Association between recurrent miscarriages and organochlorine pesticide levels

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Clinical Biochemistry 43 (2010) 131 – 135

Association between recurrent miscarriages and organochlorine pesticide levels Rahul Pathak a , MD. Mustafa a , Rafat S. Ahmed a , A.K. Tripathi a , Kiran Guleria b , B.D. Banerjee a,⁎ a

Environmental Biochemistry and Immunology Laboratory, Department of Biochemistry, University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, India b Department of Obstetrics and Gynaecology, University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, India Received 28 May 2009; received in revised form 18 August 2009; accepted 22 September 2009 Available online 3 October 2009

Abstract Objectives: Recurrent miscarriage (RM) is a challenging medical problem because of its unknown pathogenesis and etiology in most of the cases. Recent studies suggest the role of persistent environmental pollutants such as organochlorine pesticides (OCPs) in the etiology of RM. The present study was conducted to investigate possible associations of OCPs in the pathogenesis of RM. Design and methods: Blood OCP levels were analyzed in women with RM (cases) and women with normal full term delivery with live birth (controls) by using a gas chromatograph equipped with an electron capture detector. Results: A statistically significant association (p = 0.01) was observed between blood γ-HCH levels and women with recurrent miscarriages. Conclusions: This study suggests that high blood levels of γ-HCH may be associated with risk of RM. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Recurrent miscarriages; Organochlorine pesticides; γ-HCH; Endocrine disruption

Introduction Recurrent miscarriage (RM) is defined as three or more consecutive miscarriages before 20th week of gestation. RM is a challenging medical problem because of its least known pathogenesis and etiology in most of the cases. Both fetal and maternal factors are found to be associated with pathophysiology of RM. Fetal factors include genetic or developmental abnormalities, while uterine pathology, endocrine dysfunction, anti-phospholipid syndrome and thrombophilic disorders have been identified as maternal aspects [1]. The role of other factors like infections, hormonal imbalances, parity, menstrual disorders, nutritional deficiencies, psychological trauma, stress life events alcohol and caffeine intake have been studied earlier but the results are inconsistent [2,3]. A positive association between smoking and occupational exposure to pesticides with increased ⁎ Corresponding author. Fax: +91 11 22590495. E-mail address: [email protected] (B.D. Banerjee).

risk of abortion/miscarriages has been reported in recent years [4–6]. There are also indications that exposure to pesticides may contribute to adverse reproductive outcomes [7]. In women with RM, intensive diagnostics can identify the definite cause only in few cases [3]. Hence, further environmental factors must be identified that may affect pregnancy and play an etiological role in the pathogenesis of recurrent miscarriages. Recent studies suggest role of persistent environmental pollutants such as organochlorine pesticides (OCPs) in the etiology of adverse reproductive effects [8,9]. In India, dichlorodiphenyltrichloroethane (DDT) and hexachlorocyclohexane (HCH) were banned for use in agriculture since 1998 and 1989, respectively, but are still used for control of vectors in public health [10]. However, since OCPs are lipophilic in nature and have long half lives of months to years, they tend to accumulate in adipose tissues and also biomagnify through the food chain [11]. As a result, OCPs can still be detected in ecosystem e.g., in water, soil, air, food items etc. creating a persistent exposure risk to human. Significant levels of many

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OCPs have been found in humans body tissues like blood, placenta, amniotic fluid, and in secretions like semen, breast milk, etc. [12-14]. OCPs can induce endocrine dysfunctions, immunological changes, oxidative stress and DNA damage and may be associated with adverse effects [15–17]. Previous studies have reported high levels of DDT isomers and its metabolites in women with spontaneous abortion [3,8,9]. However, studies regarding role of other OCPs in RM are scarce. The present study was conducted to investigate possible association of OCPs in the pathogenesis of RM. Thus, the levels of OCPs were compared between women with repeated miscarriages and women having normal vaginal full term delivery with live birth. Materials and methods Cases and controls Thirty women (case) with a history of at least three recurrent miscarriages before the 20th week of gestation and attended Guru Teg Bahadur Hospital associated with University College of Medical Sciences, Dilshad Garden, Delhi between 2005 and 2008 were included in this study. An equal number of women (thirty) undergoing normal vaginal labor at term with live birth were recruited in the control group. Women with hormonal disorders (hyperprolactinemia, hyperandrogenemia, luteal insufficiency), uterine abnormalities (uterus fibroids, uterus bicornis, uterus subseptus), chromosomal translocation, antiphospholipid syndrome, immunological causes of miscarriages, anemia, hypertension, bacterial vaginosis, TORCH infections, toxemia of pregnancy, renal disease, heart disease, diabetes, urinary track infections, metabolic disorders, tuberculosis, smoking, alcohol consumption or chronic drug intake and having complications during pregnancy and/or delivery were excluded from both the groups. The spouses of these women were also non-diabetic with normal karyotype, normal sperm count and normal sperm morphology. The women we included in this study were of relatively homogenous group and they were similar in terms of demographical characteristics such as age, weight, BMI, food habits, drinking water supply, living style and socioeconomic status (Table 1). We have excluded potentially confounding factors such as women of farming communities, occupational exposure to pesticides and industrial chemicals from this study. Women confirmed their participation by signing a consent form and this study was approved by the institutional ethical clearance committee for human research. Collection of samples Blood samples (1 mL) were collected in EDTA containing vial. All the tests were performed within 12 h of sample collection. Extraction of OCPs from blood samples and quantification All the chemicals used in the process were of high-purity grade. The HPLC-grade solvents were checked for any

Table 1 Demographical characteristics of women with RM (case) and control women. Case (n = 30)

Control (n = 30)

Mean ± SD

Mean ± SD

Maternal Age (years) Maternal weight (kg) BMI (kg/m2)

25.6 ± 1.29 49.39 ± 3.44 19.64 ± 2.51

25.9 ± 1.62 51.5 ± 2.74 19.81 ± 2.76

Food habits Vegetarian Non-vegetarian

21 9

20 10

Drinking water supply Government Source Private Source

29 1

28 2

Living style Slum Market Area Colony

1 16 13

1 18 11

Socioeconomic status High Middle Low

4 25 1

5 24 1

n = Number of samples.

contamination before extraction. OCP residues extraction was done by using HPLC- grade hexane and acetone (2:1) according to method of Bush et al [18]. Blood (1 mL) was taken in a 50-mL flask for extraction of organochlorine pesticides. Hexane (6 mL) and acetone (3 mL) were added and the contents were shaken at room temp for 30 min in a mechanical shaker. The extract was centrifuged for 10 min at 2000 rpm and the clear top layer of hexane was collected in a clean test tube. The remaining portion was again extracted twice using same process and the hexane fractions were added to the previous solvent fractions. Clean up of the samples was done by column chromatography following USEPA method 3620B. Elute was collected in a 100-mL beaker and hexane was evaporated to concentrate the sample. The concentrated residues were dissolved in hexane for further analysis. Quantification of organochlorine residue levels was done by Perkin Elmer Gas Chromatograph (GC) equipped with 63 Ni selective Electron Capture Detector. The column used was Elite-GC DB-5, 60 meter and 0.25 mm ID. The carrier gas and the make up gas was nitrogen with a 2.0-mL/min and 35-mL/min flow rate, respectively, employing the split less mode. Final extract (1 μL) was injected at a temperature of 170 °C with a hold time of 1 min. The temperature was raised from 170 °C to 225 °C at a rate of 5 °C/min with a hold time of 5 min and finally from 225° to 275° at a rate of 6 °C/min with a hold time of 15 min. The total run length was 40 min per sample. Quantitative analysis of organochlorine residues in each sample was affected by comparing the peak area with those obtained from a chromatogram of a mixed OCPs standard (Supelco, Sigma-Aldrich) of known concentration. Identification of the analytes was confirmed by spiking with known

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standards of pesticides (Supelco, Sigma-Aldrich). The detection limit of the detector was b 0.05 pg perchloroethylene with nitrogen as a carrier gas. The detection limit of the method was 4 pg/mL for each OCP. For quality control process, five blood samples in triplicate were spiked with a mixed standard of OCPs at 5 and 25 ng/mL. The average recoveries of fortified samples exceeded 95%. The case and control samples were run in the same analytical batches and further, a quality check sample was always run with each set of samples for pesticide analysis to maintain accuracy. For the internal control of our measurements, pesticide identification was confirmed by GC-MS at Central Pollution Control Board, New Delhi, Ministry of Environmental and Forests, Government of India, under our collaborative research programme as per MOU guidelines between two Institutes. Statistical analysis All the values were expressed as mean ± standard deviation and 25, 50 and 75 percentile basis. The data were analyzed by paired-sample t-test and followed by Dunnett's test for multiple comparisons by using SPSS 17.0 software. The value of p b 0.05 was considered to denote significance. Cases and control were not significantly different in terms of demographical characteristics such as age, weight, food habits, BMI, socioeconomic status, drinking water supply and smoking status; therefore, these variables were not evaluated and adjusted further. Statistician was blinded to the study design and case status and followed through out the study. Results Demographical characteristics and OCP residue levels of both the groups (study and control) are shown in Tables 1 and 2. Women with recurrent miscarriages and women with normal full term delivery with live birth were not significantly different in terms of demographical characteristics such as age, weight, BMI, food habits, drinking water supply, living style and socioeconomic status (Table 1). Data presented in Table 2 clearly show that blood samples of women with RM had higher levels of α-HCH, β-HCH, γHCH, p,p'-DDT and p,p'-DDE in comparison to control subjects. However, only significant association (p = 0.01) was found between γ-HCH levels and RM in comparison to controls in this study (Table 2).

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Discussion The present hospital based case control study was carried out to determine the association of OCP levels with RM. We have found significant difference between women with RM and control subjects with respect to blood concentration of γ-HCH. Human epidemiological data on association of OCPs with miscarriages are inconsistent and results are conflicting [3,8,19,20]. Saxena et al. [19] reported high levels of γ-HCH and other OCPs in the blood of women who had experienced premature delivery (n = 12) and/or one time spontaneous abortion (n = 8) compared with women who had undergone full-term pregnancy. However, in their study emphasis was given to premature delivery cases only and nearly eight time increased level of γ-HCH was found in cases (56.09 ± 30.86 ng/mL) in comparison to subjects analyzed in this present study (6.99 ± 3.93 ng/mL). Moreover, our inclusion/ exclusion criteria is clinically different in terms of define recurrent miscarriages i.e. women with minimum three recurrent miscarriages before 20 weeks of gestation compared to study of Saxena et al. [19] where they took samples of women with single abortion. Moreover, subjects of their study were also enrolled in the early 1980s, when HCH use in the India was at its peak and currently the use of γ-HCH is restricted to public health programmes only [10,11]. India and Romania are the only countries in the world still producing γHCH and it is estimated that global γ-HCH usage from 1950 to 2000 for agricultural, livestock, forestry, human health and other purposes amounts to around 6,00,000 tons. γ-HCH has been used as a broad-spectrum insecticide for both agricultural and non-agricultural purposes such as seed and soil treatment, foliar applications, tree and wood treatment and as anti-scabies lotion in both veterinary and human applications [21]. γ-HCH has been found in all environmental compartments, and levels in air, water, soil sediment, aquatic and terrestrial organisms and food have been measured in India [22,23]. Humans are therefore being exposed to γ-HCH as demonstrated by detectable levels in human blood, adipose tissue and breast milk as reported earlier from our lab and other studies [24,25]. The mechanism behind the possible role of γ-HCH in adverse reproductive outcomes is poorly understood. Fetotoxic, endocrine, immunotoxic and genotoxic effects of γ-HCH may be hypothesized for its possible association with RM. Reduced ovulation rate has been observed in mature female rabbits after sub chronically exposure of γ-HCH [26]. Moreover, γ-HCH

Table 2 Distribution and comparison of OCP levels (ng/mL) in women with RM (case) and control women. OCP name α-HCH β-HCH γ-HCH Total-HCH pp'-DDE pp'-DDT ⁎

Case (n = 30)

Control (n = 30)

Mean ± SD

25%

50%

75%

Mean ± SD

25%

50%

75%

p-value

4.15 ± 2.01 6.61 ± 2.05 6.99 ± 3.93 17.75 ± 5.37 2.40 ± 1.67 1.43 ± 2.51

2.25 4.69 4.58 11.69 1.52 0.00

3.66 6.98 6.95 17.96 2.14 0.00

5.24 7.58 10.32 22.18 3.98 1.98

3.09 ± 2.84 5.81 ± 3.54 4.65 ± 4.09 13.55 ± 3.59 2.18 ± 1.52 1.29 ± 0.98

1.26 2.84 1.96 9.29 0.00 0.00

1.35 5.24 2.94 14.10 2.73 1.39

5.22 8.13 6.22 16.29 3.54 1.87

0.09 0.25 0.01 ⁎ 0.06 0.68 0.76

Significantly different from control (p b 0.05).

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treated rabbits have been showed a significant accumulation in 6-day-old embryos developing in-vivo and an increase in preimplantation mortality [27]. Sircar and Lahiri [28] have also reported that after orally exposure to γ-HCH to pregnant mice during early stage caused a total absence of any implantation site and while administered later part of gestation it resulted in death of all pups. Several studies using experimental animals suggested that γHCH act as endocrine disruptor and showed both estrogenic and anti-estrogenic effects [15,29,30]. As an endocrine disruptor it may be able to react directly or indirectly with hormone structure to alter its function, change the pattern of hormone synthesis, or modulate the number of hormone receptors and their affinities for specific molecules [31–33]. Furthermore, γHCH has been found to alter level of thyroid, pituitary and sex hormones in females [16]. It has been shown that γ-HCH suppress follicle stimulating hormone and transforming growth factor β1 stimulated progesterone production. Progesterone is critical for implantation and the maintenance of human pregnancy. Therefore, compounds that impair progesterone production, increase its metabolism or block its action may alter the homeostasis of pregnancy. γ –HCH has also been found to inhibit cytochrome P450 (CYP) enzyme level [34] and steroidogenic acute regulatory protein (StAR) expression in cultured rat granulose cells [31,32]. StAR protein mediates the intramitochondrial transfer of cholesterol to the CYP 450 enzyme and CYP 450 enzyme catalyzes steroid hormone biosynthesis [35]. Impaired level of sex hormone may interfere with the implantation and can induce fetal abnormalities. These observations suggest possible mechanisms by which γ-HCH can disrupt endocrine function and may be associated with RM as observed in our study. γ-HCH may also cause fetal loss by altering immune system and cellular components. Increased cytotoxic potential has been observed with increasing levels of γ-HCH in human blood which may be directed against the fetus [16]. The production of tolerance inducing factors during early pregnancy may be disturbed due to the activation of the immune system and this may lead to the rejection of the fetus which immunologically represents allogenic material. Increased oxidative stress, enhanced lipid peroxidation and single strand DNA breaks have been observed in the fetal and placental tissues after exposure to γ-HCH [36–37]. Increased DNA damage has been reported after exposure to γ-HCH in cultured rat cells [38]. Dose-dependent increase in the frequency of chromosomal aberrations and sister chromatid exchanges, followed by significant decrease in mitotic index have also been observed in human peripheral lymphocytes after treatment with γ-HCH [39]. The result of these studies indicates that high γ-HCH levels may adversely affect the newborns. The teratogenic potential of several other exogenous substances such as heavy metals [40,41], bisphenol A [42], nicotine, organophosphorus and carbamate pesticides has been reported [4–7]. The observed results of these studies also support the hypothesis of an association between environmental exposure and miscarriages. In conclusion, our study shows an association between high blood levels of γ-HCH and women with recurrent miscarriages.

This study may highlight the reproductive effects of environmental chemicals or xenobiotics on the “course of pregnancy” and women with a history of RM may be benefited by knowing about their OCP burden in specialized clinics after consultation with physician/gynecologist. However, our study has several dependable limitations such as a small sample size. Moreover, we are also unable to determine if the association which we observed between γ-HCH and repeated miscarriages was due to exposure of the mother during pregnancy or early childhood of an individual that affected their subsequent reproductive development. Furthermore, we must emphasize that toxicity depends upon numerous additional factors such as genetic predisposition, dietary habits and contamination to other pollutants. Hence, further studies with large sample sizes which examine the relationships between OCPs exposures and recurrent miscarriages along with assessment of hormonal, genetic and immunological factors are clearly needed. Acknowledgments One of the authors, Rahul Pathak acknowledges to Indian Council of Medical Research, New Delhi for providing individual Senior Research Fellowship support. Authors are also thankful to Ministry of Environment and Forests (Govt. of India) for sanction of research project vide no. RE-19-10-2007. References [1] Szekeres-Bartho J, Balasch J. Progesterone therapy for recurrent miscarriage. Hum Reprod Updat 2008;14(1):27–35. [2] Pandey MK, Rani R, Agrawal S. An update in recurrent spontaneous abortion. Arch Gynecol Obstet 2005;272(2):95–108. [3] Korrick SA, Chen C, Damokosh AI, et al. Association of DDT with spontaneous abortion: a case-control study. Ann Epidemiol 2001;11(7): 491–6. [4] Shea AK, Steiner M. Cigarette smoking during pregnancy. Nicotine Tob Res 2008;10(2):267–78. [5] Blanco-Muñoz J, Torres-Sánchez L, López-Carrillo L. Exposure to maternal and paternal tobacco consumption and risk of spontaneous abortion. Public Health Rep 2009;124(2):317–22. [6] Settimi L, Spinelli A, Lauria L, et al. Spontaneous abortion and maternal work in greenhouses. Am J Ind Med 2008;51(4):290–5. [7] Hanke W, Jurewicz J. The risk of adverse reproductive and developmental disorders due to occupational pesticide exposure: an overview of current epidemiological evidence. Int J Occup Med Environ Health 2004;17(2): 223–43. [8] Venners SA, Korrick S, Xu X, et al. Preconception serum DDT and pregnancy loss: a prospective study using a biomarker of pregnancy. Am J Epidemiol 2005;162(8):709–16. [9] Longnecker MP, Klebanoff MA, Dunson DB, et al. Maternal serum level of the DDT metabolite DDE in relation to fetal loss in previous pregnancies. Environ Res 2005;97(2):127–33. [10] Kumar A, Dayal P, Shukla G, Singh G, Joseph PE. DDT and HCH residue load in mother's breast milk: a survey of lactating mother's from remote villages in Agra region. Environ Int 2006;32(2):248–51. [11] Siddiqui MK, Srivastava S, Srivastava SP, Mehrotra PK, Mathur N, Tandon I. Persistent chlorinated pesticides and intra-uterine foetal growth retardation: a possible association. Int Arch Occup Environ Health 2003;76(1):75–80. [12] Foster W, Chan S, Platt L, Hughes C. Detection of endocrine disrupting chemicals in samples of second trimester human amniotic fluid. J Clin Endocrinol Metab 2000;85(8):2954–7.

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