# Presentation: 228063

## Risk Factors for Female Infertility in an Agricultural Region

**Anne R. Greenelee, PhD****1**
**Tye E. Arbuckle, PhD****2**
**Po-Huang Chyou, PhD****1**

**Notes:**
1. Marshfield Medical Research Foundation, Marshfield, WI
2. Health Canada, Ottawa, Ontario
---
Anne R. Greenlee's bio
---
I would like to acknowledge my co-authors: Tye Arbuckle, Epidemiologist; and Po Chyou, Biostatistician.

## Agricultural Exposures - Risk

**Male Partner**
**Altered semen parameters**
**Poor fertilization rate**
**Early and late abortions**
**Female partner**
**Ovulatory-tubal factor**
**Time to pregnancy**
**Spontaneous abortion**
**Fetal death due to anomalies**

## Pesticide Residues in the Male Reproductive Tract

| Chemical | 2,4-D
ng/ml (ppb) | p,p’-DDE
ng/ml (ppb) | Mirex
ng/ml (ppb) |
| --- | --- | --- | --- |
| Seminal
plasma | 29.8 ± 4.8
(n=97) | 0.39 ± 0.43
(n=25) | 0.10 ± 0.8
(n=25) |

**Notes:**
 Semen 2,4-D levels (phenoxyherbicide) (97 farmers provided semen samples near the time of mixing/applying 2,4-D; Ontario, Canada)
 Arbuckle et al., 1999 "2,4-D residues in semen of Ontario farmers". Repro Tox 13:421-429.
 Semen p,p'-DDE levels (25 Ontario residents)
 WG Foster Environ Health Perspect 103 (suppl 9):63-69, 1995.
 Semen levels of Mirex (25 Ontario residents)
 Mirex (insecticide), derivative of chlordecone = kepone, banned in 1977
 WG Foster Environ Health Perspect 103 (suppl 9):63-69, 1995

## Pesticide Residues in theFemale Reproductive Tract

|  | p,p’-DDE
(ppb) | a-HCH
(ppb) | Dieldrin
(ppb) |
| --- | --- | --- | --- |
| Follicular
(18) | 3.37 ± 0.42 | 0.34 ± 0.14 | 0.13 ± 0.13 |
| Amniotic
(41) | 0.21 ± 0.17 | 0.14 ± 0.05 | NT |
| Placenta
(9) | 4.7 ± 22.3 | 17.2 ± 62.4 | NT |
| Breast Milk
(20) | 6310 ± 5900 | 859 ± 2750 | 48.7 ± 80 |

**Notes:**
 Follicular Fluid (n = 18) (IVF patients)
 Trapp et al. 1984 Fertility and Sterility 42:146 "Pollutants in human follicular fluid.
 Amniotic Fluid (n = 53, ages 36 +/- 0.5 yr, undergoing amniocentesis; Los Angeles)
 Foster et al. "Detection of EDC in samples of second trimester human amniotic fluid". J Clinical Endocrinol Metab 2000 85:2954
 Placenta (n = 9 women experiencing stillbirths, India)
 Saxena et al 1983 "A comparison of organochlorine insecticide contents in specimens of maternal blood, placenta and umbilical cord blood from stillborn and live born cases". J Tox Environ Health 11:71.
 Breast Milk (n = 20 women at 1 mo post partum, from Pueblo Yaqui, Sonora Mexico)
 Guillette et al, 1998 Anthropological approach to the evaluation of preschool children exposed to pesticides in Mexico" Environ Health Perspect 106:347-353. 

## Goal

**Case-control study to retrospectively examine relationship between *****specific***** agricultural (occupational; residential) exposures and risk of female infertility.**

**Notes:**
The study was proposed in 1996 and initiated in 1997 to explore specific agricultural exposures and risk of female infertility.

## IRB Approval

**Study approved by Clinic IRB**
**All subjects gave *****verbal*****, informed consent**
**3.5 year recruitment period, **
**   ****6/97-2/01**

## Infertility

**Unable to achieve pregnancy or bear live child after 12 months of trying.**

## Cases and Controls

**Cases --**
**18-35 years old**
**- preg (>12 mo)**
**Infertility Care **
**   ****OB-GYN at MC and Wausau Med Ctr**
**Spouse/partner**

**Controls --**
**18-35 years old**
**+ preg (<12 mo)**
**Prenatal Care **
**   ****OB-GYN at MC and Wausau Med Ctr**
**Spouse/partner**

## Infertility Conditions

**Pituitary-hypothalamic dysfunction**
**Anovulation / tubal factor / endometriosis**
**Altered menstrual cycle**
**Abnormalities of uterus, cervix, vagina**
**Recurrent miscarriage**
**Unexplained infertility**

**Notes:**
Potential cases were identified through a review of the electronic medical records of all patients seeking infertility treatment. Patients were included in the study if they were diagnosed with female infertility due to:

    * Pituitary-hypothalamic dysfunction
    * Annovulation/tubal factor/endometriosis
    * Altered menstrual cycle length
    * Abnormalities of uterus, cervix, vagina
    * Recurrent miscarriage
    * Unexplained infertility

## Infertility Diagnoses

|  | Frequency | % |
| --- | --- | --- |
| Unexplained | 292 | 57.6 |
| Endometriosis | 116 | 22.8 |
| Anovulation | 49 | 9.7 |
| Other | 26 | 5.1 |
| Pituitary-Hypothalamic | 14 | 2.8 |
| Tubal | 7 | 1.4 |
| Preg – ectopic | 2 | 0.4 |
| Cervical-Vaginal | 1 | 0.2 |

**Notes:**
 The most common diagnosis was "unspecified" followed by endometriosis and anovulation

## Exclusions

**Couple Sterility**
**Tubal ligation**
**Hysterectomy**
**Vasectomy**
**Endometriosis**
**   ****(without mention of infertility)**

**Notes:**
Couples were excluded from the study if they reported being surgically sterile due to:

    * Tubal ligation
    * Hysterectomy
    * Vasectomy

Females with a diagnosis of endometriosis without mention of fertility were also excluded.

## Survey

**Demographics**
**Occupationa/Farm/Residential exposures**
**Pesticide Use**
**Livestock pharmaceuticals**
**Source of drinking water**
**Diet/reporductive health**
**Physical/mental stressors**
**Weight and height**
**Time reviewing exposure lists**

**Notes:**
Case and control surveys consisted of 76 questions. Interviews were conducted by telephone.

Unique features:

    * male risk factors
    * physical activity levels
    * mental well being

## Data Analysis

**Multivariate logistic regression**
**Adj OR, 95% CI**
**Confounding variables**
**Maternal level of education**
**Maternal/paternal hours of passive smoke**
**Maternal/paternal time reviewing exposure lists**
**Per capita income**

**Notes:**
Conditional logistic regression models were developed for all risk factors of interest and their potential confounding variables.

Potential cofounders included:

    * Maternal level of education
    * Maternal/paternal hours of passive smoke
    * Maternal/paternal time reviewing exposure lists
    * Per capita income

## Recruitment Numbers

|  | Screened | Eligible | Ineligible/Refused | Participation Rate |
| --- | --- | --- | --- | --- |
| Case | 1,791 | 626 | 73/231 | 58.2
(322/553) |
| Control | 822 | 558 | 45/191 | 62.7%
(322/513) |

**Notes:**
This table shows the number of cases and controls that were screened, identified as eligible, contacted and the % interviewed

Half of the story - male surveys double the number completed

Total = 475 x 2 = 950

## % Recruitment to the Fertility Risk Factor Study

## Reasons for Refusing

**Lack of interest**
**Insufficient time**
**Sensitive nature of topic**
**Involved in unstable relationship**
**Uncomfortable with phone interviews**

**Notes:**
 People refusing to participate in the study were asked three questions:

1. Why they chose to not participate

2. What was their current occupation

3. Years of schooling.

The reasons for refusing were ranked in the order of most to least frequent answers.

## Interpretation Qualifiers

**Exposures**
**“****2 years before trying to conceive”**
**Central Wisconsin population**

**Notes:**
Before I begin discussing the results, you need to understand that the findings pertain to exposures that occurred "2 years before trying to conceive", NOT for lifetime exposures or exposures that occurred while trying to conceive.

Findings pertain to the population studied – Central and Northern Wisconsinites

## Infertile Case Women

|  | OR (95% CI) |
| --- | --- |
| Work outside of home | 4.8 (2.6 - 8.5) |
| HS graduate | 1.7 (1.1 - 2.6) |
| Current smoker | 1.6 (0.9 - 2.9) |
| 1-5 hr passive smoke | 1.8 (1.2 - 2.5) |
| >1 alcoholic drink/wk | 1.8 (1.2 - 2.8) |
| Steady wt gain as adult | 3.5 (2.0 - 6.1) |
| Male partner > 41 yr | 4.5 (1.2 - 16.3) |

**Notes:**
 In comparison to control women, infertile women were more likely to report: 

## Agricultural Factors

|  | OR (95% CI) |
| --- | --- |
| Mix-apply herbicides | 26.9 (1.9 – 384.8) |
| Ever-use fungicides | 3.3 (0.8 – 13.2) |

## Pesticide Use

**Mix and apply herbicides**
**Case: unk > glyphosate > 2,4,5-T > atrazine**
**Control: unk > glyphosate > 2,4,5-T > atrazine**
**Use of fungicides by either partner**
**Case : unk > chlorothalonil > captan > **
**   ****benomyl = maneb = zineb = dicofol**
**Control: Chlorothalonil > captan = maneb = unk**

## Fertile Control Women

| Live on farm, ranch or rural home | 0.5 (0.4 – 0.8) |
| --- | --- |
| Drink Central WI groundwater | 0.6 (0.4 – 0.9) |
| Consume 3 glasses milk/day | 0.3 (0.1 – 0.7) |

## Summary of Findings

**Significant Risks**
**Mix-apply herbicides**
**Ever-use fungicides**
**Alcohol, smoke, passive smoke, steady weight gain**
**Partner’s age**
**Protective Factors**
**Residing on farm, ranch, rural area**
**Private well water for drinking**
** ******** ****3 glasses of milk per day**

## Possible Mechanisms

**Hormone signaling “endocrine” disruption**
**Age-related decline in gamete quality**
**Private well water vs. municipal water – disinfection by-products**
**Milk – no direct benefit: healthy choice; conjugated linoleic acids**

**Notes:**
Animal models and in vitro assays suggest that herbicides and fungicides can contribute to female infertility by:

    * Interfering with hormone signaling
    * Disrupting cleavage and implantation of the embryo
    * Altering chromosome integrity of the conceptus or female gametes

Recent evidence suggests that aging is associated with declines in:

    * Semen volume
    * Sperm motility
    * Sperm morphology
    * NOT sperm concentration
    * Egg quality

Controlling for day-specific probability of pregnancy:

    * Male decline begins in their late 30s
    * Female decline begins in their late 20s
    * Private well vs Municipal water

Groundwater in Central WI is threatened by land application of agrochemicals, manure spreading and faulty septic systems. However, households supplied with central WI ground water were found to be at less risk for infertility when compared with households using municipal sources. It is possible that disinfection by products (trihalomethanes, haloacetic acids) may be contributing to adverse reproductive outcomes.

Milk was of interest because of the report by Cramer et all suggesting that consuming milk into adulthood may contribute to the age-related decline in female fertility, the premise being dietary galactose may have deleterious effects on ovarian function. Our finding suggest drinking 3 or more glasses of mild per day was positively correlated with female fertility. The benefits may be related to the presence of conjugated linoleic acids, microcomponents of ruminant fat. CLA may have beneficial effects on reproduction, cancer, heart disease, immunity and growth.

## Strengths and Limitations

**Strengths**
**Females and male partners participated**
**Participation rate ~ 60%**
**Behavior can be modified**
**Limitations**
**Self-reported information**
**Individual pesticides not analyzed**
**Pesticide  exposure – subcategory of infertility**

## Future Studies

**Exposures vs. subcategories of infertility**
**Job matrix variables – protective gear**
**Intervention effectiveness - smoking**

## Publications

**Poster (ISEE, Vancouver, BC; 2002)**
**Epidemiology (July, 2003)**
**Presentations**
**Morgantown, WV, 1998**
**Cooperstown, NY, 2000**
**Little Amana, IA, 2002**
**Marshfield, WI, 2002**

## Thank You!

**NIOSH**
**MMRF**
**William R. Carl, MS, MPH**
**Elaine Eaker, ScD**
**Steve Broste, MS**
**Susan Schwabe**
**Juanita Herr**
**Deb Kempf**
**Laura Wittman**
**Lorelle Benetti**

**Participants**
**Dept OB-GYN MC/Wausau**
**Patty Schnitzer, PhD**
**Andy Olshan, PhD**
**Geoffrey M. Calvert, MD, PhD**
**Frank Baker, MD, PhD**