{"paper_id":"dd7ac1e2-19f9-4dfb-a3a1-e45801e81d62","body_text":"Free radicals cause oxidative damages to the\ncell membrane lipid content ( 1 ). The role of the\nreactive oxygen species (ROS) in peroxidation\nof lipids and their interference with sperm function, ovum function, and human reproduction\nhave been reported ( 2 ). Natural byproduct of\nmetabolism is ROS which includes the superoxide anion (O 2 \n• –) and the hydroxyl radical (OH).\nROS can induce DNA fragmentation, protein\noxidation, lipid per oxidation and cellular damage ( 3 - 5 ). Within a cell, ROS is neutralized by\nthe antioxidants ( 6 ). Paraoxonase (PON) is one\nof the strong antioxidants in the serum and the\nfollicular fluid (FF). PON1 and PON3, which\nare both associated in serum with high density\nlipoprotein (HDL) cholesterol (C), protect the\nserum lipids from oxidation, probably through\ntheir ability to hydrolyze specific oxidized lipids ( 7 ,  8 ). The  PON  gene family consists of\nthree members of  PON1  gene,  PON2  gene and\nPON3 gene, encoding  PON  enzyme family ( 4 ).\nAll three genes of  PON  have been preserved in\nmammals, and this fact indicates the important\nphysiological role of this antioxidant enzyme\n( 9 ). There is a higher PON enzymatic activity\nin FF compared with serum, which has been\nattributed to PON expression and secretion by\ngranulosa cells in FF ( 10 ).\nPON3 is synthesized in the liver and carried in the\nblood in association with HDL. PON3 is also able\nto prevent the oxidation of low density lipoproteins\n(LDL) ( 11 ). Plasma concentration of PON3 is 100\ntimes less than that of PON1 ( 12 ,  13 ). In recent studies, the activity of this enzyme in FF has been reported ( 14 ) and is calculated to be three times more than\nits original concentration in serum ( 15 ). However,\nthe role of PON3 in women’s fertility has not yet\nbeen fully studied. Considering the strong antioxidant property of PON3 and its high concentration in\nthe FF, it is likely that this enzyme plays an important\nrole in the oogenesis, eggs quality and fertilization.\nIn the present study, PON3 activity and the ratio of\nantioxidant to peroxidation in the FF of women with\nmale factor infertility (MFI) and with female factor\ninfertility (FFI) were compared after ovarian stimulation, while their variation with respect to the number of oocytes, embryo cell number (ECN), embryo\nfragmentation score (EFS) and fertilization rate (FR)\nwere statistically analyzed.\n\nIn this cross-sectional study, we gained the\nagreement of the Ethical Committee of Tabriz\nUniversity of Medical Science, and all patients\ngave written informed consent. Fifty infertile couples referred to Tabriz Alzahra Women’s Hospital,\nTabriz, Iran, for infertility treatment using assist-\ned reproductive technique (ART) were selected\nwithin a three-month period. Out of 50 couples, 30\nwomen with MFI were used as the control group\n(MFI group), and the remaining 20 women with\nFFI were used as the test group (FFI group).\nAmong selected patients, 60% of infertile\npartner were male and 40% were female. Lack\nof infections and husband with no smoking habit were defined as including criteria. The long\nprotocol, gonadotropin-releasing hormone agonist (GnRHa) and human menopausal gonado-\ntropin (HMG) were used for all subjects as the\ntreatment protocol for the stimulation of ovulation ( 16 ). On the 14 th \nday of the menstrual cycle,\nthe follicles larger than 3 mm were punctured\nand the FF was extracted. After separating the\noocytes from FF, the remaining was centrifuged\nand the supernatant was kept at -86˚C for further studies. The collected oocytes were incubated at 37˚C, 5% CO 2 \nand 95% humidity for\n4 hours, and then were used for  in vitro  fertilization (IVF). In couples with abnormal sperm\nparameters such as low sperm count, low sperm\nmotility and morphological defects, intracytoplasmaic sperm injection (ICSI) was used for\noocytes fertilization, otherwise IVF was performed. Identification of zygotes was carried\nout 18 hours after insemination through the appearance of two pronuclei (2PN). All embryos\nwere recultured in standard culture medium\n(ISM1) at 37˚C, 5% CO 2 \nand 95% humidity for\n24 hours. On the second day of the culture, the\nmorphology of the embryos was determined using ECN and EFS ( 17 ,  18 ). In addition, FR was\nused as an oocyte fertilization index. FR was\ncalculated as the number of fertilized oocytes/\nnumber of mature oocytes ×100.\nPON3 has a unique ability to metabolize the lipophilic agents such as lovastatin and simvastatin ( 19 ).\nPON3 activity was determined through monitoring of the conversion of simvastatin (SV) to β,δ-\ndihydroxyacid simvastatin (SVA) using reverse\nphase-high performance liquid chromatography\n(RP-HPLC) method as described before ( 20 ) with\nslight modification as follows: SV (120 µM) was\nincubated with FF at 37˚C for 60 minutes, and the\nreaction was terminated by addition of acetonitrile. The sample was centrifuged at 6000 rpm for\n6 minutes, and the amount of SVA in the supernatant was analyzed using a high-performance\nliquid chromatography (HPLC) system (Waters\nAssociates, Norwich, Cheshire, UK) which consisted of a Waters 515 pump, Waters 717 plus Autosampler, Waters 2487, and Dual λ Absorbance\nDetector. The mobile phase was a mixture of 100\nmM monopotassium phosphate (pH=4.5)/ acetonitrile (27/73 v/v). Separations were performed on a\nC18 column (Phenosphere-LUNA, 5 μM, 250×4.6\nmm) with a C18 guard column (Perfectsil Target\nODS- 3 , 5 μM, 10×4 mm). The effluent was monitored by ultraviolet (UV) detection at 239 nm at a\nflow rate of 1 ml/minute.\nMDA levels in FF were determined by the\nthiobarbituric acid (TBA) method and expressed\nas nmol MDA formed/mL FF ( 20 ). Briefly, 0.5\nml FF was shaken with 2.5 ml of 20% trichloroacetic acid (TCA) in a 10 ml centrifuge tube.\nThen, 1ml of 0.67% TBA was added to the mixture, shaken, and heated in a boiling water bath\nfor 60 minutes and it was cooled rapidly. MDA\ncontent in the serum was spectrophotometerically determined at 532 nm. The calibration\ncurve was plotted with 0.1 to 20 μmol/L tetraethoxypropane (TEP).\nTotal antioxidant status (TAS) was measured in\nFF using a commercial kit (Randox Laboratories,\nFrance). The assay was performed by incubation of 2,2'-azino-di-( 3 -ethylbenzthiazoline\nsulphonate) (ABTS) with a peroxidase (methmyoglobin) and hydrogen peroxide to develop\na relatively stable blue-green color, which subsequently measured at 600 nm ( 21 ). Trolox, a\ntraditional standard for TAS measurement, was\nused to calculate Trolox molar equivalent ( 22 ).\nHDL-C level was measured after extraction of\nthe particles from FF by means of phosphotungstic\nsolution and centrifugation. The amount of cholesterol in the supernatant was determined spectrophotometrically by cholesterol oxidase (Pars\nAzmon, Iran).\nAll data were expressed as mean ± SD. The\nnormality test showed that all data were normal\nor nearly normally distributed. Statistical comparisons were performed using t test. ANOVA\nwas used to evaluate the differences between the\nmeans of more than two groups. A value of p<0.05\nwas considered statistically significant. Statistical\nanalysis was carried out by Statistical Package for\nthe Social Sciences (SPSS; version 16, SPSS Inc.,\nChicago, USA).\n\nThe general information of the population study is\nshown in table 1. In the test group, 68% of the patients had normal ovaries, 18% had polycystic ovaries and 14% had abnormal ovaries (small ovaries,\novaries which were operated upon, and no eggs were\nproduced), 4% of the test subjects had abnormal uteruses (small uterus and endometriosis) and 30% suffered from abnormal menstrual cycles.\nGeneral information of the studied women\nPCOS; Polycystic ovary syndrome.\nPON3 activity in the FF of the MFI group\nwas found to be significantly (p<0.001) higher than that in the women with FFI (4.7 ± 0.8\nvs. 3.8 ± 0.7 µmol/min/ml). In contrast, the\nconcentration of MDA, a lipid peroxidation\nindicator, in the FF of MFI group was 3.1 ±\n1.4 nmol/ml compared to the value of 4.2 ±\n1.7 nmol/ml measured in FF of women with\nFFI (p=0.024). Therefore, the ratio of antioxidant to peroxidation, which was evaluated as\nPON3/MDA value in the control group, was\nalso higher than the corresponding value in\nthe women with FFI. No statistically significant difference was found in the HDL-C level\nin the FF of both groups ( Table 2 ). Although\nthere was no significant difference in the TAS\nlevels in the women with MFI and FFI, the ratio of PON3 to TAS in two groups were statistically different (p= 0.013).\nBiochemical factors measured in the FF based\non the number of oocytes are displayed in table\n3. No significant difference was observed in the\nstudied factors with respect to the number of oocytes.\nFollicular fluid biochemical parameters in the studied population\nMFI; Male factor infertility, FFI; Female factor infertility and TAS; Total antioxidant status.\nFollicular fluid biochemical parameters according to the oocyte numbers in studied women\nTAS; Total antioxidant status. Values are expressed as mean ± SD.\nComparison of number of the oocytes (t=1.32,\np=0.13), EFS (t=0.81, p=0.41), ECN (t=0.89,\np=0.32) and FR (t=1.52, p=0.11) between women\nwith MFI and FFI by t test showed no significant\ndifference between IVF and ICSI techniques. The\nrelations between FF biochemical parameters with\nEFS, ECN and FR were assessed using regression\nanalysis and the results have been summarized\nin table 4. A significant negative correlation was\nfound between PON3 activity (r=-0.65, p=0.02)\nand PON3/MDA (r=-0.63, p=0.001) with EFS,\nwhereas there was a positive correlation between\nEFS and MDA (r=0.55, p=0.002) which indicates\nthat an increase in antioxidant/peroxidation value\nis accompanied with an increase in the embryo\nquality. No significant correlation was found between ECN and FF biochemical parameters. A\nnegative correlation between FR and MDA (r=-\n0.42, p=0.02), while a positive relations between\nFR and PON3 activity (r=0.56, p=0.004), HDL-\nC (r=0.35, p=0.041) and PON3/MDA (r=0.59,\np=0.001) could be indicative of the beneficial\neffects of antioxidant in the success of ART. The\nsame pattern was also observed when correlations\nwere examined separately for women with MFI\nand FFI.\nCorrelation of follicular fluid biochemical factors with embryo quality and fertilization rate\nEFS; Emberyo fragmentation score, ECN; Emberyo cell number, FR; Fertilization rate and TAS; Total antioxidant status.\nThe numbers in brackets are p values.\n\nIt has been shown that the concentration of lipidic hydroperoxides and active substances of\nthiobarbituric acid in the FF is lower than serum\nin women who underwent IVF. This confirms the\npresence of a suitable antioxidant in oocyte’s environment prior to ovulation ( 23 ). The concentration of this enzyme in FF is much higher than its\nconcentration in serum. The results of this study\nshows that PON3 activity in FFI is significantly\nlower in comparison with the MFI group. According to a study by Closshey et al. ( 15 ) the level of\nPON3 activity in the FF in 14 infertile women who\nunderwent IVF was higher in comparison with serum. Moreover, it was shown that there is a significant positive correlation between PON3 and the\nrate of laboratory pregnancy and fertility. These\nresults match the results of this study. These findings along with high ratio of PON3/TAS in MFI\ngroup indicate the important role of PON3 in FF\nand in oogenesis.\nPON3 is synthesized in the liver, attached to\nHDL and carried by the fluids in the body ( 11 ). It\nhas been shown that HDL is the only lipoprotein\nwhich is present in FF ( 24 ). Therefore, it seems\nthat HDL-C concentration in FF is associated with\ngrowth, oocyte maturation and rate of fertility in\nIVF ( 25 ). In the present study, no significant difference was found between the HDL-C levels of\nFF in MFI and FFI groups. Moreover, there was no\nsignificant association between HDL-C concentration and the number of oocytes. Contrary to the\nresults of this study, Browne et al. have shown that\nthe level of HDL-C of FF affects the number and quality of oocyte during stimulation of ovulation\n( 26 ). It has been shown that older ages are associated with reduced amount of HDL apolipoprotein,\nwhich is accompanied by reduction of the number\nof mature oocytes in women ( 27 ). Although there\nare some reports verifying the role of HDL in oogenesis, further studies are needed on this issue.\nIn our study, no significant difference was observed in the concentration of TAS in the two\ngroups. In similar studies, no significant difference\nwas found in the TAS levels in the women with\nFFI who suffered from endometriosis when compared with the TAS concentration of the MFI ( 28 ,\n 29 ). This is in line with the findings of this study.\nIn present study, high level of PON3/TAS ratio in\nthe control group (MFI group) and high level of\nMDA in FFI group indicate that PON3 plays an\nimportant role in the prevention of follicular oxidative stress. Furthermore, high level of MDA in\nFF of women with FFI could also be suggestive of\nthe above-findings. This finding is similar to the\nfindings of Yildrim et al. ( 30 ) who have shown\nthat the lipidic peroxidation in the FF of the FFI\ngroup with polycyctic ovarian syndrome (PCOS)\nis much higher than that of the MFI group.\nThe results obtained in the present study show\nthat the PON3/MDA ratio in the women with FFI\nis significantly lower than the corresponding value in the women with MFI (p=0.002). It could be\nstated that the ratio of antioxidant to peroxidation\nin the FF is a suitable factor for assessing the oxidative stress in the follicles.\nPON3 is a strong antioxidant in FF. Closshey\net al. ( 15 ) have reported that high PON3 activity\ninside follicle could probably be due to being produced locally in follicle. According to Browne et\nal. ( 26 ), the origin of the enzyme is granula-generating cells. In this study, for the first time, it was\nshown that PON3 activity in the FF of the women\nwith FFI is lower than that in women with MFI.\nNow, it is known that PON3 is able to prevent\nLDL oxidation ( 10 ). This enzyme can utilize the\nproducts of lipids oxidation as substrates, and thus,\nreduces the severity of oxidative stress in the cell\n( 31 ,  32 ). Therefore, it is more likely that PON3\nhave some important roles in the growth and maturation of the oocytes.\nOn the other hands, we were not able to find any\nsignificant difference between PON3 activity and\nthe number of oocytes. Plachot et al. ( 33 ) have\nstated that the rate of fertilized oocytes is associated with not only the number but also the quality of\noocytes. Thus, high level of PON3 activity during\nthe growth period, maturity and quality of oocytes\nplays a vital role. Moreover, current study showed\nthat the ratio of PON3 to MDA, as an indicator of\nantioxidant to peroxidation, in the FF of FFI group\nis lower than that of MFI group. Therefore, the antioxidant and peroxidation status in FF could be\ncorrelated with female infertility.\nA significant negative relation between PON3\nand PON3/MDA with EFS, and a positive relation\nbetween these parameters with FR may indicate\nthat PON3 plays an important role in fertilization\nand the quality of embryo. A high level of PON3\nin FF could be indicative of its specific role in development and maturation of good oocyte which,\nin turn, can lead to a healthy embryo.\nThe role of PON3 in fertility has not received\nenough attention. Browne et al. ( 34 ) have reported\na significant negative association between HDL-C\nand EFS; however, they could not find any significant relation between EFS and PON3 activity in\nFF. Although the negative relation observed in our\nstudy between HDL-C and EFS was not statistically significant, we were able to show a significant positive relation between FR and HDL-C in\nFF. This may indicate the importance of HDL in\nfertilization which has also been reported by others ( 35 ). It has been shown that HDL and the proteins present in the structure of HDL could have a\ncytoprotective effects on oocyte and surrounding\ngranulosa cells ( 36 ). As PON is one of the important antioxidant components of HDL and PON3\nconcentration in FF is much higher than its level\nin blood, it is likely that the local role of PON3\nis much more dominant. It should be noted that\nstudied variables including embryo quality and\nfertilization rate may be affected differentially in\nIVF and ICSI patients. However, because of our\nlimited number of patients, it was not possible to\nperform two separate analyses for IVF and ICSI\ngroups.\n\nOur findings confirm that PON3, as an antioxidant potential in follicular fluid, has a major role\nin regulating fertility and maintaining embryonic growth. Thus, PON3 could be a valuable therapeutic target to improve the success rate of ART.","source_license":"CC-BY-4.0","license_restricted":false}