Evaluation of the Protective Effects of CoQ 10 on Ovarian I/R Injury: An Experimental Study

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Original Article

Gynecol Obstet Invest 2013;76:100–106 DOI: 10.1159/000353425

Evaluation of the Protective Effects of CoQ 10 on Ovarian I/R Injury: An Experimental Study

Ali Ozler a Abdulkadir Turgut a Neval Yaman Görük a Ulas Alabalik b

Mustafa Kemal Basarali c Fatih Akdemir d

Departments of a Obstetrics and Gynecology, b Pathology, and c Medical Biochemistry, School of Medicine, and d Veterinary Faculty, Department of Animal Nutrition, Dicle University, Diyarbakır , Turkey

Conclusions: Conservative surgery (detorsion) was found to provide inadequate protection to ovarian tissue. The results of this study suggest that CoQ 10 could be useful for the pro-tection of ovarian tissue before conservative surgery.

© 2013 S. Karger AG, Basel

Introduction

Adnexal torsion is defined as total or partial rotation of the ovary, the fallopian tube, or both, around its vas-cular axis [1] . Torsion is rarer than other causes of acute abdominal pain requiring surgery. However, it is a com-mon gynecologic surgical emergency, with a prevalence of 2.7% [2] . Ischemia and reperfusion (I/R) injury is a major cause of adnexal tissue damage after torsion and detorsion. If treatment is delayed, the ischemia may result in necrosis of the ovary. Rapid diagnosis and ear-ly surgical treatment are the most successful strategies for preserving fertility and preventing the loss of the ovary.

A pathologic process called reperfusion injury is caused by detorsion, performed to maintain proper cir-culation to the ovary. During reperfusion, depending on the supply of excess molecular oxygen to ischemic tissue,

Key Words

Adnexal torsion · Coenzyme Q 10 · Oxidative stress

Abstract

Background: The aim of this study was to investigate the protective effects of coenzyme Q 10 (CoQ 10 ) on ovarian isch-emia/reperfusion injury in an experimental rat adnexal tor-sion model. Methods: 48 female adult Wistar albino rats, weighing 220–250 g, were randomly equally divided into six groups (n = 8): sham, torsion, detorsion, sham+CoQ 10 , torsion+CoQ 10 , and detorsion+CoQ 10 groups. Bilateral ad-nexal torsion was performed for 3 h in all groups, except the sham and sham+CoQ 10 groups. Bilateral adnexal detorsion was performed on the detorsion and detorsion+CoQ 10 groups. CoQ 10 was injected intraperitoneally 30 min before the sham operation, torsion, and detorsion. Results: The tor-sion and detorsion groups had significantly higher histolog-ic evaluation scores, as well as higher MDA levels, TOS values, and oxidative stress index values than the sham group. A strong correlation between total histologic evaluation scores for ischemia/reperfusion injury and the oxidative stress in-dex was found. The mean oxidant marker levels and histo-pathologic scores for the ovarian tissue significantly de-creased after using CoQ 10 , which is a potent antioxidant.

Received: October 18, 2012 Accepted after revision: May 30, 2013 Published online: July 23, 2013

Ali Özler, MD Department of Obstetrics and Gynecology Medical Faculty of Dicle University TR–21280 Diyarbakır (Turkey) E-Mail draliozler @ gmail.com

© 2013 S. Karger AG, Basel0378–7346/13/0762–0100$38.00/0

www.karger.com/goi

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Effects of CoQ 10 on Ovarian I/R Injury Gynecol Obstet Invest 2013;76:100–106DOI: 10.1159/000353425

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acute ischemic injury is worsened by the products of ox-idative stress (free radicals and reactive oxygen species) [3, 4] . Various markers have been used as antioxidant markers (such as superoxide dismutase, glutathione, and myeloperoxidase activity) and oxidative stress markers (such as malondialdehyde and nitric oxide) to evaluate I/R injury in rat ovarian tissues in previous studies [3, 5, 6] . However, separate evaluation of these markers was not only impractical but also insufficient for a compre-hensive assessment. Total antioxidant status (TAS) has been used to estimate overall antioxidant status [7] . Like-wise, total oxidant status (TOS) is measured to determine the overall oxidation state [8] . The oxidative stress index (OSI), calculated as the ratio of TOS to TAS, is consid-ered a more accurate index of oxidative stress in the tis-sue because it is a comprehensive measurement of TAS and TOS.

Coenzyme Q 10 (CoQ 10 ), involved in the mitochondri-al electron transport chain, is a strong antioxidant which provides membrane stabilization [9] . Because the major-ity of CoQ 10 is in the quinol form in the cell membranes [10] , it can be an effective antioxidant [11] . Many studies on the inhibitory effects of CoQ 10 on lipid peroxidation exist in the literature [12–14] . In addition, CoQ 10 has been found to be as effective as α-tocopherol against free oxygen radicals [15] . CoQ 10 is a cofactor in the produc-tion of adenosine triphosphate by oxidative phosphoryla-tion [9] . Its functions, such as salvaging free oxygen radi-cals, inhibiting lipid peroxidation, and increasing use of oxygen in energy production, played an important role in the selection of CoQ 10 . Many studies have investigated the protective effects of CoQ 10 against I/R injury in vari-ous tissues [12–14] . The successful results obtained from previous studies of CoQ 10 administration encouraged us to attempt such treatment with a model of ovarian I/R injury. Therefore, the aim of the present study was to in-vestigate the protective effects of CoQ 10 on I/R injury in an experimental rat adnexal torsion model.

Materials and Methods

A total of 48 female adult Wistar albino rats, weighing 220–250 g, were used in this study. The rats were obtained from Dicle University’s Experimental Animal Laboratory (Dusam) after per-mission was granted by the Dicle University Ethics Committee for Animal Research (Dühadek). The rats were kept for at least 7 days under appropriate temperature and humidity conditions and a 12-hour light cycle, and provided with sufficient fluids and feed. The rats were initially numbered randomly and then randomly equally divided into six groups (n = 8): sham (I), torsion (II), detorsion (III), sham+CoQ 10 (IV), torsion+CoQ 10 (V), and detorsion+CoQ 10

(VI). The numbers and the group numbers of the rats were re-corded by one of the investigators. The investigators responsible for the biochemical and histological evaluations were blind to the randomization until the end of the study.

Each rat was weighed and anesthetized with intramuscular ketamine hydrochloride (50 mg/kg Ketalar; Eczacibasi, Istanbul, Turkey) and xylazine hydrochloride (10 mg/kg Rompun; Bayer Türk İlaç Ltd, Istanbul, Turkey). Sterile conditions were provided for the surgical procedure. Laparotomy was performed with a midline abdominal incision of 2.5 cm. In the sham group, after the uterine horns and adnexa were observed for 1 min, the abdominal wall was closed with 3/0 silk sutures (sham operation). Re-lapa-rotomy was performed 3 h later, and both ovaries were surgically removed. The adnexal torsion procedure was carried out as fol-lows: the adnexa were rotated 360 o in a clockwise direction, in-cluding the tubal and ovarian vessels, and then fixed to the ab-dominal wall [3] . In the torsion group, after bilateral adnexal tor-sion (ischemia) was performed for 3 h, both ovaries were surgically removed with re-laparotomy. In the detorsion group, bilateral adnexal torsion (3-hour ischemia) and bilateral detorsion (3-hour reperfusion) were carried out. After a total time of 6 h, both ovaries were surgically removed. In the sham+CoQ 10 group, CoQ 10 (10 mg/kg; Sigma Chemical Co., St. Louis, Mo., USA) was injected intraperitoneally 30 min before the sham operation.

The route of administration and dose of CoQ 10 reported suc-cessful against I/R injury of various tissues in earlier experimental studies were preferred in this study [13, 16] . Oral and intraperito-neal administrations of CoQ 10 to rats have been compared in pre-vious studies [17, 18] . Oral administration of CoQ 10 increases its concentration in the liver, but not in other organs, such as the heart, kidney, skeletal muscle, and brain. In addition, intraperito-neal administration is more suitable than oral administration for the ovarian torsion model, which requires the rapid antioxidant effects of CoQ 10 . After the 3-hour period following the sham op-eration, both ovaries were surgically removed. In the torsion+CoQ 10 group, CoQ 10 was injected intraperitoneally 30 min before per-forming the adnexal torsion. After an ischemia period of 3 h, both ovaries were surgically removed. In the detorsion+CoQ 10 group, bilateral adnexal torsion (3-hour ischemia) and bilateral detorsion (3-hour reperfusion) were carried out. CoQ 10 was injected intra-peritoneally 30 min before the onset of the reperfusion process. After a total time of 6 h, both ovaries were surgically removed. Af-ter these surgical procedures had been performed, the rats were sacrificed. One of the ovaries was cleaned of adherent soft tissues and stored in a freezer at –80 o C for biochemical analysis; the other ovary was transferred to 10% formaldehyde solution for histologic examination. All of the samples were delivered to the laboratories in sequentially numbered containers.

Histological Evaluation The ovarian tissues were fixed in 10% neutral buffered formalin

solution for 48 h, dehydrated, cleared, and embedded in paraffin, as is usual. Serial tissue sections, 4 μm thick, were cut using a mi-crotome (Leica RM2125RTS) and stained with hematoxylin and eosin for general morphological observation. All sections were in-vestigated with a light microscope (Nikon Eclipse 80i), and the pieces were photographed. At least five microscopic areas were examined to score the specimens semiquantitatively. The criteria for ovarian injury were follicular cell degeneration (granulosa cells), vascular congestion, hemorrhage, and inflammation (neu-

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trophil infiltration). Each specimen was scored for each criterion using a scale ranging from 0 to 3 (0: none; 1: mild; 2: moderate; 3: severe) [19] . The ovary sections were analyzed in a blinded fashion by the same pathologist.

Biochemical Steps and Analyses Ovarian tissues for the estimation of tissue oxidant and anti-

oxidant levels were prepared at 4 ° C. The tissues were weighed and cut into small pieces and homogenized in 10 volumes of ice-cold phosphate buffer solution (50 m M /l, pH 7.0) using a homogenizer (Ultra-Turrax T8 dispersing homogenizator; IKA-Werke GmbH, Staufen, Germany). The homogenate was centrifuged at 15,000 rpm for 10 min at 4 ° C to obtain a supernatant. Supernatant sam-ples were used to determine TOS and TAS.

Measurement of Malondialdehyde (MDA): MDA levels were es-timated by the double heating method of Draper et al. [20] . Accord-ing to the method, spectrophotometric measurement of the color generated by the reaction of thiobarbituric acid (TBA) with MDA was performed. The concentration of MDA was calculated using the absorbance coefficient of the MDA-TBA complex (absorbance coefficient of 1.56 × 105 cm –1 M –1 ) and expressed as μmol/l. All of the MDA analytical steps were performed in the Biochemistry De-partment Laboratory (Dicle University Medical Faculty).

Measurement of Paraoxonase (PON-1) Activity: PON-1 was quantified spectrophotometrically via the modified Eckerson meth-od [21] using 0.1 M glycine-NaOH buffer pH 10, 1 m M calcium chlo-ride, 0.3 M sodium chloride, and 2 m M paraoxone in 1 ml of the reac-tion mixture containing 50 μl of serum. The reaction was followed for 5 min at 37 ° C by monitoring the appearance of p -nitrophenol at 405 nm. One unit of PON-1 activity is defined as 1 nmol of p -nitro-phenol formed per minute under the above assay conditions.

Measurement of TAS: The TAS of supernatant fractions was determined using a novel automated measurement method devel-oped by Erel [7] . In this method, hydroxyl radical, which is the most potent biological radical, is produced. The antioxidative ef-fect, initiated by the produced hydroxyl radical, of the sample against potent free radical reactions is then measured. The assay has excellent precision values of lower than 3%. The results are ex-pressed as nmol trolox Eq/mg protein.

Measurement of TOS: The TOS of supernatant fractions was determined using a novel automated measurement method devel-oped by Erel [8] . Oxidants present in the sample oxidize the fer-rous ion- o -dianisidine complex to produce ferric ion. The oxida-tion reaction is enhanced by glycerol molecules, which are abun-dantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide and the results are expressed in terms of nmol H 2 O 2 Eq/mg protein.

Determination of OSI: The percent ratio of the TOS level to the TAS level was accepted as the OSI. The OSI value was calculated according to the following formula [22] : OSI (arbitrary unit) = TOS (nmol H 2 O 2 Eq/mg protein)/TAS (nmol trolox Eq/mg pro-tein). The results are expressed as arbitrary units.

Statistical Analysis Means and standard deviations were used to describe numeri-

cal variables. The Kolmogorov-Smirnov test was used to evaluate the distribution pattern of the data. Statistical comparisons be-

tween groups were performed using the Mann-Whitney U test. The correlation coefficient was determined using the Spearman test. The statistical package SPSS for Windows 15.0 (SPSS, Inc., Chicago, Ill.) was used to analyze the data. p values <0.05 were ac-cepted as significant.

Results

The histopathologic evaluation scores are presented in table 1 . Histologic ovarian specimens of the torsion and detorsion groups had higher scores for vascular conges-tion, hemorrhage, and inflammatory cell infiltration, when compared with the sham group (p < 0.05). In addi-tion, the mean score for degeneration of the antral fol-licular cells, indicating acute tissue damage, was signifi-cantly higher in the torsion and detorsion groups than in the sham group (1.8 ± 0.8 and 2.1 ± 0.6 vs. 0.6 ± 0.4, re-spectively; p < 0.05). Total histologic evaluation scores, determined by collecting the histopathologic scores of four ovarian injury parameters, were significantly higher in the torsion and detorsion groups than in the sham group (7.1 ± 2.2 and 9.5 ± 1.0 vs. 3.2 ± 1.5, respectively; p < 0.05 and p < 0.01, respectively).

The MDA level, TOS, TAS, and OSI values as well as the PON-1 enzyme activities in the rat ovarian tissues of the groups are shown in table 2 . The torsion and detor-sion groups had significantly higher MDA, TOS, and OSI values compared to the sham group (p < 0.05). PON-1 enzyme activity was significantly higher in the torsion group compared to the sham group (p < 0.05). A strong correlation between the total histologic evaluation scores and the OSI values, which are total scores of histological evaluation and measurement of oxidative stress, was found in the sham, torsion, and detorsion groups (r = 0.701, p < 0.001).

As seen in table 2 , the mean MDA, TOS, and OSI values in the ovarian tissues of the rats treated with CoQ 10 were significantly decreased compared to the control groups (sham, torsion, and detorsion groups; p < 0.05). Similarly, except for vascular hemorrhage, the histopathologic evaluation scores in the rats treated with CoQ10 were decreased compared to the control groups (p < 0.05). Comparison of the levels of oxidative stress markers and histological evaluation scores for the treated and untreated groups are shown in tables 1 and 2 . Comparisons of histologic morphology in the ovaries of the treated and untreated groups are illustrated in figure 1 .

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Table 2. MDA, TOS, TAS, and OSI levels as well as the PON-1 enzyme activities in the rat ovarian tissues of all groups (n = 8)

Histopathologic scores Controlgroups(n = 8)

Mean±SD 95% CI for mean (lower–upper)

p values Treatment groups(n = 8)


p values

TOS, mmol H2O2 Eq/g protein

Sham 14.3±2.1 12.06–16.14 – Sham+CoQ10 10.5±3.3 7.72–13.29 0.012*Torsion 25.5±3.1 22.94–28.15 0.001* Torsion+CoQ10 17.9±1.6 16.48–19.31 0.001#Detorsion 30.8±5.1 26.61–35.15 0.001* Detorsion+CoQ10 20.2±5.3 15.73–24.6 0.005+

TAS, mmol trolox Eq/g protein


OSI Sham 29.5±12.8 18.78–40.18 – Sham+CoQ10 20.2±16.5 6.34–34.0 0.011*Torsion 58.5±15.4 45.63–71.47 0.005* Torsion+CoQ10 32.2±5.5 27.55–36.85 0.016#Detorsion 143.8±43.2 107.71–179.88 0.012* Detorsion+CoQ10 66.7±38.5 34.57–99.02 0.021+

MDA, nmol/g protein Sham 13.4±5.1 9.21–17.72 – Sham+CoQ10 7.9±0.69 7.32–8.48 0.001*Torsion 21.1±2.8 18.75–23.44 0.012* Torsion+CoQ10 16.9±2.6 14.69–19.12 0.016#Detorsion 22.9±4.7 18.92–26.93 0.012* Detorsion+CoQ10 20.1±2.9 17.56–22.57 0.021+

PON-1 activity, U/mg protein


* When compared with the sham group. # When compared with the torsion group. + When compared with the torsion group.

Table 1. Histopathologic evaluation scores of the rat ovarian tissues in all groups (n = 8)

Histopathologic scores Controlgroups(n = 8)


p values Treatment groups(n = 8)


pvalues

Vascular congestion Sham 1.6±0.5 1.19–2.05 – Sham+CoQ10 1.4±0.7 0.70–2.15 0.688*Torsion 2.6±0.7 2.00–3.24 0.010* Torsion+CoQ10 2.8±0.3 2.50–3.20 0.562#Detorsion 2.7±0.4 2.36–3.13 0.002* Detorsion+CoQ10 2.8±0.4 2.40–3.26 0.717+

Hemorrhage Sham 0.5±0.3 0.20–0.91 – Sham+CoQ10 0.0±0.0 0.00–0.00 0.035*Torsion 1.3±0.9 0.60–2.14 0.040* Torsion+CoQ10 0.5±0.7 –0.15–1.29 0.087#Detorsion 2.8±0.3 2.57–3.17 0.000* Detorsion+CoQ10 1.5±0.8 0.62–3.37 0.002+

Inflammatory cell Sham 0.5±0.3 0.21–0.89 – Sham+CoQ10 0.0±0.0 0.00–0.00 0.035*infiltration Torsion 1.2±0.4 0.86–1.63 0.015* Torsion+CoQ10 0.1±0.3 –0.20–0.49 0.002#

Detorsion 1.7±0.4 1.36–2.13 0.002* Detorsion+CoQ10 0.6±0.4 0.12–1.20 0.005+

Cellular degeneration Sham 0.6±0.4 0.19–1.05 – Sham+CoQ10 1.0±0.5 0.46–1.53 0.209*Torsion 1.8±0.8 1.17–2.57 0.006* Torsion+CoQ10 1.0±0.5 0.46–1.53 0.042#Detorsion 2.1±0.6 1.58–2.66 0.001* Detorsion+CoQ10 1.0±0.6 0.33–1.66 0.011+

Total score Sham 3.2±1.5 1.92–4.57 – Sham+CoQ10 2.4±1.1 1.38–3.47 0.340*Torsion 7.1±2.2 5.25–8.99 0.003* Torsion+CoQ10 4.5±1.5 3.17–5.96 0.035#Detorsion 9.5±1.0 8.60–10.39 0.001* Detorsion+CoQ10 6.0±2.0 3.91–8.09 0.002+

* When compared with the sham group. # When compared with the torsion group. + When compared with the torsion group.

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Discussion

I/R injury due to ovarian torsion and detorsion leads to biochemical and morphological changes in the ovarian tissue. In this study, a histopathological evaluation of I/R injury was performed using a cellular scoring system. Sev-eral studies in the literature have evaluated ovarian I/R injury, but not quantitatively [3, 23] . In the present study,

ovarian specimens from the torsion group had higher scores for vascular congestion, hemorrhage, inflamma-tory cell infiltration, and follicular cell degeneration when compared to the sham group. The detorsion group had the highest score for four ovarian injury parameters. Pre-vious studies using a cellular scoring system have report-ed more ovarian damage after detorsion than due to the torsion [24, 25] . Ischemia, which causes insufficient blood

Fig. 1. Hematoxylin and eosin-stained ovarian sections. a , b In the sham group ( a ) and sham+CoQ 10 group ( b ), normal ovarian tissue architecture (×100). c , d . In the torsion group, ovarian sections show-ing severe vascular congestion (arrows) ( c ) (×100), and an antral fol-licle structure containing severe degeneration of granulosa cells (ar-rows), as well as a large number of apoptotic cells ( d ) (×400). e , f In the torsion+CoQ 10 group, ovarian sections showing mild vascular congestion (arrow) ( e ) (×100), and an antral follicle structure con-taining mild degeneration of granulosa cells (arrow), as well as a small

number of apoptotic cells ( f ) (×400). g , h In the detorsion group, ovar-ian sections showing severe hemorrhage (star) ( g ) (×100), and an antral follicle containing severe degeneration of granulosa cells (ar-rowheads), as well as a large number of apoptotic cells, vascular con-gestion (arrows), and hemorrhage (star) ( h ) (×400). i , k In the detorsion+CoQ 10 group, ovarian sections showing moderate vascular congestion (arrows) and mild hemorrhage (star) ( i ) (×100), as well as an antral follicle structure containing mild degeneration of granulosa cells (arrows) and a small number of apoptotic cells ( f ) (×400).

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flow and insufficient oxygen support, leads to a decrease in the production of ATP, an increase in the production of lactic acid, and the accumulation of lipid peroxidase in tissue [5] . The production of free oxygen radicals and the accumulation of activated neutrophils, which release re-active oxygen species, are induced by reperfusion [26] . These reactive oxygen species lead to a further increase in the cellular damage in ischemic tissues, through the per-oxidation of lipids in mitochondrial and cell membranes [5] . In this study, the total scores for cellular damage were well correlated with the oxidative stress in the torsion and detorsion groups.

MDA, the end product of lipid peroxidation, as a per-oxidized polyunsaturated fatty acid, is an important marker of oxidative stress. PON-1, which hydrolyzes lip-id peroxides in oxidized lipoproteins, is an important marker for evaluating the antioxidant defense system. In previous studies which investigated oxidative damage in various tissues, an increase in the serum levels of MDA and a decrease in PON-1 activity were reported [3, 27, 28] . In addition, for the first time, we measured both TAS and TOS using new measurement methods developed by Erel [7, 8] to more accurately assess oxidative stress. We found increased MDA, TOS, and OSI values as markers of oxidative stress, and decreased levels of TAS and PON-1 activity as markers of antioxidants in the torsion and detorsion groups compared to the sham group. These re-sults indicate increased oxidative stress and decreased an-tioxidation in ovarian torsion.

CoQ 10 has been used for the prevention and treatment of I/R injury in many organs, and successful results have been reported [12–14] . To our knowledge, the effects of CoQ 10 on ovarian I/R injury have not been investigated previously. CoQ 10 , involved in the electron transport chain, is known to scavenge free radicals and contribute to antioxidant defenses in vivo [9] . Previous studies have demonstrated that administration of CoQ 10 as a potent antioxidant medication increases superoxide dismutase

levels, reduces levels of NO and MDA scavenging free radicals, and inhibits lipid peroxidation [13, 14] . In an experimental brain I/R model, Kalayci et al. [14] found that levels of MDA, one of the products of lipid peroxida-tion, were significantly lower in the treatment group, which received a single dose of 10 mg/kg CoQ 10 intra-peritoneally, than in the control group. However, they found no significant difference between the levels of PON enzyme activity of the different groups. In a different ex-perimental study, Erol et al. [13] analyzed the efficacy of CoQ 10 administration on experimental testicular I/R in-jury, and they reported that testicular MDA levels de-creased after the treatment.

In agreement with the above-mentioned studies, we found that administration of CoQ 10 treatment before conservative surgery (detorsion) led to a significant de-crease in MDA levels. In addition, in our study, PON-1 activity in ovarian tissue increased after administration of CoQ 10 . Antioxidant status (including superoxide dis-mutase, glutathione, PON-1, and myeloperoxidase activ-ity) and oxidative status (including MDA and nitric oxide levels) were evaluated comprehensively by measuring the TAS and the TOS and calculating the OSI. Concordant with the results of previous studies, the oxidative status could be reduced and the antioxidant status could be in-creased using CoQ 10 treatment.

In conclusion, conservative surgery (detorsion) was found to provide inadequate protection to ovarian tissue. The results of this study suggest that CoQ 10 may reduce I/R injury in ovarian tissue due to the potent antioxidant activity of CoQ 10 . However, these results need to be con-firmed by further clinical trials prior to recommending CoQ 10 as treatment against ovarian I/R injury.

Disclosure Statement

The authors have no conflicts of interest to disclose.

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Evaluation of the Protective Effects of CoQ 10 on Ovarian I/R Injury: An Experimental Study