1 Department of Occupational Medicine, Aarhus University Hospital, DK-8000, Aarhus, 2 Department of Gynaecology and Obstetrics, Aarhus University Hospital, DK-8200, Aarhus, 3 The Fertility Clinic, Copenhagen University Hospital, Herlev, DK-2730, Herlev, 4 The Fertility Clinic, Copenhagen University Hospital, Rigshospitalet, DK-2100, Copenhagen and 5 Danish Epidemiology Science Centre, Aarhus University, DK-8000, Aarhus, Denmark
6 To whom correspondence should be addressed at Åbyhojvej 17, DK-8210 Aarhus V, Denmark. e-mail: henrik.hjollund{at}dadlnet.dk
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Abstract |
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Key words: abortion/IVF/male mediated/occupation/pesticides
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Introduction |
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It is now well documented that in humans some 30% of implanted embryos are lost, and around two-thirds of these are lost before the woman knows that she is pregnant (Rothman and Greenland, 1998). Few studies have included data on pre-clinical abortion (Wilcox et al., 1988
; Zinaman et al., 1996
; Bonde et al., 1998
; Hjollund et al., 2000
; Cho et al., 2002
), but such studies are time consuming and expensive. IVF patients provide opportunities to follow a large number of pregnancies from the start, making analyses of even rare exposures possible. The male partner is not medically treated and is less likely to alter occupational activities before semen donation. Consequently, IVF pregnancies constitute an interesting model for research into paternal risk factors for spontaneous abortion.
The aim of this study of pregnancies identified in the Danish In Vitro Fertilization (DIVF) Registry was to investigate the survival of early pregnancies following paternal exposure to pesticides.
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Materials and methods |
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For each treatment cycle, the following information is available: indication for IVF treatment, origin of egg and sperm, fertilization method, date of initiation, aspiration and ET, information on the HCG analysis, and whether or not a clinical pregnancy was detected. In the year 2000, there were six public and 11 private fertility clinics. From 1994 to 2000, there were 46 614 treatments given to 15 013 women (Danish National Board of Health, 2002). Information from the DIVF register previously has been linked to register information on birth and spontaneous abortion for descriptive purposes (Danish National Board of Health, 1998
, 2002).
Information on pregnancy outcome
Information of pregnancy survival until clinical detection was collected from the DIVF register, while data on pregnancy outcome were collected from other registers (the Danish hospital register and the Danish birth register) by linkage with the womans CPR number. Diagnoses coded as spontaneous abortion, induced abortion, ectopic pregnancy and mola (ICD-10 codes DO00DO06) were classified as termination of a pregnancy (see Appendix 1). If the difference between gestational age calculated from the treatment cycle and the gestational age registered in the health register was 2 weeks, the event was assigned to this treatment cycle and the pregnancy was assigned the calculated gestational age as the date of termination of the pregnancy: if no such record was found, the pregnancy was classified as a spontaneous abortion and was assigned a gestational age of 7 weeks. Home deliveries are registered in the Birth Register, but a few births may escape registration, e.g. if the woman gives birth outside the country. To account for this, women with a clinical pregnancy but no information on either abortion or birth were linked to their CPR. In 362 of these apparently early subclinical abortions, the woman had a child with a birth date within the expected time of delivery and this child had Danish citizenship at birth. These cycles were deleted since it could not be confirmed if the children were outcomes of the cycles in question or from treatments abroad, or a child adopted later by the woman (see Appendix 2).
Treatment with donated eggs, fertilization with donor sperm or thawed eggs were excluded. We also excluded treatment cycles ending with ectopic pregnancies, mola or induced abortion (1.9% of pregnancies). Treatments after 31 December 2000 were excluded to insure complete follow-up in the health registers. The criteria for selected cycles are listed in Table I. A total of 5879 women had at least one pregnancy fulfilling the criteria.
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For the remaining women, the following information was retrieved from the Danish Central Population Register: postal address, actual marital status, CPR numbers of any current and any previous spouses and the date of changes in marital status, and CPR number of any man living at the same address as the IVF-treated woman on the day the file was processed (October 2001) as well as at the time of her first HCG-positive treatment cycle. The identity of the father is not recorded in the DIVF register, but Danish legislation demands that couples treated with IVF should be cohabiting at the time of treatment. If the woman was married at the date of the first treatment, the spouse was considered to be the father (70.6%). If the woman was unmarried but cohabiting with one man at the time of the first treatment, the cohabiting man was considered to be the father (29.4%). Otherwise the woman was excluded. Four men appeared as partner to more than one woman, and these couples were excluded. For ethical reasons, only couples who were still married or cohabiting in 2001 were selected to receive the questionnaire. A few couples had declined contact from researchers (1.2%). A total of 5879 couples who had received their first treatment after January 1, 1996 were included (Table I).
Basic questionnaire information
The basic questionnaire addressed the situation in October 2001 and was divided into a section for the mother and a section for the father. The questionnaire had 40 items on education, job, trade, specific occupational exposures as well as smoking, drinking and other lifestyle issues. The participants were asked to indicate if their actual job or trade and lifestyle had changed during the last 5 years. The women were also asked questions on reproductive history prior to their first IVF treatment. The questionnaires were mailed on October 29, 2001, and 4007 useable questionnaires (response rate 68.2%) were received after one reminder. The characteristics of the couples are shown in Table II.
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Results |
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The proportion of miscarriage before the 28th gestational week was 19.7, 19.7, 21.3 and 22.2% in the non-mutually exclusive groups of herbicide-, fungicide-, insecticide- and growth retardant-exposed groups, respectively. In the unexposed reference group, 28.4% of pregnancies ended in a spontaneous abortion. The survival curve of pregnancies by application of herbicides, fungicides, insecticides and growth retardants is shown in Figure 1, together with the two reference groups. The survival was consistently higher among farmers compared with the reference groups.
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The outcome of pregnancy was also categorized according to whether or not the father sprayed pesticides or growth retardants in the month when he supplied the semen: 1 month before; 2 months before; and 3 months before. The largest departure from unity [risk rate ratio 0.5) was found with a 2 month lag time [95% confidence interval (CI) 0.21.4].
Also, the risk of a hospitalized abortion was at its lowest among those exposed, although this was not statistically significant (data not shown). Similar results were found in analyses of clinically detected pregnancies only (n = 2515, 6.7% abortions).
In supplementary analyses, the following pregnancies were excluded, but with limited effect on the risk estimates: a reported IVF treatment before the first entry in the DIVF registry (n = 143), a reported spontaneous abortion before the womans first IVF treatment (n =696), centres with <200 recorded pregnancies (n = 327), private IVF centres (n = 848), indications other than tubal factor (n = 1814), no indications of male factor (n = 1619), only one, or more than two embryos transferred (n = 531) and pregnancies resulting from ICSI (n = 1002). Likewise, unchanged estimates were found in the following presumably highly exposed pregnancies (see Table III ): if the father (i) sprayed more >40 h in the last 12 months; (ii) did not use gloves when mixing chemicals; (iii) used a tractor without an air filter; (iv) used a manually controlled sprayer; and (v) had the same job characteristics in both 1996 and 2001 (data not shown).
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Discussion |
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Validity of the IVF model
The validity of our results depends on the comparability of IVF pregnancies and normal pregnancies with respect to mechanisms leading to spontaneous abortion. Spontaneous abortion occurred in 30% of reference pregnancies. This corresponds well to the proportion of spontaneous abortion in other IVF studies (Liu and Rosenwaks, 1991
) and in normal pregnancies, when early pregnancy losses are included (Rothman and Greenland, 1998
). On the other hand, in IVF pregnancies, normally two or more embryos are transferred (Danish National Board of Health, 2002
). If more than one embryo is implanted successfully and results in an HCG-positive pregnancy, a spontaneous abortion is only registered in the case of death of all fetuses. Thus, we overestimate the survival of the individual fetus and, although we obtained similar findings when stratified on the number of transferred embryos, it should be acknowledged that in >80% of cases, more than one embryo was transferred. Little is known about the physiological processes involved in human gestation in the period from implantation of the blastocyst until a pregnancy is detectable, and the initial adhesion of the blastocyst has never been observed (Robertson et al., 1985
; Norwitz et al., 2001
). Moreover, mechanisms responsible for early embryonal loss may be different in IVF pregnancies and natural pregnancies, and findings based upon IVF data perhaps cannot be generalized to pregnant women in general.
Sources of bias
Information on exposure was collected retrospectively in relation to the outcome, and the scope of the study was known to the participants. Information bias as well as selection bias may therefore play a role. The pregnancy survival was highest among respondents to the basic as well as to the specific questionnaires. At least with respect to the basic questionnaire, this does indicate selection. The same mechanisms should, however, be operating in the three other groups who received a specific questionnaire, and analyses with these groups as reference revealed results similar to the main analyses. Active selection of eggs, sperm and embryos for transfer may represent the most important limiting factor for the external validity of the findings.
In conclusion, we found no indication that paternal exposure to pesticides and growth retardants confers a risk to pregnancy survival in IVF pregnancies. Exposure to pesticides does occur among Danish tractor drivers, mainly through dermal exposure (Larsen et al., 1998b), but bioactive exposure to potentially harmful pesticides in Denmark is believed to be low. The findings are restricted to countries with similar standards of protection.
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Acknowledgments |
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Appendix 1 |
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Appendix 2 |
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References |
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Submitted on November 18, 2003; accepted on January 6, 2004.