Risk Factors for Female Infertility in an Agricultural Region

  • Greenlee, Anne R.;
  • Arbuckle, Tye E.;
  • Chyou, Po
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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 Controls
18-35 years old 18-35 years old
- preg (>12 mo)
 + preg (<12 mo)
Infertility Care OB-GYN at MC and Wausau Med Ctr Prenatal Care OB-GYN at MC and Wausau Med Ctr
Spouse/partner 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
  • Occupational/Farm/Residential exposures
  • Pesticide Use
  • Livestock pharmaceuticals
  • Source of drinking water
  • Diet/reproductive 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 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
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

1. Marshfield Medical Research Foundation, Marshfield, WI
2. Health Canada, Ottawa, Ontario

Disclaimer and Reproduction Information: Information in NASD does not represent NIOSH policy. Information included in NASD appears by permission of the author and/or copyright holder. More

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