Study area

Guadeloupe consists of two main islands, Grande Terre and Basse Terre, separated by a narrow sea channel. Agriculture is the main economic activity and banana is the principal crop. The banana plantations are mostly located on the rainy slopes of Basse-Terre and intensive pesticide use controls insects, nematodes and fungi in these plantations. The National Park of Guadeloupe encompasses the central part of Basse-Terre and includes the Natural Reserve of Grand Cul-de-Sac Marin. While we are aware that no area can be totally pollution-free, this park has historically been protected from direct pollution by humans and provides an opportunity for research on wildlife less strongly affected by pollution.

Human study

The study was carried out from 1999 to 2001. The study population consisted of 20- to 50-year-old male workers from Basse-Terre regularly followed up by the Guadeloupe Occupational Health Service. Information meetings were organized in the field, at which the men were informed that the study protocol included the collection of a semen sample, a blood sample, and physical and andrological examinations. Basic data, such as age and current job were collected for all men, including those who later declined participation in other aspects of the study. Each participating worker was given an appointment at the University Hospital of Pointe à Pitre. Blood samples were collected and medical examinations carried out, and information about occupational history and past and present exposure to chemical and physical agents was collected in face-to-face interviews. An indemnity of 80 Euros was paid to each participant who gave written informed consent and the Ethics Committee of Guadeloupe approved this study.

During the hospital visit, subjects underwent a physical and andrological examination, performed by a single physician unaware of the subject's exposure status. A detailed examination of external genitalia and staging of secondary sexual development were carried out according to WHO recommendations [14]. Testicular volumes were evaluated using the Lambert formula: volume (cm³) = 0.71 × major axis (cm) × minor axis (cm)². Abnormalities in testis thermoregulation are known to alter semen characteristics, so scrotal temperatures were measured in naked subjects in the supine position [15]. Demographic data, medical, surgical, urogenital and reproductive history, recent illnesses, fever and treatment and lifestyle data were collected using a standardized questionnaire during a face-to-face interview with the physician.

Pesticide exposure status was assessed with a questionnaire. Information was collected yearly concerning occupational and non occupational exposure to chemical substances (pesticides, solvents, metals and diesel exhaust fumes) or physical agents (radiant heat and ionizing radiation). The number of years of use and annual frequency of use was recorded for each category of chemical substance or physical agent. We asked about whether pesticides were applied in a banana plantation, sugar cane plantation or other agricultural setting, and distinguished between the agricultural use of pesticides and kitchen garden activity or other domestic exposure. One occupational physician specifically trained in agricultural activities assessed job descriptions blindly, to assess the degree of correlation between job tasks and declared occupational exposures.

Semen samples were collected by masturbation in the hospital after a period of sexual abstinence for 2 to 7 days. Semen analysis was performed according to WHO guidelines [16], at the Pathology Laboratory of the University Hospital of Pointe à Pitre. Semen samples were analyzed by 2 technicians blind to exposure status and specifically trained on site. Intra- and inter-technician variations in the assessment of sperm concentration and motility were evaluated several times before and during the study and were considered satisfactory, the coefficients of variation being typically around 5 to 10%. Sperm morphology was assessed from smears after Shorr staining, using the modified David classification [17], by a single technician at the Reproductive Biology Laboratory, Cochin University Hospital, Paris.

Blood samples were collected between 8 and 9 a.m. and serum samples were stored at -20°C until the analysis. FSH (follicle-stimulating hormone) and LH (luteinizing hormone) levels were determined by immunoradiometric assay (Immunotech, Marseilles, France). The dose-response curve of FSH was calibrated against the WHO international standard of 94/632, and the LH dose-response curve was calibrated against the 2^nd IS international standard of 80/552. Total testosterone concentration was measured by radioimmunoassay (Immunotech, Marseilles, France) and dimeric inhibin B concentration was assessed by enzyme-linked immunosorbent assay (Oxford Innovation Ltd, Oxford, UK). Results were expressed as concentrations for testosterone (ng/ml), for inhibin B (pg/ml) and for FSH and LH (IU/l). Between- and within-assay coefficients of variation were < 8 % for FSH and LH, < 11 % for testosterone and < 17 % for inhibin B.

We analyzed data for age, weight, height, body mass index, smoking habits (past or current smoker vs. non smokers), alcohol consumption (current drinker vs. non drinker), recent illness (yes vs. no), fever or medical treatment during the past 3 months (yes vs. no), fertility status scored as at least one live-born child (yes vs. no) and, for fertile participants, the total number of children and time to pregnancy for the most recent child, days of sexual abstinence before semen analysis, testicular volume and scrotal temperature. In addition, the month in which the semen analysis was performed was noted, to account for a possible seasonal effect. The categories spring/summer vs. autumn/winter were compared and, as there are no significant differences in temperature or day length between seasons on Guadeloupe, we also considered summer/autumn (wet season) vs. winter/spring (dry season). The semen characteristics and hormones studied were: pH, semen volume (ml), sperm concentration (millions/ml), total sperm count (millions, the product of semen volume and sperm concentration), sperm viability (the percentage of live spermatozoa), "a"-type progressive motility (the percentage of rapid and progressive spermatozoa), "a"+"b"-type progressive motility (the percentage of rapid and slow progressive spermatozoa), sperm morphology (the percentage of morphologically normal spermatozoa) and the multiple anomaly index (mean number of morphological defects per abnormal spermatozoa). Semen characteristics were also categorized as dichotomous variables, using WHO reference values [16] as thresholds: 2 ml for seminal volume, 20 millions/ml for sperm concentration, 40 millions for total sperm count, 25% for "a"-type progressive motility and 50% for "a" + "b"-type progressive motility. The threshold selected for sperm morphology was the median percentage of morphologically normal spermatozoa in non exposed men (13%).

Animal study

We selected two sites extremely different in terms of agricultural activity. The "pesticide-exposed area", located in Neuf-Chateau (south-east Basse-Terre), has been exclusively devoted to banana cultivation since the mid-20th Century. The "pesticide-free area" was Fajou Islet in the Grand Cul-de-Sac Marin lagoon, within the marine reserve of the National Park.

Trapping and field operations took place between the 18th and the 28th of January 2000, a period of high reproductive activity in French West Indies rodent populations. Ship rats (Rattus rattus), Norwegian rats (Rattus norvegicus) and Javanese mongoose (Herpestes auropunctatus), all alien species in the French West Indies [18], were trapped using Manufrance^© live traps baited with a mixture of peanut butter, oat flakes and sardine oil [19]. The trapped animals were killed in the field by intrathoracic pentobarbital injection. Blood was collected immediately by heart puncture. Animals and blood samples were stored in a container at + 4°C and transferred within 45 minutes to a laboratory specifically installed for this study.

Mammals were sexed and males selected, weighed and dissected. The testes, epididymis, ventral prostate and seminal vesicles were removed and weighed. One testis per animal was fixed by immersion in Bouin's solution for histology, the second testis and one epididymis being immediately frozen at – 20°C. Both eye balls were removed and stored in 10% formaldehyde at room temperature for more than 3 months to tan eye lenses. Eye lenses were cleaned and dried at 105°C until their weight stabilized. Lens pair weight was used as a surrogate for age [20]. Each fixed testis was embedded in paraffin, and 5 μm sections were cut, stained with hematoxylin and eosin and examined under a light microscope [21]. The number of sperm in the testis and caudal epididymis was assessed as previously described [22]. The total number of sperm was defined as the sum of testicular and epididymal sperm reserves. The gonadosomatic index was calculated as (testis weight/body weight) × 100.

Serum fractions were stored at -20°C until use. Rat FSH levels were determined by immunoradiometric assay (BiocodeHycel, Belgium) and rat LH levels, by radioimmunoassay (BiocodeHycel, Belgium). Dose-response curves for FSH and LH were calibrated against the corresponding NIH standard. Total rat testosterone levels were determined by radioimmunoassay (Immunotech, Marseilles, France). Results were expressed as concentrations (ng/ml for testosterone, IU/l for FSH and LH). The intra- and inter-assay coefficients of variation were < 6, 7 and 8 % for testosterone, FSH and LH, respectively.

Statistical analysis

We used chi-squared or Fisher's exact tests for categorical variables and unpaired t-tests (equal variances) and analysis of variance/covariance for continuous variables, in both human and animal studies. We normalized distributions by applying a log10 transformation or a square-root transformation and then used the Kolmogorov-Smirnov test to check that the resulting data were normally distributed. The effect of exposure on the categorized human seminal outcomes was assessed, taking possible confounding factors into account, by logistic regression analysis to produce odds ratios (OR) and 95 percent confidence intervals (95% CI). Factors were considered confounding if their p values in univariate analysis were below 0.2, and were included in the final model if they modified the odds ratio of the outcome by more than 10 percent [23]. The potential confounding variables evaluated were age, alcohol and tobacco consumption, solvent exposure, past history of genital infection, sexual abstinence, and season of semen analysis. Age and duration of abstinence were systematically included as known confounding factors for semen variables. Linear regression was used to assess the relationship between eye lens weight, body weight and total sperm reserve in the animal study. All analyses were carried out using the Statview software package (SAS Institute Inc., Cary, USA). All tests were two-sided, and the results were considered significant if P < 0.05.

Human Study

In total, 101 of the 594 men approached for this study (17%) agreed to participate. There was no difference in age or proportion of banana plantation workers and workers not employed by banana plantations between those agreeing to and declining participation (data not shown). Six men who agreed to participate were excluded for one of the following reasons: past chemotherapy for Hodgkins disease, current treatment for infertility, orchidectomy, current active genital infection or sexual abstinence for less than 48 hours before semen analysis. Forty-two were employed as banana plantation workers and regularly used pesticides as part of their job. They worked on 13 plantations but reported having applied pesticides in 37 different plantations over the course of their careers, corresponding to more than 80% of the local area under banana. We listed the chemical products used in the banana plantations from the notebooks used to record pesticide applications. The pesticides in use at the time of the study belonged to the organophosporus (cadusaphos, ethoprophos, isazophos, pyrimiphos-ethyl, terbufos) and carbamate (aldicarb) classes. The median duration of pesticide use was 14 years for banana workers. The mean annual frequency of application was 30 days, and was similar for all banana plantation workers. A group of 45 manual and office workers enrolled in the study did not apply pesticides in either occupational or non occupational circumstances. Finally, eight men were in contact with pesticides under miscellaneous conditions, including occupational exposure in sugarcane fields or vegetable root plots or domestic applications in kitchen gardens. This group was not analyzed further due to the small number of subjects and their heterogeneity in terms of the chemical family of pesticides used and the frequency and intensity of application.

Animal Study

In total, 120 mammals were trapped: 75 in banana plantations and 45 in Fajou Islet. The very small number of trapped mongooses (n = 9) and the smaller numbers of Norwegian rats in Fajou Islet than in banana plantations (n = 19) ruled out the further analysis of these species. We captured 92 ship rats, and the proportion of males and females did not differ significantly between the sites (p = 0.29). The relationships between eye lens weight (a surrogate of age), body weight and sperm reserve were used to distinguish sexually mature from immature male ship rats. Five animals with a body weight less than 100 g (the cut-off generally used to distinguish between adult and non adult ship rats) with no epididymal and testicular spermatozoa had a dry eye lens weight below 20 mg. Three other animals with an absence of epididymal spermatozoa but with testicular spermatozoa had eye lens weights below 21 mg. For these eight rats, testis histology indicated that there was no, or only focal, spermatogenesis. We therefore used a threshold eye lens weight of 21 mg w to distinguish sexually mature ship rats from immature ones.