N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model

ARTICLE IN PRESS Reproductive BioMedicine Online (2016) ■■, ■■–■■

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N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model

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Gulcin Sahin Ersoy a,*, Meryem Eken b, Reshef Tal c, Deniz Oztekin d, Belgin Devranoglu b, Ecmel Isik Kaygusuz e, Ozge Cevik f

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Department of Obstetrics and Gynecology, Kartal Dr. Lutfi Kirdar Education and Research Hospital, Istanbul, Turkey; Department of Obstetrics and Gynecology, Zeynep Kamil Education and Research Hospital, Istanbul, Turkey; c Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA; d Department of Obstetrics and Gynecology, Tepecik Education and Research Hospital, Izmir, Turkey; e Department of Pathology, Zeynep Kamil Education and Research Hospital, Istanbul, Turkey; f Department of Biochemistry, Cumhuriyet University, Faculty of Pharmacy, Sivas, Turkey b

* Corresponding author. E-mail address: [email protected] (G Sahin Ersoy).The study was presented in abstract format at the 71st Annual Meeting of the American Society for Reproductive Medicine, Baltimore, MD, 17–21 October, 2015. Dr Gulcin Sahin Ersoy received her MD degree from Ankara University in 2004. After completing her residency programme at Izmir Tepecik Education and Research Hospital in 2011 she started to work in Kartal Dr. Lutfi Kirdar Education and Research Hospital as an obstetrics and gynaecology specialist. Currently she is taking time out from working in Turkey and is working as a postdoctoral research fellow at the Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine. Her special interests include ovarian reserve, endometrial receptivity and implantation.

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This study evaluated the effects of N-acetylcysteine (NAC) and enoxaparin on ovarian tissue preservation, ovarian reserve and oxidative damage following ovarian torsion/detorsion injury. Rats were divided into four groups (n = 6/group): control; ischaemia/ reperfusion (I/R); I/R + NAC; I/R + enoxaparin. Twenty-four hours after detorsion, ovarian tissues were collected for histopathological analysis and measurement of tissue 8-OHdG, GSH, MDA, MPO and SOD concentrations, as well as pre- and post-operative circulating AMH concentrations. Administration of NAC resulted in more pre-antral follicles compared with enoxaparin treatment and haemorrhage and follicle cell degeneration were more pronounced in I/R + enoxaparin group than I/R + NAC group. Both NAC and enoxaparin led to a significant reduction in ovarian tissue 8-OHdG (P = 0.004 and P = 0.01, respectively) and MPO (P = 0.013 and P = 0.023, respectively) concentrations compared with I/R group, indicating a protective effect against I/R oxidative damage. Only NAC-treated animals showed a significant increase in GSH and SOD concentrations and decrease in MDA concentrations compared with I/R group (P = 0.007, P = 0.024 and P = 0.026, respectively). These results indicate that NAC is more effective than enoxaparin in minimizing ovarian damage and preserving ovarian reserve following ovarian torsion.

Abstract

© 2016 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.

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KEYWORDS: anti-Müllerian hormone, enoxaparin sodium, N-acetylcysteine, ovarian ischaemia-reperfusion, ovarian reserve, torsion

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http://dx.doi.org/10.1016/j.rbmo.2016.03.009 1472-6483/© 2016 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.

Please cite this article in press as: Gulcin Sahin Ersoy, Meryem Eken, Reshef Tal, Deniz Oztekin, Belgin Devranoglu, Ecmel Isik Kaygusuz, Ozge Cevik, N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.03.009

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G Sahin Ersoy et al.

Introduction

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Ovarian torsion is a gynaecologic emergency with a prevalence of 2.7% and is believed to have an adverse effect on ovarian reserve even if the diagnosis is made promptly (Hibbard, 1985). Ovarian tissue ischaemia begins immediately following the obstruction of the ovarian artery. After spontaneous or surgical detorsion of the ovary, reperfusion occurs, inducing the expression of oxidative stress products such as oxygen species-free radicals and inflammatory cells, which are regarded as the main factors responsible for the subsequent damage to the ovaries. Therefore, reperfusion is thought to be the primary cause of the damage to the ovarian tissue (Cadirci et al., 2010; Ergun et al., 2010). Ozler et al. (2013) have shown that the ovarian damage-related decrease in ovarian reserve continues during the reperfusion phase concomitant with the efflux of free radicals and the aftermath of inflammatory responses. Consequently, they have suggested that antioxidant treatments may complement surgery and be beneficial in preserving the ovarian reserve in this setting. Previous studies have shown that treatment with antioxidant drugs can prevent ovarian ischaemia/reperfusion (I/R) injury (Kaya et al., 2014; Kumtepe et al., 2010). N-acetylcysteine (NAC) is an aminothiol, a precursor of glutathione known to act as a potent antioxidant by scavenging free-radicals (Danilovic et al., 2014; Orhan et al., 2006). N-acetylcysteine has been demonstrated to be protective of I/R tissue injury in various organs (Cuzzocrea et al., 2000; Turkmen et al., 2012), including the ovary (Usta et al., 2008). However, its effects on ovarian reserve following I/R are unknown. Enoxaparin sodium, a low molecular weight heparin, is a well-known anticoagulant agent and has been shown to preserve the ovarian reserve following ovarian I/R injury in a rat ovarian torsion model (Kaya et al., 2014). It is also an antioxidant agent but its effects on oxidative stress following I/R injury in the ovary have not been studied. Moreover, it is unknown which treatment may more efficacious in terms of protection of the ovary and its follicular pool following I/R injury. We postulated that treatment with either NAC or enoxaparin would result in protection of the ovary and its egg reserve and that both agents would reduce oxidative stress in the ovary following ovarian torsion/detorsion injury in an experimental rat model.

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Materials and methods

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Animals

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In total, 24 Wistar albino adult (12-week-old) female rats weighing between 230 and 250 g were obtained from Acibadem University Experimental Animal Laboratory. All experimental procedures and protocols were approved by the Local Animal Ethics Committee of Acıbadem University on 10 November 2014 (approval number: 2014/22) and were performed according to the National Health and Medical Research Council guidelines for the care of experimental animals. This study was reported in accordance with the ARRIVE (Animal Research: Reporting of In vivo Experiments) guidelines (McGrath et al., 2010).

The rats were randomly assigned to standard cages with two animals per cage and maintained in the laboratory under controlled environmental conditions on a 12 h light/dark cycle at a room temperature at 21 ± 1°C and free access to water and food.

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Chemicals

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N-acetylcysteine (Sigma-Aldrich, US) and enoxaparin sodium (Clexane, Sanofi Aventis, France) were purchased for the experimental procedure. Ketamine hydrochloride (Ketasol, Richter Pharma, Austria) and Xylazine (Rompun, Bayer Healthcare, Germany) were purchased for anaesthesia.

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Experimental groups

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Following the determination of oestrus stage of each rat by performing daily vaginal smears, they were randomly divided into four groups. All surgical procedures in the different groups are detailed in the “Surgical procedures” section. Group I: control group (n = 6) underwent sham surgery. Group II: ischaemia/reperfusion (I/R) (n = 6) underwent the bilateral ovarian torsion-detorsion procedure. Group III: ischaemia/reperfusion + NAC (I/R + NAC) (n = 6) underwent the ovarian torsion-detorsion procedure and received 30 mg/kg NAC intraperitoneally 30 min before laparotomy. Group IV: ischaemia/reperfusion + enoxaparin sodium (I/R + enoxaparin) (n = 6) underwent the ovarian torsion-detorsion procedure and received 0.5 mg/kg enoxaparin sodium subcutaneously 30 min before laparotomy.

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Surgical procedures

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A pilot study was initially performed to determine the durations of torsion and detorsion that would result in ovarian reserve change in our model. We chose a 3 h duration of torsion based on the study by Ozler et al. (2013), which reported decline in ovarian reserve in their ovarian torsion rat model. Our preliminary findings confirmed the change in ovarian reserve after 3 h-torsion followed by 24 h-detorsion determined histologically and by measuring anti-Müllerian hormone (AMH) concentrations. Animals were anaesthetized by administering 60 mg/kg ketamine hydrochloride and 10 mg/kg xylazine intramuscularly. For measurement of pre-ischaemia AMH concentrations, 1 ml blood sample was drawn from the jugular vein of each rat. The surgical field was shaved and disinfected with povidone-iodine solution. A 3-cm midline lower abdominal incision was performed and the bilateral uterine horns and ovaries were identified. In the control group (group I), following the observation of ovaries for 1 min, surgery was ended. In the other groups (groups II–IV), bilateral ovarian I/R was carried out. This procedure was performed as follows: 3 h of ischaemia by twisting the ovary with its accompanying tubo-ovarian vessels through one complete circle in a clockwise direction and clamping the pedicle with 20–25 grams-pressure bulldog clamps (Vascu-statt, Scanlan, USA); the 3-h phase of ischemia was followed by a

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Please cite this article in press as: Gulcin Sahin Ersoy, Meryem Eken, Reshef Tal, Deniz Oztekin, Belgin Devranoglu, Ecmel Isik Kaygusuz, Ozge Cevik, N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.03.009

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ARTICLE IN PRESS N-acetylcysteine preserves ovarian reserve in ovarian torsion 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198

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24-h period of reperfusion (detorsion). Following the execution of detorsion the abdominal incision was closed in two layers. At the end of the reperfusion period 1 ml blood samples were drawn from the jugular vein for measurement of postreperfusion AMH concentrations; thereafter, the animals were killed and their ovaries were harvested. Ovarian tissue samples were vertically divided into two halves. One half was transferred into 10% neutral-buffered formalin solution (10% formaldehyde, 4 g of NaH2PO4, 6 g of Na2HPO4 in solution of per litre) for histological analysis. The other half of the ovary was cleaned of retroperitoneal white adipose tissue and stored rapidly in a −80°C freezer for biochemical analysis.

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Serum AMH measurement

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All collected blood samples were centrifuged at 2500 g and 4°C for 10 min, and sera were separated and stored at −80°C until assayed. Serum concentrations of AMH were quantified using enzyme-linked immunosorbent assay (ELISA) antiMüllerian Hormone Kit according to the manufacturer’s instructions (Bioassay Technology, Shanghai). The lower limit of sensitivity of the AMH ELISA was 0.051 ng/ml. The degree of precision of the ELISA system in terms of intra-assay and inter-assay coefficients of variance were <10% and <12%, respectively.

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Tissue 8-OHdG measurement

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Tissue samples were collected and genomic DNA was immediately extracted using commercial PureLink® Genomic DNA Extraction Kit according to the manufacturer’s protocol (Invitrogen, US). The samples were stored at −80°C for the determination of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Measurement of tissue 8-OHdG concentrations was performed via competitive ELISA using OxiSelect ™ Oxidative DNA Damage ELISA kit according to the manufacturer’s instructions (Cell Biolabs, USA).

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Tissue GSH and MDA measurement

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Ovarian samples were homogenized with 150 mmol/l KCl for the determination of malondialdehyde (MDA) and glutathione (GSH) concentrations. The MDA concentrations were assayed for products of lipid peroxidation by monitoring thiobarbituric acid reactive substance formation as described previously (Beuge and Aust, 1978). Lipid peroxidation is reported as nmol MDA/mg protein. GSH measurements were performed using metaphosphoric acid (Na2HPO4) for protein precipitation following the Ellman procedure (Beutler, 1975). Briefly, after centrifugation at 2000 g for 10 min, 0.5 ml of supernatant was added to 2 ml of 0.3 mol/l Na2HPO4 solution. 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. GSH concentrations were expressed in μmol GSH/mg protein.

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Tissue MPO measurement

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The myeloperoxidase (MPO) activity was measured in Q3 ovarian tissues as previously described by Hillegass et al.

3 (1990). Tissue samples were homogenized with potassium buffer (20 mmol/l KH2PO4, pH 7.4) containing 0.5% (w/v) hexadecyltrimethylammonium bromide. MPO activity was assessed by measuring the H 2 O 2 -dependent oxidation of o-dianisidine dihydrochloride. One unit of enzyme activity was measured at 460 nm and the results were expressed as U/mg protein.

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Tissue SOD measurement

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Superoxide dismutase (SOD) activity in the ovarian tissue samples was measured as previously described (Mylroie et al., 1986). Homogenized tissues were treated with 0.39 mmol/l riboflavin and 6 mmol/l o-dianisidine dihydrochloride in potassium phosphate buffer (pH 7.5). Absorbances were measured at 460 nm and results were expressed as U/mg protein.

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Histological analyses

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Following the fixation of ovarian tissue in 10% neutralbuffered formalin solution for 24 h, a routine tissue-processing procedure was performed, and the samples were embedded in paraffin. Paraffin wax blocks were cut into 5-μm-thick sections (Leica RM2125RTS, Leica Biosystems, Nussloch, Germany) and stained with haematoxylin and eosin. The main parameters of ovarian injury were determined as interstitial oedema, vascular dilatation, haemorrhage, polymorphonuclear leukocyte (PNL) infiltration and follicle cell degeneration (Kara et al., 2012). Each specimen was scored on a scale of 0 to 3 according to damage severity (0: none; 1: mild; 2: moderate; and 3: severe). The semiquantitative histologic scoring of the ovarian sections was performed by a single observer who was blinded to the study protocol. All sections were examined and photographed with a light microscope (Olympus BX-50, Olympus Corp, Tokyo, Japan). At least five microscopic areas were examined to assess each specimen. The histologic method for the follicle count was defined according to the study of Durlinger et al. (1999). The follicles were classified into the following groups based on the mean diameter: primordial follicles (diameter <20 μm), pre-antral follicles (20–220 μm), small antral follicles (221–310 μm), and large antral follicles (311–370 μm) (Kaya et al., 2014). The ovarian sections were analysed in a blinded fashion by the same observer.

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Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay The existence of cell death in the follicles was evaluated by using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling (TUNEL) analysis (ApopTag® Plus Peroxidase In situ Apoptosis Kit, Millipore, Cat No: S7101, USA). The staining was performed according to the manufacturer’s instructions. Briefly, tissue sections mounted on glass slides were deparaffinized, rehydrated through alcohol gradient and treated with 20 μg/ml proteinase K (37°C, 20 min). For blocking of endogenous peroxidase, 3% H2O2 in methanol was used. After rinsing with PBS,

Please cite this article in press as: Gulcin Sahin Ersoy, Meryem Eken, Reshef Tal, Deniz Oztekin, Belgin Devranoglu, Ecmel Isik Kaygusuz, Ozge Cevik, N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.03.009

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G Sahin Ersoy et al.

sections were incubated with TdT equilibration buffer for 30 min. Then slides were covered with TdT enzyme solution and incubated for 1 h at 37°C. STOP solution was dropped on the sections for 5 min. Following washing in PBS, antidigoxigenin conjugate was applied to the sections for 30 min in a humidified chamber. The slides were rinsed in PBS buffer, and the entire tissue sections were covered with DAB solution for 10 min. The nuclei were counterstained with Mayer’s haematoxylin, dehydrated and cover-slipped. TUNEL-positive cells are stained brown and are indicative of apoptosis. Some of the ovary tissue sections were used as negative controls by replacing treatment with TdT with distilled water. Negative control slides stained with TUNEL technique failed to show any immunoreactivity. All the sections were examined with BX51 Olympus microscope (Olympus, Tokyo, Japan) and photographed by DP72 camera system (Olympus, Tokyo, Japan). Semi-quantitative scoring of follicular apoptosis was performed following counting TUNEL-positive cells as follows: 0, positive in <5% of the cells; 1, positive in 5–25% of the cells; 2, positive in 26–50% of the cells; 3, positive in >50% of the cells/follicles.

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Statistical analysis

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The variables were examined using the Kolmogorov–Smirnov test to find out whether or not they are normally distributed. The data with normal distribution were analysed with one-way ANOVA whereas data not normally distributed were evaluated with Kruskal-Wallis test. When overall significance was observed in ANOVA, pairwise post-hoc tests were performed using Tukey’s test. The Mann-Whitney U-test was performed to test the significance of pairwise differences after the Kruskal-Wallis test. Since our study is restricted to a small number of comparisons and is based on a clearly stated a-priori hypothesis (i.e. that treatment with either NAC or enoxaparin would result in protection of the ovary and its egg reserve and

that both agents would reduce oxidative stress in the ovary following ovarian I/R injury), we did not perform correction for multiple comparisons, as previously recommended to decrease the likelihood of Type II error (Schulz and Grimes, 2005). Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 17 (SPSS Inc., Chicago, IL, USA). A P-value of <0.05 was considered statistically significant.

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Results

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The experimental study was completed without any adverse events and specimens were collected from each animal for histological and biochemical analyses. Ovarian tissue GSH and SOD concentrations were significantly decreased in rats subjected to I/R compared with the control group (P = 0.001 and P < 0.001, respectively). Whereas the I/R + enoxaparin group showed no significant change in ovarian GSH and SOD concentrations compared with the I/R group, the NAC-treated rats demonstrated a significant (P = 0.007 and P = 0.024, respectively) increase in their concentrations compared with the I/R group, indicating less oxidative damage (Table 1). In agreement with this, ovarian tissue MDA and MPO, which signify oxidative damage, were significantly decreased in the NAC-treated rats compared with the I/R group (P = 0.026 and P = 0.013, respectively). In the enoxaparin-treated rats, MPO level was also significantly (P = 0.023) decreased while MDA level was not significantly different compared with the I/R group. Similarly, 8-OHdG concentrations were significantly higher in the I/R group (1.89 ± 0.36 ng/μg DNA) than in the control, I/R + NAC (1.13 ± 0.03 ng/μg DNA) and I/R + enoxaparin (1.23 ± 0.10 ng/μg DNA) groups (P = 0.004, P = 0.004 and P = 0.01, respectively), indicating more severe oxidative DNA damage in the I/R group, while no significant difference in 8-OHdG concentrations were noted between the I/R + NAC and I/R + enoxaparin groups.

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Table 1 The levels of 8-hydroxydeoxyguanosine, malondialdehyde, glutathione, superoxide dismutase, myeloperoxidase and histologic damage and TUNEL scores of the experimental groups.

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Control

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I/R + NAC

I/R

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P-value

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8OHdG (ng/μg DNA) GSH (μmol/mg protein) MDA (mmol/mg protein) MPO (U/mg protein) SOD (U/mg protein) Histologic score Oedema Dilatation Haemorrhage PNL Degeneration TUNEL scoring

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1.07 2.54 11.11 1.85 4.59

± ± ± ± ±

0.04 0.26 1.83 0.49 1.06

1.89 1.79 14.8 4.52 2.3

± ± ± ± ±

0.36b 0.36b 1.6b 1.53c 0.84c

1.13 2.42 12.16 2.92 3.61

± ± ± ± ±

0.03a,e 0.22e 0.92d 0.6d 0.41d

1.23 2.01 12.84 3.05 3.09

± ± ± ± ±

0.10b,d 0.29a 1.41 0.51d 0.21b

<0.001 0.001 0.003 <0.001 0.001

0.50 0.33 0 0.33 0 0.63

± ± ± ± ± ±

0.54 0.51 0 0.51 0 0.45

1.83 2.33 2.33 1.67 2.17 2.70

± ± ± ± ± ±

0.75a 0.81b 0.81b 1.21 0.98b 0.17b

1.33 1.67 1.17 0.67 0.67 1.55

± ± ± ± ± ±

0.51 0.51b 0.40b,d 0.81 0.51d 0.35a,e

1.33 1.50 2.17 0.83 1.67 2.18

± ± ± ± ± ±

0.51 0.54a 0.40b,g 0.75 0.51b,f 0.21b,e,f

0.020 0.003 <0.001 NS 0.001 <0.001

Data shown mean ± SD. P-values in the column for comparison across all groups. 8OHdG = 8-hydroxydeoxyguanosine; GSH = glutathione; I/R = ischaemia/reperfusion; MDA = malondialdehyde; MPO = myeloperoxidase; NAC = N-acetylcysteine; NS = not statistically significant; PNL = polymorphonuclear leukocyte; SOD = superoxide dismutase; TUNEL = terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling. a P < 0.05, bP < 0.01, cP < 0.001 versus C group; dP < 0.05, eP < 0.01, versus I/R group; fP < 0.05, gP < 0.01 versus I/R + NAC group.

Please cite this article in press as: Gulcin Sahin Ersoy, Meryem Eken, Reshef Tal, Deniz Oztekin, Belgin Devranoglu, Ecmel Isik Kaygusuz, Ozge Cevik, N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.03.009

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ARTICLE IN PRESS N-acetylcysteine preserves ovarian reserve in ovarian torsion 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401

402

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Histological analysis of ovarian tissue revealed no statistically significant difference in PNL infiltration scores between groups, but significantly greater oedema (P < 0.05), vascular dilatation (P < 0.01), haemorrhage (P < 0.01) and follicular cell degeneration (P < 0.01) were detected in the I/R group compared with the control group (Figure 1 and Table 1). Notably, the I/R + NAC group demonstrated significantly less haemorrhage and follicular cell degeneration compared with both I/R (P = 0.019 for haemorrhage, P = 0.016 for follicular cell degeneration) and I/R + enoxaparin groups; the haemorrhage score was 1.17 ± 0.40 in the I/R + NAC group versus 2.17 ± 0.40 in the I/R + enoxaparin group (P = 0.006); and the follicular cell degeneration scores were 0.67 ± 0.51 and 1.67 ± 0.51 in the I/R + NAC and I/R + enoxaparin groups, respectively (P = 0.014, Table 1). No statistically significant differences were noted between the I/R groups in terms of tissue oedema and vessel dilatation. Follicular apoptotic cell death status was evaluated with TUNEL assay (Table 1 and Figure 2). When compared with the I/R group, a decrease in TUNEL-positive cells was seen in I/R + NAC and the I/R + enoxaparin groups. In addition, the TUNEL-positive cell number in the follicles was significantly higher (P < 0.05) in the I/R + enoxaparin group than the I/R + NAC group (Figure 2c and d). In comparison to the pre-operative period, the serum AMH concentrations were significantly decreased post-operatively in I/R, I/R + NAC and I/R + enoxaparin groups compared with

5 controls (P < 0.001 for all groups). However, the reduction in AMH concentrations in the I/R + NAC and I/R + enoxaparin groups were significantly lower than the decline in the I/R group (P < 0.01; Table 2). Consistent with the AMH level data, the average quantity of follicles at all developmental stages decreased in the I/R group compared with controls (Table 2). The NACtreated rats had significantly greater average number of primordial (P = 0.004) and pre-antral (P = 0.003) follicles compared with the I/R group. Moreover, in the I/R + enoxaparin group the pre-antral follicle counts were significantly lower than the I/R + NAC group (P = 0.04). No statistically significant differences were observed in the small antral and large antral follicle counts between the control, I/R + NAC and I/R + enoxaparin groups.

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425

Discussion

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The aim of our study was to evaluate and compare the antioxidant and cytoprotective effects of NAC and enoxaparin on the ovary and its follicular reserve using histopathological and biochemical techniques. We showed that a distinct histopathological injury and a reduction in the ovarian reserve occurred after torsion/detorsion. However, NAC and enoxaparin sodium showed a significant protective effect against the I/R injury. The comparison of the protective effects of NAC with

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Figure 1 Light microscopic appearance of ovarian morphology (haematoxylin and eosin; ×200). (A) In the control group, primordial follicle (thin arrow), pre-antral follicles (thick arrows), small antral follicle (triangle), large antral follicle (arrowhead), normal vascular structure (star). (B) In the ischaemia/reperfusion group, follicular cell degeneration (thin arrrow), haemorrhage (thick arrow), vascular dilatation (arrowhead), polymorphonuclear leukocytes (star). (C) In the ischaemia/reperfusion + enoxaparin group, haemorrhage (thick arrows), vascular dilatation (arrowhead), follicular cell degeneration (thin arrow). (D) In the ischaemia/reperfusion + NAC group, follicular cell degeneration (thin arrows), minimal vascular dilatation (arrowhead), polymorphonuclear leukocytes (star) and minimal haemorrhage (thick arrow).

Please cite this article in press as: Gulcin Sahin Ersoy, Meryem Eken, Reshef Tal, Deniz Oztekin, Belgin Devranoglu, Ecmel Isik Kaygusuz, Ozge Cevik, N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.03.009

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ARTICLE IN PRESS 6

436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469

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G Sahin Ersoy et al.

Figure 2 Histomorphological evaluation of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining in ovaries following I/R injury. Representative light micrographs counterstained with Mayer’s haematoxylin are shown. (a) Control group showing no apoptotic cells; (b) ischaemia/reperfusion (I/R) group showing numerous apoptotic cells in the stroma and follicles; (c) I/R + NAC group showing only few apoptotic cells; (d) I/R + enoxaparin group demonstrating more apoptotic cells but less than I/R only group. Arrows indicate TUNEL-positive cells (dark brown nucleus).

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Table 2

Follicle counts, pre- and post-operative anti-Müllerian hormone concentrations of the experimental groups.

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Control

I/R

I/R + NAC

I/R + Enoxaparin

P-value

8.74 ± 0.78 8.77 ± 0.89 0.02 ± 0.49

9.34 ± 1.03 4.49 ± 0.41c −4.85 ± 0.76c

8.88 ± 0.82 6.40 ± 0.84c,e −2.48 ± 0.52c,d

9.55 ± 0.89 6.26 ± 0.53c,e −3.29 ± 0.69c,e

NS <0.001 <0.001

± ± ± ± ±

0.001 0.002 NS NS 0.003

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AMH (ng/ml) Pre-operative Post-operative AMH reduction Follicle counts primordial Pre-antral small antral large antral corpus luteum

12 6.83 2.83 1.83 4.67

± ± ± ± ±

4.94 2.63 0.75 0.40 1.03

3.50 2.17 1.67 1.17 2.50

± ± ± ± ±

1.04b 0.75b 0.51 0.40 0.83b

7.83 5.33 2.33 1.67 3.67

± ± ± ± ±

1.60e 1.36e 0.51 0.51 1.03

5.67 3.67 2.17 1.67 2.83

2.73a 1.03d,f 0.98 0.81 0.75a

Data are expressed as mean ± SD. P-values are in the column for comparison across all groups. AMH = anti-Müllerian hormone; I/R = ischaemia/reperfusion; NAC = N-acetylcysteine; NS = not statistically significant. a P < 0.05, bP < 0.01, cP < 0.001 versus C group; dP < 0.05, eP < 0.01, versus I/R group; fP < 0.05 versus I/R + NAC group.

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enoxaparin sodium has demonstrated that the administration of NAC provided better antioxidative protective effect and resulted in less histopathological damage to the ovary and better preservation of primordial and pre-antral follicles. Detorsion surgery is the most effective and successful method in reduction of ovarian damage and preservation of ovarian tissue in the setting of torsion (Sagsoz et al., 2002; Taskin et al., 1998), and it should be accomplished promptly in order to avoid the subsequent loss of ovarian function.

However, following detorsion surgery, the ovary is reperfused with oxygenated blood, causing an increase in the generation of reactive oxygen species (Ergenoglu et al., 2013). These free radicals initiate the lipid peroxidation process in mitochondrial and cell membranes resulting in apoptosis and cellular dysfunction (Paller et al., 1984). Therefore, the addition of an antioxidative treatment to the conservative surgery may contribute to the protection of the ovarian tissue and reserve. Several antioxidative agents have been suggested to be protective in the setting of ovarian I/R injury (Incebiyik et al.,

Please cite this article in press as: Gulcin Sahin Ersoy, Meryem Eken, Reshef Tal, Deniz Oztekin, Belgin Devranoglu, Ecmel Isik Kaygusuz, Ozge Cevik, N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.03.009

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ARTICLE IN PRESS N-acetylcysteine preserves ovarian reserve in ovarian torsion 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541

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2015; Kumtepe et al., 2010; Kurt et al., 2015; Yigiter et al., 2011). NAC is an antioxidative agent that includes a thiol group. As a result of its antioxidative activity, the intracellular glutathione synthesis is augmented and free oxygen radicals are scavenged (Inci et al., 2007). Moreover, NAC also has an antiapoptotic effect and can promote cell survival (Zafarullah et al., 2003). NAC was introduced as a mucolytic drug in the 1960s. It easily penetrates cell membranes and, unlike cysteine, the rate-limiting amino acid in glutathione synthesis, has very low toxicity (Cay et al., 2006). Thus, from a clinical point of view, NAC is an attractive drug because of its low cost, scarce side effects and because there is considerable experience of its use in critically ill patients (Nitescu et al., 2006). Usta et al. (2008) examined the effects of NAC in a rat model of ovarian torsion/detorsion. They have concluded that the infusion of a 30 mg/kg dose of NAC 30 min before reperfusion decreases the MDA concentrations and the total tissue damage score. However, they did not evaluate the effects of NAC on the ovarian reserve following ovarian torsion/ detorsion injury. Our data are consistent with the findings of Usta et al. (2008) in terms of the antioxidative ovarian protective effects of NAC following ovarian torsion. Moreover, we show that this antioxidative protection translates to better preservation of ovarian reserve as determined by follicular counts and AMH concentrations. Enoxaparin sodium, a low molecular weight heparin in the form of a sulphated glycosaminoglycan, is widely used as an anticoagulant agent for both acute thrombosis and its prophylaxis and is known to have minimal side effects (Caliskan et al., 2014). The protective effects of low molecular weight heparins on I/R injuries of tissues like brain, myocardium, liver and lower limb have been reported in previous studies (Deepa and Varalakshmi, 2003; Poyrazoglu et al., 2006; Yassin et al., 2002; Zhang et al., 2007). In addition, its protective effects on ovarian I/R injury in terms of preservation of ovarian reserve has been recently reported by Kaya et al. (2014). They have demonstrated that the administration of enoxaparin sodium twice, 2 h before and 24 h after ovarian torsion, prevented the decline of large antral follicle count and AMH concentrations in an experimental ovarian torsion/detorsion injury model in rats. Since enoxaparin sodium is known to have antioxidant properties in addition to its anticoagulant effects, we wished to study its antioxidative effects in the ovarian I/R model and compare its effects with another potent antioxidative agent, NAC. Reactive oxygen species give rise to the deformation of DNA-protein crosslinks and alteration of the bases (Nagai et al., 2008). 8-OHdG is one of the most stable DNA bases and a wellestablished biomarker of oxidative damage in DNA (Doetsch and Cunningham, 1990). Therefore, in the present study the 8-OHdG concentrations were measured in order to accurately estimate the free radical-mediated oxidative damage. Our data showed that the 8-OHdG concentrations were significantly lower in the I/R + NAC and I/R + enoxaparin groups than the I/R group, but no significant difference was detected between the NAC and enoxaparin treatment groups. Similarly, there was no statistically significant difference between the I/R + NAC and I/R + enoxaparin groups in terms of the endogenous oxidative stress enzymes. However, the NAC-treated rats demonstrated a significant increase in GSH

7 and SOD concentrations compared with the I/R group, while the I/R + enoxaparin group did not, indicating less oxidative damage following NAC treatment. Histological evaluation revealed that haemorrhage and follicular cell degeneration scores were significantly higher in the I/R + enoxaparin group than the I/R + NAC group. We believe that the anticoagulant property of enoxaparin sodium may have been responsible for the detection of a significant difference in histological scores, especially for haemorrhage. NAC treatment also resulted in greater preservation of primordial and pre-antral follicles compared with enoxaparin treatment. This may also be related to differences in extent of tissue damage between the two treatments. In the present study, pre-operative and post-operative AMH concentrations have been used as a hormonal indicator of the ovarian reserve. AMH is a dimeric glycoprotein that is exclusively produced by the granulosa cells of the pre-antral and antral follicles (Feyereisen et al., 2006). It has been suggested as an early, reliable measure of the ovarian follicular reserve under different clinical conditions (Broer et al., 2014; Gnoth et al., 2008). It is also known that the combination of AMH with antral follicle count is the superior method for the evaluation of ovarian reserve (de Vet et al., 2002; Kwee et al., 2008; Seifer and Maclaughlin, 2007). The decline of AMH concentrations in experimental ovarian torsion/detorsion injury has been reported in previous studies (Caliskan et al., 2014; Ozler et al., 2013). Similarly, we have found that postoperative serum concentrations of AMH were significantly lower than the pre-operative concentrations in I/R, I/R + NAC and I/R + enoxaparin groups. However, the reduction of AMH values was greatest in the I/R group. Although no statistically significant difference was found between the I/R + NAC and I/R + enoxaparin groups in terms of the reduction of AMH values, this reduction was slightly lower in the I/R + NAC group. Consistent with the AMH data, the total counts of primordial, pre-antral follicles and corpora lutei were found to be decreased in the I/R group when compared with control group. In terms of small antral and large antral follicles neither the I/R + NAC nor the I/R + enoxaparin groups have demonstrated any difference compared with the control group and each other. However, the primordial and pre-antral follicle counts were significantly lower in the I/R + enoxaparin group compared with the I/R + NAC group. Since AMH is produced by pre-antral but mostly small antral follicles, it is not surprising that this difference was not reflected by AMH concentrations, which showed only a non-significant trend towards less decrease in the I/R + NAC group. Ovarian tissue cryopreservation is a promising technology for preserving fertility in paediatric and adult cancer patients undergoing gonadotoxic chemotherapy or radiotherapy as well as in women with benign disease such as premature ovarian failure (Donnez and Dolmans, 2015; Kim, 2010; Skaznik-Wikiel et al., 2011). However, the loss of follicles and ultimate survival of the ovarian autograft is thought to be related to the degree of I/R injury (Donnez et al., 2006; Kim et al., 2001). An initial period of ischaemia followed by reperfusion occurs as part of the ovarian tissue reimplantation process, resulting in generation of excess reactive oxygen species that can damage the transplanted ovarian tissue (Mahmoodi et al., 2015). The findings of our study may therefore also be applicable for protection of transplanted ovarian autografts in the setting of ovarian tissue cryopreservation.

Please cite this article in press as: Gulcin Sahin Ersoy, Meryem Eken, Reshef Tal, Deniz Oztekin, Belgin Devranoglu, Ecmel Isik Kaygusuz, Ozge Cevik, N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.03.009

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ARTICLE IN PRESS 8 A potential limitation of this study is the timing of administration of the treatment agents. We administered the drugs 30 min before ovarian torsion. However, since the diagnosis of ovarian torsion is frequently delayed in clinical practice, the protective effects on the ovary observed in this study may be different from the potential outcomes in clinical practice. A longer than 3 h duration of torsion may give rise to more detrimental and irreversible effects on ovarian reserve. Therefore, future studies are warranted to evaluate different durations of torsion and to analyse long-term effects of the drugs on ovarian reserve. On the other hand, it is possible to use NAC for the prevention of anticipated I/R injury in ovarian transplantation patients since transplantation is a planned surgery but, similarly, further studies with modified doses and different timing of NAC administration are needed to optimize protocols and translate the findings to clinical trials of ovarian transplantations. In summary, the biomarkers of oxidative damage have clearly demonstrated in the present study that both enoxaparin sodium and NAC have positive antioxidative protective effects in the setting of ovarian I/R injury. In addition, NAC appears to be more effective agent than enoxaparin in terms of protection of ovarian damage and reserve, probably through its more potent antioxidant effects.

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Acknowledgement

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The authors thank Graciela Krikun, PhD (Yale University School of Medicine, New Haven, CT, USA) and Burak Ersoy, Assistant Prof. (Maltepe University School of Medicine, Istanbul, Turkey) for their assistance with manuscript review and editing.

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Q4

Uncited references

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Rodriguez-Wallberg, Oktay, 2014

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References

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832 Declaration: The authors report no financial or commercial conflicts of interest.

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Received 6 November 2015; refereed 23 March 2016; accepted 23 March 2016.

Please cite this article in press as: Gulcin Sahin Ersoy, Meryem Eken, Reshef Tal, Deniz Oztekin, Belgin Devranoglu, Ecmel Isik Kaygusuz, Ozge Cevik, N-acetylcysteine leads to greater ovarian protection than enoxaparin sodium in a rat ovarian torsion model, Reproductive BioMedicine Online (2016), doi: 10.1016/j.rbmo.2016.03.009

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