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Clinical Biochemistry 43 (2010) 131–135

Association between recurrent miscarriages and organochlorinepesticide 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 2009Available online 3 October 2009

Abstract

Objectives: Recurrent miscarriage (RM) is a challenging medical problem because of its unknown pathogenesis and etiology in most of thecases. Recent studies suggest the role of persistent environmental pollutants such as organochlorine pesticides (OCPs) in the etiology of RM. Thepresent 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 moreconsecutive miscarriages before 20th week of gestation. RM is achallenging medical problem because of its least knownpathogenesis and etiology in most of the cases. Both fetal andmaternal factors are found to be associated with pathophysiol-ogy of RM. Fetal factors include genetic or developmentalabnormalities, while uterine pathology, endocrine dysfunction,anti-phospholipid syndrome and thrombophilic disorders havebeen identified as maternal aspects [1]. The role of other factorslike infections, hormonal imbalances, parity, menstrual dis-orders, nutritional deficiencies, psychological trauma, stress lifeevents alcohol and caffeine intake have been studied earlier butthe results are inconsistent [2,3]. A positive association betweensmoking and occupational exposure to pesticides with increased

⁎ Corresponding author. Fax: +91 11 22590495.E-mail address: banerjeebd@hotmail.com (B.D. Banerjee).

0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemistsdoi:10.1016/j.clinbiochem.2009.09.019

risk of abortion/miscarriages has been reported in recent years[4–6]. There are also indications that exposure to pesticidesmay contribute to adverse reproductive outcomes [7]. In womenwith RM, intensive diagnostics can identify the definite causeonly in few cases [3]. Hence, further environmental factors mustbe identified that may affect pregnancy and play an etiologicalrole in the pathogenesis of recurrent miscarriages.

Recent studies suggest role of persistent environmentalpollutants such as organochlorine pesticides (OCPs) in theetiology of adverse reproductive effects [8,9]. In India,dichlorodiphenyltrichloroethane (DDT) and hexachlorocyclo-hexane (HCH) were banned for use in agriculture since 1998and 1989, respectively, but are still used for control of vectors inpublic health [10]. However, since OCPs are lipophilic in natureand have long half lives of months to years, they tend toaccumulate in adipose tissues and also biomagnify through thefood chain [11]. As a result, OCPs can still be detected inecosystem e.g., in water, soil, air, food items etc. creating apersistent exposure risk to human. Significant levels of many

. Published by Elsevier Inc. All rights reserved.

Table 1Demographical characteristics of women with RM (case) and control women.

Case (n=30) Control (n=30)

Mean±SD Mean±SD

Maternal Age (years) 25.6±1.29 25.9±1.62Maternal weight (kg) 49.39±3.44 51.5±2.74BMI (kg/m2) 19.64±2.51 19.81±2.76

Food habitsVegetarian 21 20Non-vegetarian 9 10

Drinking water supplyGovernment Source 29 28Private Source 1 2

Living styleSlum 1 1Market Area 16 18Colony 13 11

Socioeconomic statusHigh 4 5Middle 25 24Low 1 1

n=Number of samples.

132 R. Pathak et al. / Clinical Biochemistry 43 (2010) 131–135

OCPs have been found in humans body tissues like blood,placenta, amniotic fluid, and in secretions like semen, breastmilk, etc. [12-14]. OCPs can induce endocrine dysfunctions,immunological changes, oxidative stress and DNA damage andmay be associated with adverse effects [15–17]. Previousstudies have reported high levels of DDT isomers and itsmetabolites 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 possibleassociation of OCPs in the pathogenesis of RM. Thus, the levelsof OCPs were compared between women with repeatedmiscarriages and women having normal vaginal full termdelivery with live birth.

Materials and methods

Cases and controls

Thirty women (case) with a history of at least three recurrentmiscarriages before the 20th week of gestation and attendedGuru Teg Bahadur Hospital associated with University Collegeof Medical Sciences, Dilshad Garden, Delhi between 2005 and2008 were included in this study. An equal number of women(thirty) undergoing normal vaginal labor at term with live birthwere recruited in the control group. Women with hormonaldisorders (hyperprolactinemia, hyperandrogenemia, luteal in-sufficiency), uterine abnormalities (uterus fibroids, uterusbicornis, uterus subseptus), chromosomal translocation, anti-phospholipid 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 andhaving complications during pregnancy and/or delivery wereexcluded from both the groups. The spouses of these womenwere also non-diabetic with normal karyotype, normal spermcount and normal sperm morphology. The women we includedin this study were of relatively homogenous group and theywere similar in terms of demographical characteristics such asage, weight, BMI, food habits, drinking water supply, livingstyle and socioeconomic status (Table 1). We have excludedpotentially confounding factors such as women of farmingcommunities, occupational exposure to pesticides and industrialchemicals from this study. Women confirmed their participationby signing a consent form and this study was approved by theinstitutional ethical clearance committee for human research.

Collection of samples

Blood samples (1 mL) were collected in EDTA containingvial. All the tests were performed within 12 h of samplecollection.

Extraction of OCPs from blood samples and quantification

All the chemicals used in the process were of high-puritygrade. The HPLC-grade solvents were checked for any

contamination before extraction. OCP residues extractionwas done by using HPLC- grade hexane and acetone (2:1)according to method of Bush et al [18]. Blood (1 mL) wastaken in a 50-mL flask for extraction of organochlorinepesticides. Hexane (6 mL) and acetone (3 mL) were addedand the contents were shaken at room temp for 30 min in amechanical shaker. The extract was centrifuged for 10 min at2000 rpm and the clear top layer of hexane was collected in aclean test tube. The remaining portion was again extractedtwice using same process and the hexane fractions were addedto the previous solvent fractions. Clean up of the samples wasdone by column chromatography following USEPA method3620B. Elute was collected in a 100-mL beaker and hexanewas evaporated to concentrate the sample. The concentratedresidues were dissolved in hexane for further analysis.Quantification of organochlorine residue levels was doneby Perkin Elmer Gas Chromatograph (GC) equipped with63Ni selective Electron Capture Detector. The column usedwas Elite-GC DB-5, 60 meter and 0.25 mm ID. The carriergas and the make up gas was nitrogen with a 2.0-mL/minand 35-mL/min flow rate, respectively, employing the splitless mode. Final extract (1 μL) was injected at a temperatureof 170 °C with a hold time of 1 min. The temperature wasraised from 170 °C to 225 °C at a rate of 5 °C/min with ahold time of 5 min and finally from 225° to 275° at a rateof 6 °C/min with a hold time of 15 min. The total runlength was 40 min per sample.

Quantitative analysis of organochlorine residues in eachsample was affected by comparing the peak area with thoseobtained from a chromatogram of a mixed OCPs standard(Supelco, Sigma-Aldrich) of known concentration. Identifica-tion of the analytes was confirmed by spiking with known

133R. Pathak et al. / Clinical Biochemistry 43 (2010) 131–135

standards of pesticides (Supelco, Sigma-Aldrich). The detectionlimit of the detector was b0.05 pg perchloroethylene withnitrogen as a carrier gas. The detection limit of the method was4 pg/mL for each OCP. For quality control process, five bloodsamples in triplicate were spiked with a mixed standard of OCPsat 5 and 25 ng/mL. The average recoveries of fortified samplesexceeded 95%. The case and control samples were run in thesame analytical batches and further, a quality check sample wasalways run with each set of samples for pesticide analysis tomaintain accuracy. For the internal control of our measure-ments, pesticide identification was confirmed by GC-MS atCentral Pollution Control Board, New Delhi, Ministry ofEnvironmental and Forests, Government of India, under ourcollaborative research programme as per MOU guidelinesbetween two Institutes.

Statistical analysis

All the values were expressed as mean±standard deviationand 25, 50 and 75 percentile basis. The data were analyzed bypaired-sample t-test and followed by Dunnett's test for multiplecomparisons by using SPSS 17.0 software. The value ofpb0.05 was considered to denote significance. Cases andcontrol were not significantly different in terms of demograph-ical characteristics such as age, weight, food habits, BMI,socioeconomic status, drinking water supply and smokingstatus; therefore, these variables were not evaluated andadjusted further. Statistician was blinded to the study designand case status and followed through out the study.

Results

Demographical characteristics and OCP residue levels ofboth the groups (study and control) are shown in Tables 1and 2. Women with recurrent miscarriages and women withnormal full term delivery with live birth were not signi-ficantly different in terms of demographical characteristicssuch as age, weight, BMI, food habits, drinking water supply,living style and socioeconomic status (Table 1). Datapresented in Table 2 clearly show that blood samples ofwomen with RM had higher levels of α-HCH, β-HCH, γ-HCH, p,p'-DDT and p,p'-DDE in comparison to controlsubjects. However, only significant association (p=0.01) wasfound between γ-HCH levels and RM in comparison tocontrols in this study (Table 2).

Table 2Distribution and comparison of OCP levels (ng/mL) in women with RM (case) and

OCP name Case (n=30)

Mean±SD 25% 50% 75%

α-HCH 4.15±2.01 2.25 3.66 5.24β-HCH 6.61±2.05 4.69 6.98 7.58γ-HCH 6.99±3.93 4.58 6.95 10.32Total-HCH 17.75±5.37 11.69 17.96 22.18pp'-DDE 2.40±1.67 1.52 2.14 3.98pp'-DDT 1.43±2.51 0.00 0.00 1.98

⁎ Significantly different from control (pb0.05).

Discussion

The present hospital based case control study was carriedout to determine the association of OCP levels with RM. Wehave found significant difference between women with RM andcontrol subjects with respect to blood concentration of γ-HCH.Human epidemiological data on association of OCPs withmiscarriages are inconsistent and results are conflicting[3,8,19,20]. Saxena et al. [19] reported high levels of γ-HCHand other OCPs in the blood of women who had experiencedpremature delivery (n=12) and/or one time spontaneousabortion (n=8) compared with women who had undergonefull-term pregnancy. However, in their study emphasis wasgiven to premature delivery cases only and nearly eight timeincreased level of γ-HCH was found in cases (56.09±30.86 ng/mL) in comparison to subjects analyzed in thispresent study (6.99±3.93 ng/mL). Moreover, our inclusion/exclusion criteria is clinically different in terms of definerecurrent miscarriages i.e. women with minimum threerecurrent miscarriages before 20 weeks of gestation comparedto study of Saxena et al. [19] where they took samples ofwomen with single abortion. Moreover, subjects of their studywere also enrolled in the early 1980s, when HCH use in theIndia was at its peak and currently the use of γ-HCH isrestricted to public health programmes only [10,11]. India andRomania are the only countries in the world still producing γ-HCH and it is estimated that global γ-HCH usage from 1950to 2000 for agricultural, livestock, forestry, human health andother purposes amounts to around 6,00,000 tons. γ-HCH hasbeen used as a broad-spectrum insecticide for both agriculturaland non-agricultural purposes such as seed and soil treatment,foliar applications, tree and wood treatment and as anti-scabieslotion in both veterinary and human applications [21]. γ-HCHhas been found in all environmental compartments, and levelsin air, water, soil sediment, aquatic and terrestrial organismsand food have been measured in India [22,23]. Humans aretherefore being exposed to γ-HCH as demonstrated bydetectable levels in human blood, adipose tissue and breastmilk as reported earlier from our lab and other studies [24,25].

The mechanism behind the possible role of γ-HCH inadverse reproductive outcomes is poorly understood. Fetotoxic,endocrine, immunotoxic and genotoxic effects of γ-HCH maybe hypothesized for its possible association with RM. Reducedovulation rate has been observed in mature female rabbits aftersub chronically exposure of γ-HCH [26]. Moreover, γ-HCH

control women.

Control (n=30)

Mean±SD 25% 50% 75% p-value

3.09±2.84 1.26 1.35 5.22 0.095.81±3.54 2.84 5.24 8.13 0.254.65±4.09 1.96 2.94 6.22 0.01 ⁎

13.55±3.59 9.29 14.10 16.29 0.062.18±1.52 0.00 2.73 3.54 0.681.29±0.98 0.00 1.39 1.87 0.76

134 R. Pathak et al. / Clinical Biochemistry 43 (2010) 131–135

treated rabbits have been showed a significant accumulation in6-day-old embryos developing in-vivo and an increase inpreimplantation mortality [27]. Sircar and Lahiri [28] have alsoreported that after orally exposure to γ-HCH to pregnant miceduring early stage caused a total absence of any implantationsite and while administered later part of gestation it resulted indeath of all pups.

Several studies using experimental animals suggested that γ-HCH act as endocrine disruptor and showed both estrogenic andanti-estrogenic effects [15,29,30]. As an endocrine disruptor itmay be able to react directly or indirectly with hormonestructure to alter its function, change the pattern of hormonesynthesis, or modulate the number of hormone receptors andtheir affinities for specific molecules [31–33]. Furthermore, γ-HCH has been found to alter level of thyroid, pituitary and sexhormones in females [16]. It has been shown that γ-HCHsuppress follicle stimulating hormone and transforming growthfactor β1 stimulated progesterone production. Progesterone iscritical for implantation and the maintenance of humanpregnancy. Therefore, compounds that impair progesteroneproduction, increase its metabolism or block its action may alterthe homeostasis of pregnancy. γ –HCH has also been found toinhibit cytochrome P450 (CYP) enzyme level [34] andsteroidogenic acute regulatory protein (StAR) expression incultured rat granulose cells [31,32]. StAR protein mediates theintramitochondrial transfer of cholesterol to the CYP 450enzyme and CYP 450 enzyme catalyzes steroid hormonebiosynthesis [35]. Impaired level of sex hormone may interferewith the implantation and can induce fetal abnormalities. Theseobservations suggest possible mechanisms by which γ-HCHcan disrupt endocrine function and may be associated with RMas observed in our study.

γ-HCH may also cause fetal loss by altering immune systemand cellular components. Increased cytotoxic potential has beenobserved with increasing levels of γ-HCH in human bloodwhich may be directed against the fetus [16]. The production oftolerance inducing factors during early pregnancy may bedisturbed due to the activation of the immune system and thismay lead to the rejection of the fetus which immunologicallyrepresents allogenic material. Increased oxidative stress,enhanced lipid peroxidation and single strand DNA breakshave been observed in the fetal and placental tissues afterexposure to γ-HCH [36–37]. Increased DNA damage has beenreported after exposure to γ-HCH in cultured rat cells [38].Dose-dependent increase in the frequency of chromosomalaberrations and sister chromatid exchanges, followed bysignificant decrease in mitotic index have also been observedin human peripheral lymphocytes after treatment with γ-HCH[39]. The result of these studies indicates that high γ-HCHlevels may adversely affect the newborns. The teratogenicpotential of several other exogenous substances such as heavymetals [40,41], bisphenol A [42], nicotine, organophosphorusand carbamate pesticides has been reported [4–7]. The observedresults of these studies also support the hypothesis of anassociation between environmental exposure and miscarriages.

In conclusion, our study shows an association between highblood levels of γ-HCH and women with recurrent miscarriages.

This study may highlight the reproductive effects of environ-mental chemicals or xenobiotics on the “course of pregnancy”and women with a history of RM may be benefited by knowingabout their OCP burden in specialized clinics after consultationwith physician/gynecologist. However, our study has severaldependable limitations such as a small sample size. Moreover,we are also unable to determine if the association which weobserved between γ-HCH and repeated miscarriages was due toexposure of the mother during pregnancy or early childhood ofan individual that affected their subsequent reproductivedevelopment. Furthermore, we must emphasize that toxicitydepends upon numerous additional factors such as geneticpredisposition, dietary habits and contamination to otherpollutants. Hence, further studies with large sample sizeswhich examine the relationships between OCPs exposures andrecurrent miscarriages along with assessment of hormonal,genetic and immunological factors are clearly needed.

Acknowledgments

One of the authors, Rahul Pathak acknowledges to IndianCouncil of Medical Research, New Delhi for providingindividual Senior Research Fellowship support. Authors arealso thankful to Ministry of Environment and Forests (Govt. ofIndia) for sanction of research project vide no. RE-19-10-2007.

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