003; Swan et al., 2005; Main et al., 2006). We designed a prospective study to determine the incidence of cryptorchidism in the Nice area (France), and to assess in utero and lactational exposure to xenobiotics in a population never tested before, in order to determine if there is a potential relationship between prenatal exposure to EDs and cryptorchidism. We measured several persistent EDs in cord blood (CB) and colostrum of lactating women who delivered baby boys at maternity clinics in Nice and in Grasse. Nice, the capital of the Côte d'Azur, is a cosmopolitan town of 350 000 inhabitants; Grasse is a 45 000 inhabitants inland city, 40 km away from Nice, with a more homogenous ethnic population. We established scores of exposure to different chemicals and looked at relationships between exposure and cryptorchidism.
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Study design
We designed a 3 year prospective study screening newborns for cryptorchidism. Between April 2002 and April 2005, 6246 boys were born alive, at or after 34 weeks of gestational age (GA) at the maternity wards of Nice University Hospital, a level III maternity, and of Grasse General Hospital. Neonatal examination was standardized between the two wards, and diagnosis of cryptorchidism was accepted after at least two concording examinations by a senior pediatrician before discharge of the hospital (n = 102). Testicular position was defined according to Scorer criteria completed by Hack et al. (Scorer, 1964
; Hack et al., 2003) after firm but not forced traction of the testis to the most distal position along the pathway of normal descent. The following were regarded as undescended testis (UDT): non-palpable, inguinal, supra-scrotal, high scrotal and ectopic testis. Retractable testis was not included in the case or control group. For each cryptorchid boy, two strictly normal controls were matched for place and date of birth, birthweight, GA, and when possible parental origin. Seven boys with UDT were not included because their parents refused to participate. A total of 283 boys, 95 cryptorchid (83 uni- and 12 bilateral) and 188 controls (one case impossible to match), were included in this study. Babies were followed at 3 and 12 months of age to confirm the diagnosis of permanent cryptorchidism (n = 50) (Wagner-Mahler and Kurzenne, 2007).
Several xenobiotics selected for their known effects on testicular migration, possibly via anti-androgenic and/or estrogenic effects were measured in CB and maternal milk. Concentrations of xenobiotics in milk reflect maternal chemical burden, and are widely accepted as a proxy for prenatal exposure to lipophilic compounds (Needham and Wang, 2002). CB was systematically collected in two 10 ml glass tubes following placenta expulsion, centrifuged, aliquoted and stored at –70°C until analysis. Breastfeeding mothers provided a 10 ml colostrum sample (hindmilk) in a glass container between Day 3 and Day 5 post-partum which was stored at –20°C until analysis. Because of cost, only specimens from one of the two controls were used for xenobiotic analysis, the closest matching control being selected.
Study population
A total of 164 infant/mother pairs (78 out of 95 eligible cryptorchid and 86 out of 94 eligible controls) with xenobiotic measurements are included in this study, 81 pairs from Nice and 83 from Grasse. Except for the maternal place of birth, there were no significant differences between the mothers from Nice and Grasse. Mothers from Nice were more often born in North Africa and Africa, compared with the mothers from Grasse. Overall, 131 women were born in France (n = 117) or Europe (n = 14), 19 in North Africa, 7 in sub-Saharan Africa and 7 elsewhere.
From detailed parental questionnaires, we extracted for this study information about the mother [age, place of birth, origin of her parents, body mass index (BMI) before pregnancy and at delivery, job history, and exposure and obstetrical history] and paternal history of cryptorchidism. Informed consent was obtained from parents of each boy. The research project was sponsored by the Clinical Research Board of the Nice University Hospital, supported by a grant of the French Research Ministry and approved by the ethical committee of our institutions.
Xenobiotics sample collection and measurements
Analysis was performed by gas chromatography coupled with mass spectrometry at the Laboratoire de l'Environnement de l'Agglomération Niçoise, a laboratory accredited by the French Ministry of Health and the French Ministry of the Environment that undergoes periodic audit by the COFRAC, an independent French Committee of Accreditation.
Instead of 189 theoretical samples (95 cryptorchid and 94 matched controls), we had 168 sera and 135 maternal milk samples available for analysis. Missing serum samples correspond to miss in collection or insufficient volume often following a Caesarean section. Colostrum samples were not available for study when mothers decided not to nurse their babies, or did not have enough milk. Because of an upgrading of the mass spectrometer in 2003, lowering the level of detection by a factor 10, we removed from our analysis the measurements performed in October 2002 with the previous mass spectrometer (CB n = 17, milk n = 10). As a result, this analysis is based on 151 sera samples (84 controls and 67 cryporchid; 79 from Nice, 72 from Grasse) and 125 colostrum samples (69 controls and 56 cryptorchid; 62 from Nice, 63 from Grasse) from 164 infant/mother pairs, performed in four separate campaigns in April 2003 (CB n = 32, milk n = 30), September 2003 (CB n = 34, milk n = 24), April 2004 (CB n = 36, milk n = 26) and May 2005 (CB n = 49, milk n = 45).
Specimens were screened for xenobiotics including seven non-planar polychlorinated biphenyls (PCBs) congeners (PCB 28, PCB 52, PCB 101, PCB 118, PCB138, PCB 153 and PCB 180), one plasticizer (dibutylphthalate, DBP) and later its metabolite monobutylphthalate (mBP), and pesticides including dichloro-diphenyl-trichloro-ethylene (DDE). We calculated the arithmetic sum of the seven measured PCBs, PCBs.
For DBP measurements, all precautions were taken to avoid contamination at the time of the collection and storage, with the use of glass containers and material. However, contamination of specimens was unavoidable in the mass spectrometer. We assessed that contamination in various matrices and estimated it at 20 ng/ml in serum and 60 ng/g in milk (data not shown). We present here the data without correction. Later, we attempted to bypass this problem, inherent to the ubiquity of phthalates in a lab setting, by measuring mBP, the biological metabolite of DBP that is thought to be the direct toxicant (85 sera and 71 milks) (Kavlock et al., 2002).
In brief, for serum analysis, the extraction of xenobiotics was obtained using a cartridge Chromabond C18 ec/1 ml/100 mg (Macherey Nagel); 2.5 ml of serum was diluted with 2.5 ml of a demineralized water/propanol (85/15) solution, vigorously mixed and centrifugated. The column was conditioned with 2 ml of MEOH followed by 2 ml of the water/propanol solution. After centrifugation of the sample, the clear supernatant was collected and passed through the column. The column was flushed twice with 500 µl of the water/propanol solution. Elution was performed four times with 250 µl of n-hexane. The resulting extract was then analyzed by gas chromatography coupled to mass spectrometry (Agilent 6890 and 5973, Massy, France). Detection and quantitation were performed by mass spectrometry checking for specific ions corresponding to the given chemicals. Concentrations in sera were not corrected for fat content, as CB is known to be a low fat medium.
For milk specimens, fat was first extracted according to standard procedures. The median percentage of fat was 2% (range 0.5–4.8%). Then, the extracts were processed as described above for serum. Results are expressed in nanogram per gram of milk for phthalates, and for PCBs and DDE in nanogram per gram of fat to allow comparison with other studies.
Quantitation and detection thresholds were, respectively, 0.1 and 0.03 ng/ml in serum and 0.1 and 0.03 ng/g of milk or ng/g of fat. Detectable but not quantifiable chemicals were arbitrarily assigned a 0.09 ng/ml or ng/g of fat value.
The percentage of recovery varied from 80% to 96% in serum and from 75% to 96% in milk. Inter-assay coefficients of variation varied according to the chemical and the matrix from 3.4% for DDE to 13.5% for PCB 153 in serum and from 5.1% for PCB 180 to 12.4% for PCB 52 in milk. Intra-assay coefficients of variation varied from 3.9% for DDE to 10.9% for PCB 28 in serum and from 4.8% for PCB 153 to 13.3% for PCB 180 in milk.
All analyses were carried out blinded for the boy's cryptorchid status, each case and its control being analyzed during the same campaign.
Scores of exposure
To assess the degree of exposure, we used both individual chemical concentrations in CB and milk, and scores of exposure in milk, individual for each chemical, and composite, compounding the three chemical categories.
Categories of exposure were created for individual chemicals in milk: 0 was defined as unquantifiable values, whether detectable or not (negligible exposure); 1 as quantifiable values below the median of quantifiable values (low exposure) and 2 to values above the median (high exposure).
Then, the composite score of exposure in milk was calculated by adding the three individual scores of DDE, mBP and PCBs. We focused on the score in milk because concentrations of those chemicals were generally higher with a wider distribution in milk compared with CB. For a maximum score of 6, we considered the exposure low for a global score 
3), average for a score of 4–5 and heavy for a score of 6. We also established a score using only DDE and PCBs, for a maximum score of 4 to circumvent the problems of phthalates measurements (score of 4: heavy exposure, score 2: low exposure).
Statistics
Data were entered and stored on an Access file, and then transferred into STATA software (STATA/SE 9.0, STATA Corporation, College Station, TX, USA) for statistical analysis. Because of some missing samples, we could not perform statistical analysis on paired samples. Non-parametric tests of Kruskal and Wallis were used to compare continuous variables. 
2 test was used for category comparison. We also used uni- and multivariate logistic regression with adjustments for known risk factors for cryptorchidism (GA, birthweight, maternal BMI before pregnancy, maternal age, parity, season of birth and paternal history of cryptorchidism) and for the town of birth. Level of significance was set at P < 0.05.
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Study population
Patients characteristics are shown in Table I. Mothers of cryptorchid boys were not different from mothers of controls. Most pregnancies were uncomplicated. The table confirms the strict matching of the babies. There was only a slight, non-significant trend for an increase of cryptorchid babies born before 37 weeks of GA (10.3% versus 5.8%), and to have more deliveries by Caesarean section in controls (21.4% versus 11.7% NS).
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Xenobiotics concentrations in CB and maternal milk
Frequencies of quantifiable chemicals in CB and colostrum are shown in Table II. All sera and milk samples were positive for at least one of the three classes of chemicals. In general, chemicals were more often present in milk than in CB. In decreasing order, in CB, DBP was always quantifiable, mBP and PCB 153 were present in more than 80% and DDE in 68% of the samples. In milk, phthalates were always present, PCB 153 and PCB 138 in more than 90%, DDE in 88%, PCB 180 in 83% and PCB 118 in 62%.
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In milk, as shown in Table III, all the median values of all chemicals (except for PCB 138) showed a tendency to be higher in cryptorchid than in control boys, though without reaching significance. This was particularly clear for
PCB (206.3 versus 166.8 ng/g of fat, P = 0.1) and DDE (119.4 versus 80 ng/g of fat, P = 0.11), but also for mBP, despite smaller numbers of samples (17.3 versus 10.6 ng/g of fat, P = 0.17). This was not true in CB, where results expressed in nanogram per milliliter were usually similar in cryptorchid and control boys (Table II).
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Concentrations in milk from mothers of non-metropolitan French origin were different only for mothers from sub-Saharan African origin (n = 7), which usually contained higher concentrations of
PCBs (416 ± 199 ng/g of fat versus 269 ± 315 in French/European) and DDE (1035 ± 738 ng/g of fat versus 127 ± 172). There was no statistically significant effect of parity after correction for maternal age (data not shown).
Table IV shows the scores of exposure in milk for individual chemicals and using a composite score. According to our criteria, 16.9% had heavy exposure using the composite score in milk (DDE + PCBs + mBP). When using the ‘score without phthalates’, 25.6% had heavy exposure (DDE + PCBs).
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Relationship between exposure and cryptorchid status
Boys with UDT tended to have higher individual scores of exposure for
PCBs (57.1% of class 2 versus 39.1% in controls, P = 0.045), DDE (53.6% of class 2 versus 36.2%, P = 0.05) and to a lesser degree mBP (58.1% versus 40%, P = 0.13) in milk, as shown in Table IV, but not in CB (results not shown). The composite score restricted to DDE and PCB in milk showed the same trend (30.4% versus 21.7%, P = 0.05).
These results were supported by the analysis using logistic regression after adjustment for GA (<37 and 
37 weeks), birthweight, maternal BMI before pregnancy, maternal age, parity, season of birth, paternal history of cryptorchidism and city of delivery (Table V). The most robust data were observed with PCB with an odds ratio (OR) for UDT at birth of 2.74 (95% confidence intervals, 95% CI: 1.15–6.53, P < 0.022) for boys in the highest class of exposure in milk. There was a trend for the highest class of exposure to DDE in milk: OR 2.16 (95% CI: 0.94–4.98, P = 0.071), and also for the composite score at 4 versus 1 + 2, OR at 3.03 (95% CI: 0.99–9.21, close to significance P = 0.051). The trend was weaker for mBP in milk (OR of 2.13, 95% CI: 0.66–6.83, P = 0.204). Regression analysis in full term babies only (GA 37 weeks) showed statistically significant results for PCB (OR 2.72, 95% CI 1.12–6.56, P = 0.026) with the same trend for DDE and the composite score PCB + DDE. We performed a similar analysis, while studying babies who stayed cryptorchid at 3 months (n = 38 of 78 cryptorchid at birth) comparing them with the control group. Results were not significant in that group, although there was still a trend for the score of PCB (OR at 2.97; 95% CI: 0.88–9.99, P = 0.078) (Table V).
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Discussion |
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Our population
We report results on 164 mother/baby pairs recruited over a 3 year period with measurements of xenobiotics available in CB, colostrum or both. This subgroup came out of a theoretical cohort of 189 pairs (86% exhaustivity), with the remaining 26 being excluded because no sample was available for processing, or because measurements were performed in a first campaign with a less sensitive technique. This subgroup of 164 pairs did not differ from the study population (n = 283) for maternal age, BMI, parity, prior breastfeeding or the boys' place of birth. We decided on a tight matching of cryptorchid boys including for factors known from the literature to be associated with cryptorchidism (GA and birthweight), and date of birth (to take into account potential seasonal factors) or parental ethnic origin, since it is still unclear whether incidence of cryptorchidism may differ with race (McGlynn et al., 2006
). We are aware that matching on ethnicity involves both genetic and environmental factors, since non-Caucasian are more likely to be born outside metropolitan France, or to have parents born overseas. Thus, our tightly matched controls could be more exposed than the general population, leading to OR less high with our controls than with the general population. There was also no difference in the rate of primiparous mothers and in prior breastfeeding, which is a well-known way for a woman to get rid of her contaminants load (Needham and Wang, 2002; LaKind et al., 2004).
Xenobiotic concentrations in CB and maternal milk
Assessment of exposure has been reported around the world, both in developed and in developing countries, but data of exposure in France are very scarce (Luquet et al., 1975; Klein et al., 1986; Bordet et al., 1993). Our study, the first one in our area, shows that all sera and milk samples are contaminated by one or more of the tested chemicals, selected because of their known endocrine disrupting properties.
By design, our study included cryptorchid and healthy matched controls; thus it is possible that our population is not fully representative of the general population in our area. Indeed, it is possible that cryptorchid boys represent a more exposed subpopulation of newborns that skewed our results toward a more exposed population. Since control boys were closely matched to cryptorchid boys, including for parental origin, then, if parents from a given geographic or ethnic origin are more exposed, then controls too could be more exposed, skewing further our results.
Concentrations in colostrum samples were higher than in sera from umbilical cord, which was expected for lipophilic compounds. This was also true, however, for phthalates, supposedly non-lipophilic. In our study, milk was collected within the first 5 days of lactation. Percentage of fat is estimated 
3% in colostrum (Needham and Wang, 2002) which is a little higher than what we observed in our patients (median 2%), though our samples were hindmilk, where lipid content is supposedly higher (LaKind et al., 2004) than in foremilk. Klein et al. (1986) have shown in a short-term longitudinal study of the first 10 days of lactation that DDE concentrations are the highest on Days 1 and 2, dropping rapidly afterwards. However, it is difficult to extrapolate about the long-term depuration process (elimination into milk of maternal chemical load). Indeed, this process is complex, depending on maternal body burden, milk composition, time of collection, etc. For this reason, comparison between studies rests in part on normalization of concentrations for fat content (Lakind et al., 2004).
DBP and mBP contamination is ubiquitous. It is estimated that most of the contamination comes from food intake (Kavlock et al., 2002). Although DBP is not persistent, concentrations remain high because of continuous exposure. Indeed, phthalates, including DBP, are present in many objects of our daily environment. Because of potential contamination during lab analysis, concentrations of DBP are not fully reliable, despite the stringent ‘phthalates free’ conditions of collection, storage and analysis of our samples. For that reason, we used later in this study, its metabolite, mBP, as a better representative of this chemical contamination. However, it appears now that enzyme activity (that degrades DBP into mBP) was still possible ex vivo, since our samples were not collected on phosphoric acid (which neutralizes metabolizing enzymes ex vivo), as it has since been recommended (Calafat et al., 2004). Although this is a potential bias, it would have affected similarly samples from cryptorchid boys and controls, thus allowing in our opinion valid comparisons. Colostrum concentrations of mBP in our population were similar to those observed in Finland (12 µg/l, n = 65), but higher than those in Denmark (4.3 µg/l, n = 65), both studies based on mature milk samples (Main et al., 2006), and sharing the same methodological bias. The data reported by Calafat et al. (2004) were also in the same range, using the reference measurement method.
The presence of DDE in almost 9 out of 10 women, though usually at low levels, shows that women have been contaminated by DDT, despite the ban in France in 1972, which would have occurred in most cases before their birth. Some women born in North or sub-Saharan Africa are likely to have been directly and heavily contaminated in their birth country. Indeed, the range of concentrations was wide, reflecting diversity in our population. For DDE in CB, sera concentrations were usually lower than in most published studies (Brucker-Davis et al., in preparation), particularly from countries where DDT is still used or produced (Waliszewski et al., 2001). In milk, our DDE concentrations were also lower (Waliszewski et al., 2001; Jaraczewska et al., 2006), though curiously concentrations in another part of France in the 80s were lower (Klein et al., 1986), probably reflecting a more homogenous ethnic population. Our lower DDE concentrations may be due in part to the temporal trend for decreased concentrations since DDT regulation. Of note, the milk concentrations of DDE in our study were markedly similar to those found in the prospective cryptorchid study from Denmark and Finland which shares a similar design (Damgaard et al., 2006).
Exposure to PCBs is also almost ubiquitous in our population, despite the ban on production in France in 1987, proof of their persistence in the environment and human tissues. The most frequently found congeners both in CB and in milk were the higher-chlorinated congeners PCB 153, 138, 180 and 118. PCB 153 concentrations were the highest and a good proxy for other PCB congeners. Concentrations were in the same range as other studies (Brucker-Davis et al., in preparation; Guvenius et al., 2003), with, however, variation from one area to another (Dekoning and Karmaus, 2000).
Relationship between exposure and cryptorchid status
Quantifiable CB concentrations in both cryptorchid and non-cryptorchid boys attest universal transplacental exposure during pregnancy to selected EDs known to cause cryptorchidism in experimental animals. Persistence of, or continuous exposure to, those chemicals means that most fetuses have been exposed throughout pregnancy, including during the period of testis migration. Several reports have provided indirect evidence suggesting harmful effect of exposure to EDs on human testis migration (Garcia-Rodriguez et al., 1996; Hosie et al., 2000). Contamination of milk, a good proxy for in utero exposure, is even higher. However, most authors agree that the benefits of breastfeeding outweigh the risks of contamination (LaKind et al., 2004; Pronczuk et al., 2004), because (i) maternal milk is the optimal food containing also protecting antibodies; (ii) breastfeeding creates a unique bond fostering neuropsychological development and (iii) the alternative, infant formula, may also be contaminated.
The literature is scarce on direct measurement of chemical exposure in cryptorchid boys. To our knowledge, only one prospective case–control study with a design similar to ours, the Danish–Finnish study, has been published reporting on different classes of ED (Damgaard et al., 2006; Main et al., 2006, 2007). Our results, like theirs, support the ED hypothesis. Indeed, in our study, though the differences between cryptorchid and controls are modest, they all go in the same direction. First, median concentrations in milk of the most frequently found chemicals tend to be higher in cryptorchids, though not statistically significantly. That this trend is observed at all despite a very close matching of our population, including parental origin, supports our hypothesis: indeed, the close matching is likely to include common environmental exposure, making the finding of differences more difficult. Furthermore, we have found some interesting, though weak correlations between cryptorchid status at birth and scores representing exposure to xenobiotic in milk (class of DDE, PCB and the composite score integrating PCBs and DDE), as well as a trend for mBP. Those three classes of chemicals may act through different mechanisms, and it is interesting that the resulting effect is statistically significant. However, only in vitro or experimental in vivo studies could assess the combined effects of PCBs, DDE and phthalate, and study their potential additive or synergistic effect. We performed the same statistical analysis in pairs from French/European origin only to see if women from overseas origin could skew our data. We found the same trends, though usually slightly more significant (data not shown). Last, it is worth noting that our results are statistically significant for boys cryptorchid at birth, but not at 3 months of age. It is possible that numbers are too small at 3 months to find significant differences.
PCBs, in our study, seem to have the most statistically significant effects. Well known for their neurotoxicant effects in development (Schantz et al., 2003), they also have estrogenic properties (e.g. PCB 153) and anti-androgenic activity (including PCB 118, and to a lesser degree PCB 153 and 138). At 50 pmol, PCB 118 completely antagonizes the stimulation of the androgen receptor (AR) by di-hydro-testosterone, whereas PCB 138 and PCB 153 are only partial antagonists (Schrader and Cooke, 2003). Thus, they are candidates for the disruption of male reproductive development. However, so far the evidence in the literature is weak. Therefore, our data suggesting an effect should draw closer attention to the potential effects of this class of chemicals on testis migration.
Our results for DDE reach borderline significance; they are at odds with those of Bhatia et al. (2005). However, that study reported on DDE measurements in maternal blood from a historic bank of 75 cryptorchid boys and 283 controls born between 1959 and 1967 (Child Health and Development Study). Unfortunately, no milk was available for this study, though milk is a more relevant matrix for these lipophilic compounds. Longnecker et al. reporting data from another historical US cohort (the Collaborative Perinatal Project) on third trimester maternal DDE concentrations, concluded that for boys in the highest category of DDE exposure compared with those in the lowest, the OR for cryptorchidism was consistent with a modest association (OR 1.3), although overall, their results were inconclusive (Longnecker et al., 2002). Comparison with such study is difficult, because the diagnosis of cryptorchidism was established differently (about half of the boys in their cohort were not diagnosed at birth) and again the data relate to maternal blood (and not milk) collected four decades before our study. On the other hand, our results are consistent with those of Damgaard et al. (2006), which found also a trend for higher DDE concentrations in cryptorchids compared with controls, though it was not statistically significant (97.32 versus 83.76 ng/g of fat, P = 0.26). Our results are also consistent with evidence from wildlife (Facemire et al., 1995) and experimental animals (Gray et al., 2001; Veeramachaneni et al., 2007), keeping in mind, however, that our population was exposed only to background levels. The mechanisms of action of DDE have been well studied. DDE displays AR antagonism in vivo and in vitro. It is a known AR ligand, like vinclozolin and procymidone, but less potent (Gray et al., 2001, 2006). In vivo, it causes alterations of androgenic pathways, though cryptorchidism seems a species-specific feature (Gray et al., 2006; Veeramachaneni et al., 2007).
Regarding mBP, our results are modest, showing a trend only, reflecting the fact that our numbers were smaller (n = 71 versus 125 for other chemicals). However, anecdotally, it is worth noting that among our 164 pairs, only four mothers self-reported professional exposure to phthalates in the detailed questionnaire, their sons being cryptorchid in all four cases. Indeed, phthalates are good candidates for male genital malformations (Kavlock et al., 2002). DBP, the parent compound of mBP, has a different profile of toxicity than DDE. It does not seem to compete with androgens for binding to AR. However, in vivo, in utero exposure causes anti-androgenic effects, including cryptorchidism, via a reduction of insL3 mRNA and testosterone levels (Gray et al., 2006). Swan et al. (2005) have shown that anogenital distance is shortened in boys more exposed to phthalates. Boys with prenatal maternal urine mBP concentrations in the highest quartile had an OR for shorter anogenital index of 10.2 (95% CI 2.5–42.2) compared with boys in the lowest quartile. Interestingly, there was a good correlation between cryptorchidism and anogenital distance (R2 = 0.23, P = 0.007) (Swan et al., 2005). Though Main et al. (2006) failed to find an association between cryptorchidism and phthalate monoesters concentrations, including mBP (10.25 µg/l in cryptorchid versus 9.09 in controls), they found interesting correlations with hormone levels at 3 months of age: mBP had negative correlations with free testosterone levels (r = –0.22, P = 0.03), positive correlation with SHBG concentrations (r = 0.272, P = 0.01) and with LH to free testosterone ratio (r = 0.2, P = 0.05). Those trends should trigger more prospective studies on phthalates monoesters and TDS.
Like the Danish/Finnish project (Damgaard et al., 2006; Main et al., 2006, 2007), we found a trend toward greater contamination in cryptorchid boys, supporting the hypothesis of environmental factors in the occurrence of cryptorchidism. It is striking that our results are consistent with those of that study. Indeed, Damgaard et al. published data on pesticides concentrations in 130 specimens of mature milk from 62 mothers of cryptorchid boys and 68 controls. They measured the concentrations of 27 persistent organochlorine pesticides, including DDE. Most of the quantifiable pesticides had higher median concentrations in milk of cryptorchid boys than in controls, though without reaching significance except for transchlordane. However, the eight most abundant pesticides (including pp'-DDE) were combined; their levels in breast milk were statistically higher in boys with cryptorchidism. Of course, their results, as ours, have not identified a single culprit among the chemicals, but only a trend toward higher exposure. Fetuses are exposed to a mixture of thousands of compounds: thus, what is observed is the integration of those exposures, potentially compounding synergistic and antagonistic effects of those chemicals. Therefore, it would be naïve to expect a causal link between concentrations of a single chemical and cryptorchidism. Thus, our results, together with those of Damgaard et al. (2006), could validate the concept of a marker of a population at risk. Higher concentrations of selected chemicals could imply either a higher exposure or a default in detoxification, e.g. via a decreased enzyme activity in genetically susceptible individuals. A polymorphism of enzymes, such as cytochrome 450 has been reported previously (Kavlock et al., 2002; Cummings and Kavlock, 2004; Gueguen et al., 2006).
Last, the use of persistent organic pollutants such as PCBs or DDE is now restricted in many countries, leading to an encouraging temporal trend for decreased exposure and human tissue concentrations (Solomon and Weiss, 2002). However, phthalate use is so widespread that regulation is extremely complex and needs more hard data. Furthermore, there are many widely used pesticides or chemicals with potential endocrine disrupting properties not screened in our study, which would deserve close attention, such as other phthalates, bisphenol A, polybrominated diphenyls ethers, to name a few.
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These results are the first assessment of prenatal exposure to EDs in our area, more known for tourism than for agriculture or industrial activities. We confirm ubiquitous in utero and lactational exposure of fetuses and infants to three different classes of xenobiotics with ED properties: DDE, PCBs and phthalates. Furthermore, we found evidence in our population that even at background exposure, children in the highest tier of exposure to PCBs, in milk (but not in CB), are more likely to be born with UDT. Our results are similar for DDE, nearing significance, but are somewhat weaker for mBP, possibly because of smaller numbers. Higher concentrations of selected xenobiotics in cryptorchid boys could be a marker of a higher global exposure and/or of a decreased capacity to metabolize and eliminate xenobiotics in general. Since a single, isolated compound is unlikely to be responsible for cryptorchidism, research should turn to assess the effects of mixture of chemicals. A long-term follow-up of our cohort is planned to assess future risks of testis cancer and infertility, in relation to their exposure.
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This project was funded by the Ministry of Research and was promoted by the CHU of Nice.
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The Cryptorchidism Study Group from Nice Area includes also: Mrs Patricia Pacini (from the Laboratoire de l'Environnement de l'Agglomération Niçoise, 06200 Nice, France), Drs Mireille Boda-Buccino (Endocrinology Department, Hôpital l'Archet 1, CHU Nice, 06200 Nice, France), Laure Bornebusch (Pediatrics Department, CHG Grasse, 06135 Grasse, France), Pierre Azuar (Obstetrics Department, CHG Grasse, 06135 Grasse, France), Muriel Dupuy (Pediatrics Department, CHU Nice, 06200 Nice, France), Catherine Meneguz (Pediatrics Department, CHG Grasse, 06135 Grasse, France), Camille Tommasi (Pediatrics Department, CHG Grasse, 06135 Grasse, France), Christian Pradier (Department of Public Health, CHU Nice, 06200 Nice, France), and the head midwives of the two departments [Mrs Sandra Maccagnan (Obstetrics Department, Hôpital l'Archet 2, CHU Nice, 06200 Nice, France), Françoise Court (Obstetrics Department, CHG Grasse, 06135 Grasse, France) and Annie Lecuyer (Obstetrics Department, CHG Grasse, 06135 Grasse, France)].
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We thank the participating families, the two teams of midwives for their sustained involvement, the research assistants including Delphine Mesmin, Pélagie Thibaut, and the technical staff of the Laboratoire de l'Environnement de l'Agglomération Niçoise.
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See Appendix for authors of Cryptorchidism Study Group from Nice. 
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Submitted on December 17, 2007; resubmitted on April 3, 2008; accepted on April 17, 2008.
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