Semen quality inSemen quality in relation to exposure to relation to exposure to

currently used pesticidescurrently used pesticides

Shanna H. Swan, PhDShanna H. Swan, PhDUniversity of Missouri-Columbia

6th International Symposium onEnvironmental Endocrine Disrupters

Sendai, JapanDecember 5, 2003

Background (1)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)Background (1)

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

Background (2)Background (2)

After 1992, many publications reported significant geographic variation in sperm concentration

These uncontrolled comparisons These uncontrolled comparisons were difficult to interpretwere difficult to interpret

132 x 106 /m lNe w Y ork, NY

101 x 10 6/m lMinne apolis, MN

73 x 10 6/m lLos Angeles, CA

Fisch, 1996

Sperm concentration in three US citiesSperm concentration in three US cities

Background (3)Background (3)

Well-controlled, multi-center studies were needed to demonstrate real geographic variation in semen quality

A world-wide collaboration ofA world-wide collaboration ofmulti-center studies was begun in 1997.multi-center studies was begun in 1997.

One of these compared four European citiesOne of these compared four European cities

Sperm concentration in Sperm concentration in Copenhagen, Denmark was 74% Copenhagen, Denmark was 74%

of that in Turku, Finland.of that in Turku, Finland.

(Jorgensen et. al, 2001) 52505048Normal morphology (%)67685863Motile sperm (%)

1131028884Concentration (106/ml) TurkuEdinburghParisCopenhagen

The Study for Future The Study for Future FamiliesFamilies (SFF)(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.

The First Four Clinical Centers The First Four Clinical Centers of the Study for Future Familiesof the Study for Future Families

Ne w Y ork, NY

M inneapolis , M N

Columbia, MO

Los Angeles, CA

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 RecruitmentSFF Recruitment::

Study Population Characteristics Study Population Characteristics Men with at least 1 semen sampleMen with at least 1 semen sample

Center MO CA MN NY Total Participants 208 189 217 44 658 Mean age (yr) 30.8 30.5 32.3 35.7 31.5 College/Tech (%) 56.3 30.8 74.2 73.2 56.0 Non-Caucasian (%) 14.9 71.4 12.0 31.6 31.3 Smoked >10 cig/day (%) 13.5 2.7 4.6 2.4 6.8 Recent fever (%) 3.4 1.6 3.2 2.4 2.8 Hx of STD’s (%) 11.5 12.2 13.8 14.6 12.7

Comparing semen Comparing semen quality among centersquality 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

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Concentration % Motile Sperm Total Motile Count % Normal Sperm

MOCANYMN

Summary of Semen ParametersSummary of Semen Parameters

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*P-Value for MO vs. all other centers <.001

Semen Quality by CenterSemen Quality by Center

Sperm Concentration (106/ml)

Volume (gm)

% Motile sperm

Total Motile Count (106)

% Normal sperm

MOMO 58.758.7 3.93.9 48.248.2 113.0113.0 10.810.8CA 80.8 3.6 54.5 162.2 12.2MN 98.6 3.9 52.1 200.9 11.4NY 102.9 3.3 56.4 196.4 10.9

Men in Mid-Missouri have only 56% Men in Mid-Missouri have only 56% as many moving sperm as men in as many moving sperm as men in

urban Minneapolisurban Minneapolis

Can differences Can differences between the populationsbetween the populations

at these centers explain theat these centers explain thepoor semen quality in men frompoor semen quality in men from

mid-Missouri?mid-Missouri?

No: We considered these factorsNo: We considered these factors

AgeSmokingHistory of infertilityBody mass index (BMI)History of STDS

EthnicityRecent feverAbstinence timeAnalysis time

Differences in semen quality wereDifferences in semen quality were

unchanged by adjustment for these factorsunchanged by adjustment for these factors

Can differencesCan differences between centers’ methods between centers’ methods

of semen analysisof semen analysis account for account for these differences in these differences in

semen quality?semen quality?

All labs used the same protocols and standard equipment

Technicians were centrally trained

Standard samples sent quarterly for quality control

No: Semen analyses were No: Semen analyses were tightly controlledtightly controlled

QC data for MO and MN compared QC data for MO and MN compared to “Gold standard”to “Gold standard”

49%74.6 (x 106/ml)MO

47%72.6 (x 106/ml)MN

51%81.3 (x 106/ml)Gold Standard

%Motile SpermSperm Concentration Technician

MO and MN were similar and MO and MN were similar and slightly lower than standardslightly lower than standard

We sought causes of our findings, We sought causes of our findings, looking first in MO and MN:looking first in MO and MN: Why?Why?

Sample sizes, participation rates and populations were similar in these centers Quality control results were also similarBut semen parameters differed significantly

The remainder of this talk will The remainder of this talk will compare these two centers.compare these two centers.

How does mid-Missouri How does mid-Missouri differ from Minneapolis ?differ from Minneapolis ?

% Acres in farms and use of pesticides% Acres in farms and use of pesticides

28,7044,62729,90119%1,855MN

49,8638,16260,12557%164MOHerbicidesInsecticidesFertilizer

Pesticides applied (acres)% Acres in Farms

Population Density

SFFCenter

Study hypothesisStudy 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

How did we examine this hypothesis?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)

Pesticides found more often in MNPesticides 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 Remaining analyses limited to men from mid-Missourilimited to men from mid-Missouri

MO men selected for pesticide analysisMO men selected for pesticide analysis

Cases25 men with poor semen qualityMean sperm concentration: 32.4 x 106/ml

Controls25 men with normal semen qualityMean sperm concentration:

72.2 x 106/ml

All semen parameters were significantly lower in cases

Pesticides detected more Pesticides detected more often in cases than controlsoften in cases than controls

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Five pesticides were associated Five pesticides were associated with semen quality in MO men:with semen quality in MO men:

Three were strongly associatedThree were strongly associated

Pesticide (P-value)*Alachlor (0.0003)Diazinon (0.005)Atrazine (0.02)

*Wilcoxon rank test

Two pesticides were weakly Two pesticides were weakly associated with sperm countassociated with sperm count

Pesticide (P-value)*Metolachlor (0.054)2,4-D (0.064)

*Wilcoxon rank test

2525Total Participants 30.0 (4.3-210) 212>0.7 6.3 (1.3-29.4) 8100.15-0.7

REF15 3<0.15Odds Ratio (95% CI)ControlsCasesAlachlor Level

Dose response for alachlor in MO menDose response for alachlor in MO men

Though numbers are small, trend is highly Though numbers are small, trend is highly statistically significantstatistically significant

What if a man was exposure to more than What if a man was exposure to more than one of the five associated pesticides?one of the five associated pesticides?

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Number of pesticides at high levels

How are men being exposed?How are men being exposed?

Because of pesticides’ short half-life in the body, exposure is recentMen are not farmers, so exposure is environmental; dermal exposure is unlikely

These pesticides are not volatile; inhalation is unlikely

Therefore, ingestion is the Therefore, ingestion is the likely route of exposurelikely route of exposure

Drinking water is a likely Drinking water is a likely source of exposuresource of exposure

These pesticides are commonly found in drinking water sources

These pesticides are not removed by routine water treatment

US Geological Survey, 2001

Examining pesticides and semen Examining pesticides and semen quality in a second agricultural centerquality in a second agricultural center

Iowa City, IA and Columbia, MO have:Same population densityComparable demographics

Center staff trained in all SFF methodsWe will recruit 200 IA couples for SFFResults now available on 50 IA men

Farm Acreage around Study Site

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Use of pesticides in IA is greater than MOUse of pesticides in IA is greater than MO

Total Motile Sperm by Study Site

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Semen quality in Iowa City and other SFF centersSemen quality in Iowa City and other SFF centers

What is needed?What is needed?

Urinary pesticide levels in IA menSerum levels of pesticides to examine total exposureTap water pesticide levels Replication of study in other areas and countries

Shanna Swan (U MO) Jim Overstreet (UC Davis) Principal Investigator Andrology Center Director

Erma Drobnis (U MO) Maureen Hatch (Mt. Sinai) Clinical Center Director Clinical Center Director

Bruce Redmon (U MN) Christina Wang (UCLA) Clinical Center Director 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 FamiliesThe Study for Future Families