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Reproductive Toxicology 42 (2013) 116– 124

Contents lists available at ScienceDirect

Reproductive Toxicology

jo ur nal home p age: www. elsev ier .com/ locate / reprotox

arkers of oxidative stress in follicular fluid of womenith endometriosis and tubal infertility undergoing IVF

bhay K. Singha, Ratna Chattopadhyayb, Baidyanath Chakravartyb, Koel Chaudhurya,∗

School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, IndiaInstitute of Reproductive Medicine, Salt Lake, Kolkata, West Bengal, India

r t i c l e i n f o

rticle history:eceived 19 August 2012eceived in revised form 8 August 2013ccepted 16 August 2013vailable online 29 August 2013

eywords:

a b s t r a c t

Oxidative stress and trace elements in the oocytes environment is explored in endometriosis and impacton in vitro fertilization (IVF) outcome assessed. Follicular fluid was aspirated at the time of oocyte retrievalfrom endometriosis (n = 200) and tubal infertility (n = 140) and the analytes measured using spectroscopyand HPLC. Increased concentration of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation(LPO), iron, lead, cadmium and reduced levels of total antioxidant capacity (TAC), superoxide dismutase(SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR), vitamins A, C, E, copper, zincand selenium was observed compared to tubal infertility. Increased ROS and NO in endometriosis and

xidative stressrace elementsndometriosisollicular fluidn vitro fertilization (IVF)ocyte qualityregnancy outcome

tubal infertility associated with poor oocytes and embryo quality. Increased levels of ROS, NO, LPO, cad-mium and lead were observed in women who did not become pregnant compared to women who did.Intrafollicular zinc levels were higher in women with endometriosis who subsequently became pregnantfollowing IVF.

© 2013 Elsevier Inc. All rights reserved.

. Introduction

Endometriosis is defined as the presence of functional endome-rial glands and stroma outside the uterine cavity. Clinically, onef the major concerns about endometriosis is its tendency to causenfertility. It is suggested that altered oocyte growth and matura-ion resulting in poor embryo quality with a decreased potentialo implant is one of the key causes for low in vitro fertiliza-ion (IVF) success rate in these women [1,2]. Oxidative stress isnvolved in the etiology of defective oocyte development [3]. Ourarlier report has shown that significantly increased reactive oxy-en species (ROS), high lipid peroxidation (LPO) and decreased totalntioxidant capacity (TAC) in follicular fluid correlate with poorocyte and embryo quality and low fertilization rate [4]. We alsobserved excessive ROS generation, depolarization of mitochon-rial membrane potential and significant increase in DNA damage

n granulosa cells of women with endometriosis as compared with

ontrols [5].

Follicular fluid forms the biochemical micro-environmentf the oocyte before ovulation and assists in estimating the

∗ Corresponding author at: School of Medical Science and Technology, Indiannstitute of Technology, Kharagpur, West Bengal 721302, India.el.: +91 9434341334/3222 283573; fax: +91 3222 282221.

E-mail addresses: koeliitkgp@gmail.com, koel@smst.iitkgp.ernet.inK. Chaudhury).

890-6238/$ – see front matter © 2013 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.reprotox.2013.08.005

developmental competence of female gametes [6]. It is rich in low-molecular weight metabolites that are direct or indirect regulatorsof oxidative stress and antioxidant production [7]. Low concen-trations of intrafollicular ROS are regarded as a promising markerfor predicting IVF success [8]. It is believed that alterations in NO,a prime factor for nitrosative stress [9], can disturb oocyte mat-uration and folliculogenesis, thereby influencing oocyte quality[10]. Abnormally high concentrations of free radicals disrupt thebalance between oxidants and antioxidants, promoting oxidativestress conditions [11]. In order to protect the follicles from oxida-tive insult, follicular fluid is naturally provided with an efficientantioxidant system [12] and trace elements, which are major com-ponents of antioxidant enzymes [13]. We have observed earlier thatthe chances of retrieving good quality oocytes increases consider-ably with higher total antioxidant capacity (TAC) levels in follicularfluid [4].

Several research groups have concluded that the oxidant–antioxidant balance in the oocytes environment can have a signifi-cant impact on IVF outcome in women with endometriosis [14–16].However, the definitive role of trace elements in these womenremains largely unknown. To test our hypothesis that a relation-ship exists between oxidative stress and trace elements in follicular

fluid of infertile endometriosis women, ROS, LPO, TAC, enzymaticand non-enzymatic antioxidants, and various trace elements areevaluated. Further, the possible role of these parameters on IVFoutcome is investigated.

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. Materials and methods

.1. Subject selection

This study was conducted in a tertiary care hospital, the Institutef Reproductive Medicine, Kolkata, India. Approval was obtainedrom the Institutional Research Ethics Board (date of approval:8/01/2007). Written informed consent was taken from all cou-les participating in this study. A part of the study was done at theeproductive Health Research Unit, School of Medical Science andechnology, IIT, Kharagpur, India.

A total of 340 infertile women (26–40 years) withMI < 25 kg/m2 and duration of infertility >2 years undergo-

ng IVF were recruited from February 2010 to December 2011.ut of these, 200 women with endometriosis (Stages III and IV)ere included as the study group. Endometriosis was confirmed

y laparoscopy followed by biopsy and staging done using theevised American Society for Reproductive Medicine guidelines17]. For controls, 140 normal ovulating women with tubal factornfertility (without endometriosis) were considered. Baselineollicle-stimulating hormone (FSH), luteinizing hormone (LH) andstradiol levels in serum and various IVF outcome parametersere assessed in both the groups.

Tubal infertility, in this study, refers to women who had fal-opian tube(s) removed for tubal pregnancy and proximal tubalbstruction because of low-grade infection or fimbrial occlu-ion with or without mild peritubal adhesion. Tubal infertilityssociated with gross hydrosalpingeal changes, dense pelvic adhe-ions because of endometriosis or pelvic inflammatory diseasesere excluded. Women with adenomyosis, severe pelvic adhe-

ions, thyroid disorders, hypoprolactemia, diabetes mellitus andardiovascular diseases were excluded. Only those women wereonsidered whose partners/donors were normozoospermic withemen parameters normal as per WHO guidelines [18]. It was alsonsured that women included had not received any medicationther than the standard IVF protocol since the last 3 months.

.2. Ovarian stimulation protocols

Ovarian stimulation was performed using the down-regulationrotocol with a gonadotropin-releasing hormone (GnRH) agonist.riefly, the patients were down-regulated with a GnRH agonistLupride, Sun Pharmaceuticals, Mumbai, India) mid-luteal phasenwards and, when optimally down-regulated, were stimulatedith recombinant FSH (Gonal F, Serono, Geneva, Switzerland).

ollicular size was monitored regularly by ultrasound and serumstradiol assays. Subcutaneous human chorionic gonadotropinhCG) (Pregnyl, Organon, the Netherlands) was administered whenverage diameter of the leading follicles reached at least 18 mm.he oocytes, following retrieval under transvaginal ultrasounduidance, were graded and subsequently inseminated. Embryouality was assessed before embryo-transfer, and a maximum ofhree embryos transferred to all patients approximately 48 h (4–8ell stage) after insemination. Clinical pregnancy was defined ashe presence of a gestational sac with cardiac activity as detectedy transvaginal ultrasound at 7 weeks of gestation.

.3. Grading of oocytes and embryos

Oocyte and embryo quality were assessed by the clinical embry-logists using Veeck’s criteria [19]. Briefly, grade I oocytes areefined as very immature or prophase I oocytes having tightly

acked cumulus, granular ooplasm and presence of germinalesicle. Grade II oocytes are immature or metaphase I oocytes,haracterized by tightly apposed corona cells, compacted cumu-us, more homogeneous ooplasm and absence of germinal vesicle

icology 42 (2013) 116– 124 117

or first polar body. Grade III oocytes or mature or metaphase IIoocytes are characterized by well expanded cumulus, sun-burstappearance of the corona, first polar body in perivitelline space andclear ooplasm. Post mature or grade IV oocytes were not includedin this study. After grading, oocyte–cumulus–corona complex wereselected for insemination with freshly prepared swim-up sper-matozoa of the male partner. Fertilization was assessed 14–16 hpost insemination. Fertilization was considered normal when theoocytes contained 2 pronuclei (PN). Fertilization rate was definedas the total number of fertilized oocytes/total number of oocytesinseminated.

Grade I embryos refer to even, regular, spherical, equal sizedblastomeres with moderate refractility and no/minimal fragmen-tation. Grade II embryos are characterized by uneven, irregularand unequal sized blastomeres with mild variation in refractilityand fragmentation <10%. Grade III embryos are associated with<50% fragmentation rate, defects in cytoplasm and remaining blas-tomeres with refractility associated with cell viability.

For comparison purposes, the retrieved oocytes were dividedinto two groups: the first group consisted of MII or mature oocytes(Grade III), while the other group comprised of immature or poorquality oocytes (Grades I and II). Embryos were also classified intogood (Grades I and II) and poor quality (Grade III) embryos in a simi-lar manner. The retrieved oocytes were assessed and correspondingembryos followed-up and cultured individually.

2.4. Collection and preparation of follicular fluid

A total of 840 follicular fluid samples from endometriosis(n = 200) and 686 samples from tubal infertility (n = 140) was care-fully aspirated during oocytes retrieval and collected in individualsterile containers. Follicular fluid was not collected from folli-cles <17 mm in diameter. Follicular fluid containing more thanone oocyte/no oocyte or contaminated with blood/media wereexcluded. Samples were centrifuged at 800 × g for 10 min to removecellular components; the clear supernatant divided into aliquotsand was frozen at −70 ◦C until further use. One part of the sam-ple was used immediately for ROS and NO measurement and theremaining part assessed for the trace elements and other oxidativestress parameters. The measured values were averaged for all thefollicles obtained from a given patient.

Protein concentration in follicular fluid was determined by theBradford method using the principle of reduction of Coomassie Bril-liant Blue G-250 dye (GeNeiTM Protein Estimation Kit, BangaloreGenei, India). Bovine serum albumin (BSA) was used as the proteinstandard.

2.5. Determination of oxidative stress parameters

2.5.1. Measurement of ROS generationIt was measured in freshly aspirated follicular fluid by chemi-

luminescence assay [20] using luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) as a probe. 400 �l of clear supernatant wasplaced in the cuvette of the luminometer (Sirius single tube, modelno. 0727, Berthold Technologies, Bad Wildbad, Germany), and 10 �lluminol (5 mM) in DMSO added to it. Each sample was scanned for10 min. ROS values were expressed as counted photons per second(cps).

2.5.2. Measurement of NOIt was measured in the follicular fluid as nitrite/nitrate, by

the Griess reaction [21]. In this assay, 100 �l of follicular fluid

was mixed with an equal volume of the Griess reagent (0.1% (1-naphthyl) ethylene diamine dihydrochloride, 1% sulfanilamide, and2.5% phosphoric acid). The resulting absorbance was measured at550 nm in a microplate reader (Victor X, Perkin Elmer, Boston,

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SA). The background was determined in each experiment utiliz-ng medium incubated without samples. The amount of NO in eachample was determined using a standard curve generated withnown concentrations of NO and its concentration expressed asM.

.5.3. Measurement of LPOMalonaldehyde (MDA) was measured using thiobarbituric acid

TBA) method to express LPO [22]. MDA reacts with TBA to give aed compound, which has an absorbance at 535 nm. Briefly, 1 mlliquot of frozen sample was thawed and immediately used forhe estimation. 2 ml of stock reagent (12% (w/v) trichloroaceticcid, 0.375% (w/v) TBA and 0.25 M HCl) was mixed thoroughlyith 1 ml of the sample and heated for 15 min in a boiling water

ath. After cooling, the flocculent precipitate was removed by cen-rifugation at 1000 × g for 10 min and the optical density of theupernatant determined at 535 nm using UV/vis spectrophotome-er (model JASCO V-630, Perkin Elmer, Boston, USA). The valuesere expressed as �M MDA.

.5.4. Measurement of TACIt was measured in the follicular fluid using a modified enhanced

hemiluminescence assay [23]. Follicular fluid was diluted 1:10ith deionized water. Signal reagent was prepared using a chemi-

uminescence kit (GE Healthcare, UK). A constant peak of ROSas produced using horseradish peroxidase (HRP) conjugated IgG

Santa Cruz Biotechnology, Santa Cruz, CA). The luminometer waset in the kinetic mode and 900 �l reaction mixture added tohe cuvette (100 �l signal reagent + 700 �l deionized water + 100 �l:350 diluted HRP) to have the desired chemiluminescence output.fter 100 s, 50 �l of diluted follicular fluid was added to the mixturend 10% recovery of chemiluminescence recorded. Trolox (6-ydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a wateroluble tocopherol analog, was used as a standard. The antioxidantapacity was expressed in �M Trolox equivalents.

.6. Determination of enzymatic and non enzymatic antioxidants

.6.1. SOD assayThe activity was measured by the inhibition of pyrogallol autox-

dation at 420 nm using UV/vis spectrophotometer for 3 min, asescribed earlier [24]. The enzyme activity was expressed asIU/mg protein, where 1 U is the amount of enzyme required to

ring about 50% inhibition of the autoxidation of pyrogallol. Onenit of SOD is described as the amount of enzyme required to cause0% inhibition of pyrogallol auto-oxidation per ml of assay mixture.

.6.2. Catalase assayThe activity was measured by spectrophotometric method [25].

he reduction of H2O2 at 240 nm was recorded on a UV/vispectrophotometer at 25 ◦C. The amount of enzyme activity thatecomposed 1 mmol of H2O2 per minute was defined as one unitf activity. Specific activities were expressed as mIU/mg protein.

.6.3. Glutathione peroxidase (GPx) assayQuantification of GPx activity was done by the method of Car-

one et al. (2003) [25]. The activity of the Se-dependent GPx waseasured with H2O2 (0.25 mM) as substrate. The oxidation ofADPH was monitored at 25 ◦C on a UV/vis spectrophotometer at40 nm. One unit of enzyme activity was defined as 1 mmole oflutathione (GSH) oxidized/min at 25 ◦C. Specific activities werexpressed as mIU/mg protein.

.6.4. Glutathione reductase (GR) assayThe activity was measured as previously described [25]. The

ate of NADPH oxidation was monitored at 25 ◦C with a UV/vis

icology 42 (2013) 116– 124

spectrophotometer. One unit of enzyme activity was defined as1 mmol of NADPH oxidized/min at 25 ◦C. Specific activities wereexpressed as mIU/mg protein.

2.6.5. Vitamins assayVitamins A, C and E were analyzed by reverse phase HPLC

[26,27]. Briefly, for the detection of vitamin A and E, 300 �l fol-licular fluid was mixed with equal volumes of ethyl alcohol andextracted twice in hexane. The supernatants were removed andevaporated under nitrogen and reconstituted in 300 �l of mobilephase (60% acetonitrile, 25% methanol and 15% ethylene chloride)and injected into the HPLC system (Agilent 1100 series, California,USA) with a Hypersil ODS column (4.6 mm × 250 mm, particle size5 �m). Vitamin A (Retinyl acetate, Sigma–Aldrich, St. Louis, MO,USA) and E (Alpha-tocopherol acetate, Sigma–Aldrich, USA) stan-dards were prepared at 10 mg/ml in methyl alcohol. For vitamin Cdetection, 300 �l of follicular fluid was mixed with 600 �l hexane,vortexed and then centrifuged at 2800 × g for 5 min. Next, 150 �lof ethanol:methanol (95:5, v/v) was added to it and centrifuged at1500 × g for 15 min. The lower layer (aqueous phase) was collectedand evaporated under nitrogen. After re-suspension in water and0.01% trifluoroacetic acid (TFA), 10 �l of the sample was analyzedby HPLC using a Zorbax eclipse C18 column (4.6 mm × 150 mm, par-ticle size 5 �m). The mobile phase consisted of 0.01% TFA (pH = 2.9)and methanol. Vitamin C standard (Ascorbic acid, Sigma–Aldrich,USA) was prepared in ultrapure water containing 0.01% of TFA. Theflow rate and column temperature was set at 1.0 mL/min and 30 ◦C,respectively. Absorbance data were obtained at 325 nm, 295 nmand 245 nm for vitamin A, E and C, respectively. Concentrationswere expressed in �g/l or mg/l. The inter- and intra-assay coef-ficients of variation were less than 5% and 8%, respectively, in alldeterminations.

2.7. Trace elements determination

Levels of copper, zinc, iron, selenium, lead and cadmium weredetermined by atomic absorption spectrophotometer (Varian AA-880, Techtron Pty. Ltd., Melbourne, Australia) using acetylene flamelamps. The analysis wavelengths used were 324.8, 213.9, 248.3,196.0, 283.3 and 228.8 nm, respectively. Stock standards were pur-chased from Merck, KGaA, Darmstadt, Germany and Milli-Q waterwas used to prepare all solutions. All other reagents were pur-chased from Sigma–Aldrich or Merck and were of analytical grade.The trace elements were determined after digestion with nitricacid and hydrogen peroxide. Briefly, one volume of follicular fluid(50 ml) was mixed with two volumes of aqueous 2% (v/v) TritonX-100 solution for 30 s and then centrifuged at 3000 × g for 4 min.The clear supernatant was used for analysis. Selenium was ana-lyzed by the hydride technique [28]. Briefly, follicular fluid (50 ml)were treated with an equal volume of HCl followed by heating at90 ◦C for 20 min. Palladium solution (200 mg/l) as matrix modifierand 0.2% NaBH4 in 0.05% NaOH as reducing agent were used forselenium analysis. A standard curve was constructed for each traceelement. The limit of quantification of this procedure was 0.041,0.09, 0.03, 0.023, 0.015, 0.001 �g/l for copper, zinc, iron, selenium,lead and cadmium, respectively. The concentration of each samplewas calculated as the mean of three repetitions and was expressedas �g/l.

2.8. Statistical analyses

GraphPad Prism (GraphPad Software, Inc., USA) and Graph

Pad QuickCalcs were used for statistical analysis. All data werepresented as mean ± standard deviation (SD) and median-range,wherever appropriate. All data were assessed for normality of dis-tribution using the Shapiro–Wilk test. Comparison between groups

A.K. Singh et al. / Reproductive Toxicology 42 (2013) 116– 124 119

Table 1Clinical characteristics and IVF outcome parameters of women with endometriosisand tubal infertility group.

Parameters Endometriosis;n = 200

Tubal infertility;n = 140

Age (years) 32.3 ± 2.98 31.7 ± 3.61BMI (kg/m2) 22.2 ± 2.67 21.6 ± 3.27Duration of infertility (years) 7.1 ± 2.28 6.7 ± 1.98FSH (mIU/ml) 6.5 ± 1.65 6.3 ± 1.75LH (mIU/ml) 5.4 ± 1.32 5.2 ± 0.98Estradiol (pg/ml) 48.3 ± 5.54 47.6 ± 4.54No. of follicles aspirated 8.4 ± 2.63 10.0 ± 2.81a

No. of oocytes retrieved 7.4 ± 2.67 8.3 ± 3.72b

Metaphase II (M II) oocyte 5.2 ± 1.63 6.8 ± 1.85a

Fertilization rate (%) 73.2 ± 17.62 78.6 ± 18.45b

Grade I & II embryos formed 2.28 ± 1.2 3.32 ± 1.3a

Number of embryo transferred 2.2 ± 0.6 2.3 ± 0.8Pregnancy rate (%) 34 % (68/200) 40.7% (57/140)Miscarriage rate (%) 16 % (32/200) 12.1% (17/140)

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Table 2Levels oxidative stress parameters and antioxidants (enzymatic and non-enzymatic)in follicular fluid of women with endometriosis and tubal infertility group.

Parameters Endometriosis;n = 200

Tubal infertility;n = 140

NO (�M) 41.61 ± 3.89 36.92 ± 4.12ROS (cps) 101.23 ± 10.52 96.37 ± 11.83MDA (�M) 0.92 ± 0.23 0.68 ± 0.22TAC (�M Trolox equivalent) 658.32 ± 56.52 896.25 ± 78.47SOD (mIU/mg protein) 31.21 ± 4.12 45.37 ± 3.81Catalase (mIU/mg protein) 10.36 ± 2.14 19.56 ± 3.28GPx (mIU/mg protein) 0.82 ± 0.21 2.14 ± 0.43GR (mIU/mg protein) 0.08 ± 0.006 0.2 ± 0.1Vitamin A (�g/l) 3.12 ± 0.16 4.13 ± 0.14Vitamin C (mg/l) 4.21 ± 0.13 5.52 ± 0.17Vitamin E (mg/l) 9.53 ± 0.17 12.53 ± 0.19

Abbreviations: NO, nitric oxide; ROS, reactive oxygen species; MDA, malondialde-

ean ± SD.P ≤ 0.001; bP ≤ 0.01; others not significant.

as carried out using Student’s t-test, chi-square test, one-wayNOVA with Bonferroni correction, Kruskal–Wallis test and Spear-an correlation coefficient as applicable. Statistical significanceas defined as P ≤ 0.05.

For analysis of the effect of ROS and NO levels on oocyte andmbryo quality, the results were expressed as median and range,nd significance of the differences between sub-groups calculatedsing the Kruskal–Wallis test. To determine the ROS levels in fol-

icular fluid that can discriminate between good quality and pooruality oocytes (best relation between sensitivity and specificity)e applied the receiver operating characteristic (ROC) curve (Med-alc Software, Belgium). To construct the ROC curves, we took intoccount only two categories of oocytes: good quality and poor qual-ty oocytes. They were constructed by plotting the false positiveate or (1 – specificity) on the x-axis and the true positive rate orensitivity on the y-axis. The calculation of the area under the ROCAUC–ROC) curve measures the accuracy, i.e. the ability of the ROSalue to discriminate between good and poor quality oocytes.

. Results

.1. Demographic values

The demographic values and IVF outcome parameters ofndometriosis and tubal infertility are shown in Table 1. No sig-ificant differences were observed in mean age, BMI, duration of

nfertility, serum levels of FSH, LH and estradiol on comparingoth the groups. Total number of follicular fluid aspirated, oocytesetrieved and number of MII oocytes, fertilization rate and grade

and II embryos formed were significantly less in women withndometriosis as compared with controls (P < 0.01). There was noignificant difference between pregnancy rates in the two groups34% vs 40.7%).

.2. ROS, MDA and NO

Concentrations of ROS, MDA and NO in follicular fluid are pre-ented in Table 2. A significant increase in the expression of theseolecules was observed in endometriosis as compared with con-

rols (P < 0.001). Box and whisker plots depicting the association ofntrafollicular ROS and NO levels with oocyte and embryo quality

re shown for endometriosis (Fig. 1) and tubal infertility (Fig. 2).he concentration of ROS and NO were found to be significantlyigher in follicular fluid corresponding to immature oocytes andoor quality embryos (P < 0.001) for both the groups.

hyde; TAC, total antioxidant capacity; SOD, superoxide dismutase; GPx, glutathioneperoxidise; GR, glutathione reductase.All values are significant, P < 0.001.

NO, ROS and MDA were found to be significantly less in pregnantwomen as compared with the non-pregnant cases of endometriosis(P < 0.001). The women with tubal infertility also exhibited a sim-ilar trend (P < 0.001). On comparing the pregnant women of boththe groups, these levels were significantly higher in endometriosis(P < 0.001).

3.3. ROC curve analysis

The accuracy of follicular fluid ROS level in discriminatingbetween mature and immature oocytes by ROC curve analysisis indicated in Fig. 3. In endometriosis, ROS level ≤105.36 cpsprovided maximum discrimination with 73.68% sensitivity and89.74% specificity for mature oocytes. Further, the positive andnegative predictive values were found to be 75.47% and 88.83%,respectively in these women (Fig. 3a). Similarly, in tubal infer-tility, ROS ≤100.96 cps was found to have 78.08% sensitivity and79.52% specificity. The positive and negative predictive values ofROS ≤100.96 cps were 71.76% and 84.47%, respectively in tubalinfertility (Fig. 3b). The area under the curve (AUC) for ROS inendometriosis (0.837) was found to be comparable with controls(0.811).

3.4. Enzymatic antioxidants

There was a marked decrease of TAC in follicular fluid ofendometriosis as compared with controls (P < 0.001). A significantdecrease in levels of enzymatic antioxidants (SOD, catalase, GPxand GR) was also observed (P < 0.001). These findings are summa-rized in Table 2. TAC and enzymatic antioxidant levels were foundto be comparable between the pregnant and non-pregnant womenof endometriosis. A similar trend was also observed in tubal infertil-ity. These levels were observed to be significantly less in pregnantwomen with endometriosis as compared with pregnant womenwith tubal infertility (P < 0.001).

3.5. Vitamins and trace elements

Levels of vitamins A, C and E in follicular fluid of endometriosisand tubal infertility are shown in Table 2. A significant decrease wasobserved in endometriosis as compared with controls (P < 0.001).No significant differences were seen in the vitamin levels on

comparing the pregnant women with non pregnant cases inendometriosis. The same trend was also observed in tubal infer-tility. Both, pregnant and non-pregnant endometriotic women hada significantly lower level of vitamin concentration when compared

120 A.K. Singh et al. / Reproductive Toxicology 42 (2013) 116– 124

Fig. 1. Box and whisker plots depicting the association of oocyte and embryo quality with the levels of ROS and NO in follicular fluid of women with endometriosis (n = 200).Follicular fluid concentrations of ROS of (a) follicles containing mature and immature oocytes (b) Grade I and II and grade III embryos. Similarly, follicular fluid concentrationsof NO of (c) follicles containing mature and immature oocytes and (d) Grade I and II and grade III embryos. The box plot is constructed from five values: the smallest value,first quartile, median, third quartile and maximum values (Box and whisker plots, **P < 0.001).

Fig. 2. Box and whisker plots depicting the association of oocyte and embryo quality with the levels of ROS and NO in follicular fluid of women with tubal infertility (n = 140).Follicular fluid concentrations of ROS of (a) follicles containing mature and immature oocytes (b) Grade I and II and grade III embryos. Similarly, follicular fluid concentrationsof NO of (c) follicles containing mature and immature oocytes and (d) Grade I and II and grade III embryos. The box plot is constructed from five values: the smallest value,first quartile, median, third quartile and maximum values (Box and whisker plots, **P < 0.001).

A.K. Singh et al. / Reproductive Toxicology 42 (2013) 116– 124 121

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ubal infertility group. In endometriosis, ROS cut-off value was 105.36 cps with 73.600.96 cps with 78.08% sensitivity and 79.52% specificity. The AUC for ROS in endom

ith respective pregnant and non-pregnant tubal infertility casesP < 0.001).

Levels of various intrafollicular trace elements are summarizedn Fig. 4. Selenium, zinc and copper levels were found to be signif-cantly less in endometriosis cases as compared to women withubal infertility (P < 0.001). In contrast, iron, lead and cadmiumere significantly higher in the endometriosis group of women

P < 0.001). In both endometriosis and tubal infertility groups, sele-ium, copper and iron levels did not vary significantly on comparinghe pregnant with non-pregnant cases. Expression of intrafollicu-ar zinc showed a different pattern; while significantly higher zincevels were observed in pregnant endometriosis cases as compared

ith non-pregnant ones (P < 0.001), the levels were comparableetween pregnant and non-pregnant women of the tubal infertilityroup. In endometriosis, the toxic trace elements, lead and cad-ium were observed to be significantly less in pregnant women

s compared with the non-pregnant cases (P < 0.001). A similarrend was observed in tubal infertility cases (P < 0.001). A significantecrease in selenium, zinc, copper and increase in iron, cadmiumnd lead were seen in pregnant endometriosis cases on comparingith the pregnant tubal infertility group (P < 0.001).

A statistically significant positive correlation of selenium withntioxidant enzyme GPx (r = 0.63, P < 0.001), zinc with SOD (r = 0.62,

< 0.001) and copper with SOD (r = 0.58, P < 0.001) was observedn endometriosis. Further, the trace elements cadmium (r = 0.69,

< 0.001), lead (r = 0.67, P < 0.001) and iron (r = 0.51, P < 0.001) pos-tively correlated with ROS (Fig. 5).

. Discussion

In the present study, increased oxidative stress in the follicularnvironment of women with endometriosis as compared with tubalnfertility is indicated (Table 2). Our results are supported by severalrior findings where increased levels of oxidative stress mark-rs and decreased antioxidant levels in follicular fluid of womenith endometriosis are reported [14,29]. There are only a few

eports on the intrafollicular levels of vitamin A, C and E in infertilendometriosis cases; we found the levels to be significantly reducedn these women.

Excessive generation of intrafollicular ROS has a detrimentalffect on oocytes maturation and subsequent embryo develop-ent [16,30]. Also, NO is negatively correlated with IVF outcome

arameters [31,32]. Our findings suggesting a negative association

f point for the identification of good quality oocytes in (a) endometriosis and (b)nsitivity and 89.74% specificity. Similarly, for tubal infertility ROS cut-off value wassis was 0.837, which was comparable to 0.811 of tubal infertility.

between intrafollicular ROS/NO and oocytes and embryo quality inendometriosis is in good agreement with these reports (Fig. 1a). Asimilar trend was observed when the oocyte and embryo qualitywere related with follicular fluid ROS and NO levels of tubal infer-tility (Fig. 1b) emphasizing the close association between oxidativestress and infertility, independent of the disorder.

The direct relationship between intrafollicular oxidative andnitrosative stress and immature oocytes and poor quality embryosmotivated us to plot the ROC curve to establish the cut-off levelof intrafollicular ROS within which the chances of good qual-ity oocytes formation is significant. The threshold level of ROSin endometriosis cases was estimated to be 105.36 cps. ROS lev-els beyond this limit appear to be toxic and are associated withthe formation of immature oocytes (Fig. 3). Further, AUC valueof 0.837 indicates that this is a good predictor of good qualityoocytes. Similarly, a threshold ROS level (100.96 cps) was assessedfor controls. The AUC value for controls was found to be comparablewith endometriosis (0.811), confirming its predictive accuracy forgood quality oocytes, irrespective of the condition causing infertil-ity.

Extensive studies have shown that oxidative stress in follicu-lar fluid is directly associated with poor IVF pregnancy outcome[33–35]. We found ROS, NO and LPO to be significantly higher innon-pregnant women as compared to the pregnant ones in boththe groups (Table 3), suggesting that pregnancy outcome in patientsundergoing IVF gets adversely affected by the presence of oxidativestress in the oocyte surroundings, irrespective of the disease affect-ing fertility. Despite the similar trend observed, oxidative damagewas seen to be higher in pregnant endometriosis cases; this maybe attributed to the contributory role of oxidative stress in thedevelopment and progression of the disease [36–38].

Multivitamin and mineral supplementations are thought tostrengthen the antioxidant defense system by decreasing oxidativestress of women undergoing IVF [39]. A recent study has shown thata higher overall antioxidant status in blood plasma is advantageousfor achieving pregnancy in IVF/ICSI couples [40]. Despite markedincrease in ROS, NO and LPO, we did not find significant differencesin the levels of TAC, enzymatic antioxidants and vitamins betweennon-pregnant and pregnant group of endometriosis. A similar trendwas observed in the tubal infertility cases (Table 3). Based on our

findings, we reason that though these molecules maybe effective inquenching excessive generation of free radicals, they do not seemto play a contributory role in achieving successful pregnancy fol-lowing IVF. This is an important observation in view of the fact that

122 A.K. Singh et al. / Reproductive Toxicology 42 (2013) 116– 124

fluid

svsotr

lGotsalh

Fc

Fig. 4. Concentrations of trace elements measured in follicular

everal groups are of the opinion that antioxidants, mineral anditamins supplementation may improve the IVF success rate con-iderably [28,41]. Our observation is in agreement with the reportf Mier-Cabrera et al. (2008), where vitamins C and E supplementa-ion in women with endometriosis did not improve the pregnancyate significantly [42].

Selenium, a cofactor for GPx, plays a central role in removingipid hydroperoxides and H2O2. Diminished selenium dependentPx activity in follicular fluid is associated with non-fertilizedocytes. Also, its activity in granulosa cells affects the fertiliza-ion potential of oocytes in women undergoing IVF [43]. We found

elenium and GPx activity to be significantly less in endometriosiss compared with tubal infertility. In addition, a positive corre-ation between selenium and GPx was observed. It is, therefore,ypothesized that decreased intrafollicular selenium/GPx levels in

ig. 5. Scatter plot with regression line showing correlation between follicular fluid leadmium, (e) ROS and lead and (f) ROS and iron. Correlation coefficients and P-values are

of women with endometriosis and tubal infertility. **P < 0.001.

endometriosis enhances oxidative stress in the oocyte microenvi-ronment which affects normal oocyte development.

The primary function of SOD, a copper- and zinc dependentenzyme, is to catalyze the dismutation of superoxide radicals tooxygen and H2O2. Moreover, zinc counteracts the effects of ironand copper in the Fenton reaction, which is involved in free rad-ical generation. A study on mice has shown that zinc deficiencycauses multiple defects in ovarian function during the periovula-tory period with adverse effects on oocyte maturation, cumulusexpansion, and ovulation [44]. Also, a recent study indicates thatacute dietary zinc deficiency in mice before ovulation dramatically

decreases oocyte quality and developmental potential [45]. Gaoet al. (2007) have shown that addition of copper in culture mediaaccelerates successful formation of 8-cell embryos, morulae, andblastocysts; however, overdose can lead to toxicity [46]. Decreased

vels of (a) selenium and GPx, (b) SOD and zinc, (c) SOD and copper, (d) ROS and indicated.

A.K. Singh et al. / Reproductive Toxicology 42 (2013) 116– 124 123

Table 3Follicular fluid concentrations of oxidative stress markers, enzymatic and non enzymatic antioxidants and trace elements in pregnant and not-pregnant cases of women withendometriosis and tubal infertility group. All values are expressed as mean ± SD.

Parameters Endometriosis Tubal infertility

Pregnant (n = 68) Not-pregnant (n = 132) Pregnant (n = 57) Not-pregnant (n = 83)

NO (�M) 33.43 ± 4.48a 48.47 ± 5.25b 29.64 ± 4.62c 42.61 ± 5.83d

ROS (cps) 91 ± 14.21a 113 ± 13.35b 81 ± 16.52c 107 ± 13.57d

MDA (�M) 0.6 ± 0.21a 1.3 ± 0.46b 0.4 ± 0.34c 0.91 ± 0.37d

TAC (�M Trolox equivalent) 663.34 ± 55.35a 649.21 ± 51.36a 911 ± 73.24b 886 ± 75.62b

SOD (mIU/mg protein) 31.67 ± 2.64a 32.12 ± 2.58a 46.24 ± 4.15b 44.85 ± 4.23b

Catalase (mIU/mg protein) 10.56 ± 2.43a 9.87 ± 2.71a 20.46 ± 3.42b 19.36 ± 3.51b

GPx (mIU/mg protein) 0.84 ± 0.18a 0.79 ± 0.17a 2.17 ± 0.41b 2.06 ± 0.42b

GR (mIU/mg protein) 0.080 ± 0.006a 0.079 ± 0.005a 0.21 ± 0.12b 0.19 ± 0.13b

Vitamin A (�g/l) 3.21 ± 0.17a 3.17 ± 0.16a 4.12 ± 0.14b 4.08 ± 0.13b

Vitamin C (mg/l) 4.21 ± 0.13a 4.18 ± 0.14a 5.56 ± 0.17b 5.51 ± 0.18b

Vitamin E (mg/l) 9.54 ± 0.16a 9.50 ± 0.17a 12.53 ± 0.17b 12.48 ± 0.16b

Copper (�g/l) 321 ± 33.58a 312 ± 34.12a 452 ± 40.13b 439 ± 38.47b

Zinc (�g/l) 438 ± 35.63a 381 ± 36.24a 654 ± 43.62a 641 ± 45.35b

Selenium (�g/l) 0.431 ± 0.05a 0.419 ± 0.04a 0.733 ± 0.05b 0.718 ± 0.05b

Iron (�g/l) 1452 ± 56.47a 1467 ± 61.32a 1261 ± 64.53b 1248 ± 62.34b

Cadmium (�g/l) 0.67 ± 0.14a 0.82 ± 0.13b 0.26 ± 0.15c 0.47 ± 0.16d

Lead (�g/l) 1.69 ± 0.12a 1.84 ± 0.12b 0.71 ± 0.12c 0.96 ± 0.13d

W

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niozhngpfvlicotOWett

poierowceaelteca

ithin each row, data with different superscripts are different at P < 0.001

erum copper levels in patients undergoing IVF than controls islso reported [41]. We found positive correlation of SOD with cop-er and zinc. Decrease in the activity of SOD in the present studyay be attributed to the significant copper and zinc deficiency in

he endometriosis group, as evident from our findings.Comparable levels of selenium and copper between preg-

ant and non-pregnant women of endometriosis as well as tubalnfertility indicate that these molecules do not affect pregnancyutcome in women undergoing IVF. A significant decrease ininc in non-pregnant endometriosis cases was seen. The levels,owever, remained unchanged on comparing the pregnant andon-pregnant cases of tubal infertility. These observations sug-est that zinc deficiency in follicular fluid of endometriosis is aotential risk factor for achieving successful pregnancy. Excessree iron plays a detrimental role in free radical production byirtue of Fenton reaction and can result in toxicity [47]. Iron over-oad has been demonstrated in endometriosis patients [48]. Higherntrafollicular iron levels in patients with IVF as compared withontrols are reported by Ozkaya et al. (2011) [28]. They furtherbserved that iron concentration decreased significantly in multivi-amin/mineral supplemented groups than in untreated IVF groups.xidative stress mediated non-heme iron absorption is suggested.e also observed a marked increase in iron concentration in

ndometriosis as compared with tubal infertility. It is presumedhat iron-induced oxidative stress compromises the fertility poten-ial of these women.

Cadmium and lead are the most common environment toxicollutants which slowly accumulate in the body and mediatexidative stress [49]. They induce the formation of ROS, LPO andnterfere with the antioxidant defense system, which includes thenzymes GPx, SOD and catalase [49]. Their role in endometriosisemains largely unknown [50]. Our results show an elevated levelf intrafollicular lead and cadmium in endometriosis as comparedith tubal infertility. Further, these toxic molecules positively

orrelated with intrafollicular ROS. Although inconsistent resultsxist regarding the potential relationship between toxic trace met-ls exposures and reproductive endpoints [51], there is increasingvidence suggesting a positive association between excessiveead/cadmium levels and poor IVF outcome [52–54]. A similar

rend was observed in the present study, with a direct relationshipxisting between higher lead/cadmium content and non-pregnantases in both, endometriosis and tubal infertility. This may bettributed to the fact that increased concentrations of lead and

cadmium in the follicular compartment impart a toxic effect onoocyte [55], embryo [51] and pregnancy outcome [56].

In conclusion, deficiencies of essential trace elements and asso-ciated antioxidants, and the detrimental effect of oxidative stressand reproductive toxicants in follicular environment of infertilewomen with endometriosis are investigated. The deleterious effectof intrafollicular ROS/NO on oocytes and embryo quality and IVFpregnancy prompted us to ascertain the ROS threshold level beyondwhich it appears to be toxic, and is associated with the formationof poor quality oocytes. Further, follicular levels of lead and cad-mium showed a negative association with IVF pregnancy outcome,thereby highlighting the toxicity risk of environmental pollutants.

We observed a higher intrafollicular zinc level in women withendometriosis who went on to become pregnant after IVF treat-ment. Another interesting finding of the present study whichmay have important clinical implications is that antioxidants andtrace elements present in follicular fluid do not appear to playan active role in improving IVF pregnancy outcome. Considerablescientific deliberations regarding the benefits associated with mul-tivitamin/mineral supplementation to patients undergoing IVF is,therefore, warranted.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors thank the Department of Science and Technol-ogy [grant no. 882 (Sanc.)/ST/P/S&T/9G-23/2007], Government ofWest Bengal, India. The authors thank Priyanka Banerjee, ResearchScholar, School of Medical Science and Technology, IIT Kharag-pur for assisting in the preparation of the manuscript. One of theauthors, Abhay Kumar Singh is grateful to the Government of India,Indian Council of Medical Research (Award No. 3/1/JRF/88/MPD-2007), for financial support under the ICMR-JRF scheme.

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