1. Semen quality in relation to exposure to currently used pesticides Shanna H. Swan, PhD University of Missouri-Columbia 6 th International Symposium on Environmental Endocrine Disrupters Sendai, Japan December 5, 2003

2. Background (1) In 1992 Carlsen et al. reported a 50% global decline in sperm concentration This trend, supported by re-analyses, was seen to vary geographically Background (1)

3. Since some analyses found no decline, a global trend is still disputed.

4. Background (2) After 1992, many publications reported significant geographic variation in sperm concentration These uncontrolled comparisons were difficult to interpret

5. Sperm concentration in three US cities

6. Background (3) Well-controlled, multi-center studies were needed to demonstrate real geographic variation in semen quality A world-wide collaboration of multi-center studies was begun in 1997.

7. One of these compared four European cities Sperm concentration in Copenhagen, Denmark was 74% of that in Turku, Finland. (Jorgensen et. al, 2001) 5250 48 Normal morphology (%) 67685863 Motile sperm (%) 1131028884 Concentration (10 6 /ml) TurkuEdinburghParisCopenhagen

8. The Study for Future Families (SFF) In 1998, we obtained funding for a multi-center study of pregnant women and their partners to compare semen quality and other reproductive endpoints among US cities.

9. The First Four Clinical Centers of the Study for Future Families

10. Pregnant woman with appointments at study clinics were contacted to determine eligibility 36 % of potential subjects were ineligible 55% of eligible subjects refused participation 24% of eligible men gave a semen sample SFF Recruitment:

11. Study Population Characteristics Men with at least 1 semen sample

12. Comparing semen quality among centers Semen parameters included; sperm concentration, volume, motility (motion) and morphology (shape) Initial comparisons among centers were unadjusted and used raw values Regression models, using log transformed data, were used to control for confounding

13. Summary of Semen Parameters * * * * P-Value for MO vs. all other centers <.001

14. Semen Quality by Center Sperm Concentration (10 6 /ml) Volume (gm) % Motile sperm Total Motile Count (10 6 ) % Normal sperm MO58.73.948.2113.010.8 CA 80.83.654.5162.212.2 MN 98.63.952.1200.911.4 NY 102.93.356.4196.410.9 Men in Mid-Missouri have only 56% as many moving sperm as men in urban Minneapolis

15. Can differences between the populations at these centers explain the poor semen quality in men from mid-Missouri?

16. No: We considered these factors Age Smoking History of infertility Body mass index (BMI) History of STDS Ethnicity Recent fever Abstinence time Analysis time Differences in semen quality were unchanged by adjustment for these factors unchanged by adjustment for these factors

17. Can differences between centers’ methods of semen analysisaccount for these differences in semen quality? between centers’ methods of semen analysis account for these differences in semen quality?

18. All labs used the same protocols and standard equipment Technicians were centrally trained Standard samples sent quarterly for quality control No: Semen analyses were tightly controlled

19. QC data for MO and MN compared to “Gold standard” 49% 74.6 (x 10 6 /ml) MO 47%72.6 (x 10 6 /ml) MN 51% 81.3 (x 10 6 /ml) Gold Standard % Motile Sperm Sperm ConcentrationTechnician MO and MN were similar and slightly lower than standard

20. We sought causes of our findings, looking first in MO and MN:Why? Sample sizes, participation rates and populations were similar in these centers Quality control results were also similar But semen parameters differed significantly The remainder of this talk will compare these two centers.

21. How does mid-Missouri differ from Minneapolis ?

23. % Acres in farms and use of pesticides 28,7044,62729,90119%1,855MN 49,863 8,16260,12557%164MO HerbicidesInsecticides Fertilizer Pesticides applied (acres) % Acres in Farms Population Density SFF Center

24. Study hypothesis One or more pesticides widely used in agriculture in mid-Missouri, but not in Minneapolis, MN, contributes to the difference in semen quality between these two centers

25. How did we examine this hypothesis? Men’s urine samples were assayed for pesticide metabolites by the Centers for Disease Control (CDC). CDC was blinded to men’s semen quality or city of residence. We first compared pesticide metabolites in the urine of MO and MN men. Then, we compared pesticide metabolites in two groups of MO men (cases and controls)

26. Pesticides found more often in MN Chlorpyrifos,1-Napthol and 4-Nitrophenol None were primarily used in farming None were related to semen quality These were not studied further Remaining analyses limited to men from mid-Missouri

27. MO men selected for pesticide analysis Cases 25 men with poor semen quality Mean sperm concentration: 32.4 x 10 6 /ml Controls 25 men with normal semen quality Mean sperm concentration: 72.2 x 10 6 /ml All semen parameters were significantly lower in cases

28. Pesticides detected more often in cases than controls Percent of men with pesticide > LOD

29. Five pesticides were associated with semen quality in MO men: Three were strongly associated Pesticide(P-value)* Alachlor(0.0003) Diazinon(0.005) Atrazine(0.02) *Wilcoxon rank test

30. Two pesticides were weakly associated with sperm count Pesticide(P-value)* Metolachlor(0.054) 2,4-D(0.064) *Wilcoxon rank test

31. 25 Total Participants 30.0 (4.3-210) 212>0.7 6.3 (1.3-29.4) 8100.15-0.7 REF15 3<0.15 Odds Ratio (95% CI) Controls Cases Alachlor Level Dose response for alachlor in MO men Though numbers are small, trend is highly statistically significant

32. What if a man was exposure to more than one of the five associated pesticides? Percent of men with poor semen quality Number of pesticides at high levels

33. How are men being exposed? Because of pesticides’ short half-life in the body, exposure is recent Men are not farmers, so exposure is environmental; dermal exposure is unlikely These pesticides are not volatile; inhalation is unlikely Therefore, ingestion is the likely route of exposure

34. Drinking water is a likely source of exposure These pesticides are commonly found in drinking water sources These pesticides are not removed by routine water treatment US Geological Survey, 2001

35. Examining pesticides and semen quality in a second agricultural center Iowa City, IA and Columbia, MO have: Same population density Comparable demographics Center staff trained in all SFF methods We will recruit 200 IA couples for SFF Results now available on 50 IA men

36. Use of pesticides in IA is greater than MO

37. Semen quality in Iowa City and other SFF centers

38. What is needed? Urinary pesticide levels in IA men Serum levels of pesticides to examine total exposure Tap water pesticide levels Replication of study in other areas and countries

39. Shanna Swan (U MO) Jim Overstreet (UC Davis) Principal Investigator Andrology Center Director Erma Drobnis (U MO) Maureen Hatch (Mt. Sinai) Clinical Center Director Bruce Redmon (U MN) Christina Wang (UCLA) Clinical Center Director Amy Sparks (U IA) Dana Barr (CDC) Clinical Center Director Biomarker Analyses Funded by grants from the National Institute of Environmental Health Sciences and the Environmental Protection Agency. The Study for Future Families