{"paper_id":"5e608d79-d5f1-45b0-a83d-d37e4bf2399a","body_text":"www.ogscience.org430\nIntroduction\nRemarkable progress in assisted reproductive techniques \n(ART) has improved opportunities for subfertile couples [1]. \nIntracytoplasmic sperm injection (ICSI) is a significant break -\nthrough for couples with severe male-factor infertility. How -\never, the data demonstrated a consistent increase in ICSI \ncompared with conventional in vitro fertilization (IVF) tech -\nniques. This trend was not confined to male factor infertility \nbut also to unexplained infertility, a low number of retrieved \nCan follicular fluid 8-oxo-2’-deoxyguanosine predict \nthe clinical outcomes in ICSI cycle among couples with \nnormospermia male?\nWassan Nori, MD, PhD\n1\n, Zeena Raad Helmi, MD, PhD\n2\nDepartment of Obstetrics and Gynecology, \n1\nCollege of Medicine, Mustansiriyah University, \n2\nCollege of Medicine, Al-Mustansiriyah University, \nBaghdad, Iraq\nObjective\nOxidative stress (OS) occurs when excess free radicals damage the DNA. Moreover, 8-oxo-2’-deoxyguanosine (8-\nOHdG) is a well-known biomarker for OS linked to cellular damage and gene instability. However, its role in female \nsubfertility has not been properly assessed. We aimed to examine the level of OS represented by 8-OHdG based on \nthe cause of subfertility and to test its correlation with reproductive hormones, intracytoplasmic sperm injection (ICSI) \nparameters, and outcomes.\nMethods \nA cross-sectional study examined 108 subfertile couples with endometriosis, polycystic ovary syndrome (PCOS), tubal \nfactors, and unexplained infertility undergoing ICSI treatment with two different stimulation programs. We included \ncouples whose partners had normal sperm parameters. Levels of follicular fluid (FF) 8-OHdG were correlated with the \ncauses of subfertility and fertilization rates and compared between pregnant and non-pregnant cases.\nResults\nBased on the causes of subfertility, FF 8-OHdG was the highest among endometriosis cases, followed by PCOS cases. \nFurthermore, FF 8-OHdG was higher in non-pregnant (2.37±0.75 ng/mL) vs. pregnant (1.58±0.39 ng/mL), P<0.001. A \ntwo-way analysis of variance showed that only subfertility affected ICSI outcomes, whereas the stimulation program \ndid not. FF 8-OHdG correlated positively with female age and inversely with estradiol and good-quality embryos. \nThe receiver operating characteristic estimated 8-OHdG cutoff value of 1.8 ng/mL predicted clinical pregnancies with \n86.7% sensitivity and 74.4% specificity (P<0.001).\nConclusion\nHigher FF 8-OHdG levels negatively impacted ICSI outcomes. FF 8-OHdG discriminated between cases of clinical \npregnancy with good specificity and sensitivity. Because OS can be measured and treated, this opens up a therapeutic \nand prognostic avenue for improving ICSI outcomes.\nKeywords: ICSI; Oxidative stress; 8-oxo-2’-deoxyguanosine; Polycystic ovary syndrome; Successful pregnancy \nReceived: 2022.06.16.   Revised: 2022.08.10.   Accepted: 2023.06.30.\nCorresponding author: Wassan Nori, MD, PhD\nDepartment of Obstetrics and Gynecology, College of Medicine, \nMustansiriyah University, Falastin St, Baghdad 14022, Iraq\nE-mail: Dr.wassan76@uomustansiriyah.edu.iq\nhttps://orcid.org/0000-0002-8749-2444\nArticles published in Obstet Gynecol Sci are open-access, distributed under the terms of \nthe Creative Commons Attribution Non-Commercial License (http://creativecommons.\norg/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, \nand reproduction in any medium, provided the original work is properly cited.\nCopyright © 2023 Korean Society of Obstetrics and Gynecology \nOriginal Article \nObstet Gynecol Sci 2023;66(5):430-440\nhttps://doi.org/10.5468/ogs.22170\neISSN 2287-8580\n\n\nwww.ogscience.org 431\nWassan Nori, et al. Follicular fluid 8-OHdG in ICSI cycles\noocytes, and older women, with no strong evidence of its \nsuperiority to standard IVF in cases of non-male-related infer-\ntility [2]. A possible cause for this increase may be the total \nfertilization failure encountered in conventional IVF. Never -\ntheless, the overall risk/benefit analysis supports conventional \nIVF for non-male infertility [1,2].\nOxidative stress (OS) is an imbalance between pro-oxidant \nmolecules such as reactive oxygen and nitrogen species and \nantioxidant defenses. Overall survival (OS) is crucial in male \nand female subfertility [3].\nFurthermore, 8-hydroxy-2-deoxyguanosine (8-OHdG) is a \nwell-known biomarker for OS. It is produced in response to \nDNA-base modifications. OS is vital in promoting final oocyte \nmaturation and orchestrating follicular rupture. However, the \noverproduction of reactive oxygen species (ROS) and/or the \nimbalance between OS and antioxidative mechanisms can \ncontribute to diverse infertility disorders and human diseases \nsuch as metabolic diseases, male and female infertility, poly -\ncystic ovary syndrome (PCOS), abortion, and preeclampsia \n[4,5]. \nROS are produced within the follicle as a by-product of ox-\nygen metabolism during the ovulatory cycle; DNA is the most \ncommon target for ROS and has been linked to cellular dam-\nage and gene instability. Antioxidants act as scavengers of \nthe harmful effects of ROS and play a critical role in oocyte \nmaturation [6]. Previous studies have shown that women \nwith high rates of defective oocytes have elevated follicular \nfluid (FF) 8-OHdG concentrations compared with those with \nlow rates. Others have reported that patients with PCOS \nhave reduced serum 8-OHdG levels compared with matched \ncontrols [7]. \nROS induces female subfertility via different pathways. The \ndirect pathway triggers DNA damage in the ova. Ovarian \nfollicles with excess ROS that exceed the physiological anti -\noxidant defense mechanism consequently suffer from direct \nDNA damage, thus impairing fertilization odds. Steroidogen-\nesis (follicle-stimulating hormone and estradiol) is another \npathway of female subfertility that results in decreased \noocyte quality and overall IVF success [5-7]. ROS tend to \ndeplete glutathione; the latter is needed for the decondensa-\ntion of sperm chromatin to form male pronuclei after IVF, in \naddition to supporting the blastocyte stage. Consequently, \ntheir absence causes antral follicle apoptosis and poor-quality \noocytes [8]. \nFF is an important component of the oocyte microenviron-\nment. FF accumulates in all metabolic processes throughout \noocyte development, which are needed for oocyte develop -\nment, follicular maturity, and germ cell-somatic cell connec -\ntion. Oocytes and follicular somatic cells collaborate in the \novaries to maintain correct glucose, amino acid, and lipid \nmetabolism [4]. It is reasonable to believe that the FF bio -\nchemical milieu is critical in determining oocyte quality and \nfertilization potential to produce good embryos [4,9,10].\nThe role of 8-hydroxy-2-deoxyguanosine in male infertil -\nity has been addressed; however, its role in female infertility \nhas not been well studied. Despite major leaps in assisted \nreproductive technology, we still face a major and profound \nproblem factor attributed to the imbalance of OS and anti-\noxidative defense mechanisms [11]. Therefore, it is neces -\nsary to identify the confounding factors related to OS in ICSI \ncycles. Because infertility has many causes, this study was de-\nsigned to assess OS (measured by FF 8-OHdG) based on the \ncauses of infertility as a primary aim. The secondary aim was \nto estimate FF 8-OHdG’s potential correlation with reproduc-\ntive hormones, ICSI outcomes, and the cutoff value linked to \nclinical pregnancy among couples with normal sperm param-\neters.\nMaterials and methods\nA cross-sectional study was conducted from July 2019 to \nJune 2020, and 108 subfertile couples were referred for \nICSI procedures at the IVF Infertility Center of the University \nHospital. The Ethics Committee of Mustansiriyah University/\nFaculty of Medicine approved this study (IRB 167; April 23, \n2019). The couples were briefed about the aims and proce -\ndures of the study. Informed consent was obtained from all \npatients, and the Declaration of Helsinki was followed.\n1. Enrollment criteria \nWe included only couples with normal male partners; they \nwere subsequently subdivided according to the subfertility \ncauses (mild endometriosis, PCOS), tubal blockage, and un -\nexplained infertility). Females ranged in age from 18 to 40 \nyears, with body mass indexs (BMI) ranging from 18 to 30 \nkg/m\n2\n. All recruited couples had normal male seminal fluid \nbased on World Health Organization (WHO) criteria for 2010 \nWHO criteria [12].\nThe patients’ histopathological and/or laparoscopy reports \n\nwww.ogscience.org432\nVol. 66, No. 5, 2023\nbased on the American Society of Reproduction criteria con-\nfirmed the diagnosis of mild endometriosis. Women with \nendometriosis were subgrouped into stages II-IV. None of \nthe female partners had endometriomas >3 cm in diameter. \nPCOS was confirmed using the Rotterdam criteria [13], and \nthe tubal blockage was confirmed using hysterosalpingogra-\nphy and/or laparoscopy. Unexplained infertility was defined \nas the absence of male and female abnormalities, including \nnormal ovulation, tubal patency, and seminal fluid analysis \n[14].\nExclusion criteria were as follows: 1) abnormal seminal \nfluid parameters; 2) abnormal uterine structure, congenital \nmalformation, or documented pathology, such as fibroid or \nmoderate-to-severe endometriosis; 3) associated medical \ncomorbidities, including thyroid diseases, diabetes, chronic \ninflammation, and liver and kidney disease; 4) drug intake, \nsuch as steroids; and 5) female BMI >30 kg/m\n2\n.\nThe couples were briefed about the aims and procedures \nof the study. Informed consent was obtained from all pa -\ntients, and the Declaration of Helsinki was followed. For male \npartners, the medical and surgical history was revised, and \na sample of seminal fluid was sent for analysis. It was con -\nsidered normal according to the WHO criteria 2010 [12]. For \nfemale partners, a detailed history and physical examination \nwere undertaken, including height and weight, to calculate \nthe BMI. \n2. Controlled ovarian stimulation \nDay 2 (D2) serum hormones of the menstrual cycle were \nestimated, including follicle-stimulating hormone, luteinizing \nhormone, and estradiol (follicular stimulation hormone [FSH], \nluteinizing hormone [LH], and estradiol [E2]).\nTwo protocols were used for ovarian stimulation. Female \npartners were assigned to either the antagonist or short ago-\nnist protocols based on the female’s age, BMI, and cause of \ninfertility [13].\nWe used the flexible antagonist protocol, where recom -\nbinant FSH (Merck Serono, Modugno [BA], Italy) was intro -\nduced via the subcutaneous route (SC) from D2 or 3 of the \nmenstrual cycle daily until the E2 level exceeded 500 pg/mL, \nand the dominant follicle size was 14-15 mm. Herein, cet -\nrotide 0.25 (Merck Serono, Halle, Italy) was injected SC daily \nuntil 3 dominant follicles >17 mm were achieved, where \nhuman chorionic gonadotropin (HCG) was introduced. The \nagonist protocol began on D2 of the cycle with daily admin -\nistered decapeptyl at a dose of 0.1 mg/SC and continued till \nthe triggering day. On D3, recombinant FSH was started at a \ndaily dose of 150 international unit (IU)/SC and stopped one \nday before the triggering day.\nThe ovulation was triggered by introducing SC pregnyl \n(10,000 IU) once the E2 levels exceeded 1,500 pg/mL along-\nside a minimum of 3 dominant follicles of ≥17 mm at least \n36 hours before the ova pickup day.\nThe ova was picked using ultrasound guidance, and FF \nwas retrieved 36 hours after the trigger. The oocytes were \nseparated from the FF and prepared for ICSI. Only mature \noocytes at metaphase two were used for ICSI. The FF was \ncentrifuged, and the clear supernatant was stored at -80°C \nto assess 8-OHdG as an OS biomarker. A, competitive-\nenzyme-linked immunosorbent assay Principle/Elabscience \nkit (Immuno-tech-Beckman Coulter, Webster, TX, USA) was \nused, according to the manufacturer’s instructions.\nThe fertilization rate was assessed 16-18-hour following \nsperm injection; the existence of two pronuclei (2PN) and ex-\ntrusion of the second polar body confirmed fertilization. Two \nor 3 days after injection, the number and quality of embryos \nwere examined and categorized using Veeck [15] criteria. A \ngood-quality embryo was classified as one that had achieved \nthe 4-cell stage on D2 and the 7-cell stage on D3 and had \nfragments filling less than 20% of its volume.\nOnly three top-quality embryos were transferred to sub -\nfertile women. To confirm a positive pregnancy, serum-HCG \nlevels were measured 14 days after embryo transfer. Clinical \npregnancy was defined as the presence of an intrauterine sac \nwith a viable fetal heart 28 days after embryo transfer. The \nimplantation rate was calculated as the number of positive \nHCG per total number of transferred embryos per group [16]. \nThe number of retrieved oocytes, germinal vesicles, meta -\nphase one oocytes, metaphase two oocytes (M2), 2PN, the \nquality and number of transferred embryos, and fertilization, \nimplantation, and clinical pregnancy rates were recorded for \nanalysis.\n3. Statistical analysis\nContinuous data were presented as means and standard de-\nviations, while categorical variables were presented as num -\nbers and percentages. Data normality was evaluated using \nthe Shapiro-Wilk test. Continuous data were compared using \n(one-way analysis of variance [ANOVA]). Categorical variables \nwere compared using the chi-squared test. \n\nwww.ogscience.org 433\nWassan Nori, et al. Follicular fluid 8-OHdG in ICSI cycles\nOne-way ANOVA was used to compare the outcome of \nthe ICSI program based on subfertility causes, and the FF \n8-OHdG level was correlated with continuous data obtained \nin this study using linear regression, with a calculation of the \nassociated coefficient of correlation and P-values. A two-\nway ANOVA test was used to show the effect of infertility \ncauses and the stimulation protocol on 8-OHdG levels, with \na calculation of the associated P-values. Logistic regression \nwas constructed for the cause of infertility and its correlation \nwith the successful pregnancy rate by calculating the associ -\nated odds ratio (OR) and 95% confidence intervals (CI). A \nreceiver operating characteristic (ROC) curve was constructed \nto predict the cutoff value of FF 8-OHdG associated with suc-\ncessful pregnancy and its associated sensitivity and specificity. \nP-values less than 0.05 were significant. MedCalc version 20 \nwas used for data analysis. \nThe required sample was estimated based on the following \nformula: sample size=(Z1- α/2)\n2\n×SD\n2\n/d\n2\n. Z1-α/2 is a standard \nnormal variate (1.96); standard deviation (SD)=variable ob -\ntained from earlier studies [17]; d is absolute error calculated \nby the authors; sample size=1.96\n2\n×0.4\n2\n/0.1\n2\n=3.84×0.16/0.01; \nrequired sample is 61 patients.\nResults \nA cross-sectional study recruited 108 subfertile couples with \nnormal male partners, grouped according to female subfertil-\nity cause into the endometriosis group 12/108 (11%), tubal \nfactor 22/108 (20.3%), PCOS 26/108 (24.03%), and unex -\nplained infertility 48/108 (44.4%).\nConcerning female demographic criteria, the mean age \nwas 30.48±4.83 years (range 19-40), and the mean BMI \nwas 25.72±2.23 kg/m\n2\n. Primary infertility affected 36/108 \n(33.33%) of the cases versus 72/108 (66.66%) who had \nsecondary infertility; two stimulation protocols were used; \n42/108 (38.88%) cases received the agonist protocol versus \n66/108 (61.11%) that received flexible antagonist proto -\ncol. A positive clinical pregnancy was reported in 28/108 \n(25.92%) cases.\nNone of the hormones tested on D2 (FSH, LH, or estra -\ndiol) were statistically significant in the subgroup analysis, as \nshown in Table 1. \nRegarding the ICSI parameters, only the number of re -\ntrieved oocytes and oocytes in metaphase II were statistically \nTable 1. The basal hormonal analysis of study participants on day 2, grouped according to subfertility causes\nHormonal parameter Endometriosis (n=12) Tubal factor (n=22) PCOS (n=26) Unexplained (n=48) P-value\nFollicular stimulating hormone (mlU/mL) 5.07±2.64 6.95±3.95 5.77±2.61 7.31±2.72 0.262\nLuteinizing hormone (mlU/mL) 2.80±1.84 3.56±1.89 5.43±4.57 4.19±1.98 0.219\nEstradiol (pg/mL) 18.90±11.77 36.58±17.29 34.74±16.41 31.06±11.25 0.085\nValues are presented as means±standard deviation. \nPCOS, polycystic ovarian syndrome. \n\nwww.ogscience.org434\nVol. 66, No. 5, 2023\nsignificant between each group, as confirmed by a post-hoc \ntest, with P-values of 0.002 and 0.006, respectively, as de -\nscribed in Table 2.\nThe FF 8-OHdG levels showed significant differences \nP<0.002 based on the cause of infertility. Endometriosis \nhad the highest concentration, followed by PCOS and tubal \nfactors, whereas unexplained infertility had the lowest FF \n8-OHdG levels, as shown in Fig. 1. Using logistic regression, \nwe calculated the OR for a positive pregnancy test, with the \nrespective 95% CI and P-value described in Table 3. PCOS \ncases showed a trend of high OR for positive pregnancy (2.22; \n95% CI, 0.19-25.72; P=0.52), followed by unexplained infer-\ntility, tubal factors, and endometriosis group; the latter was \ntaken as the reference group. To test the influence of differ-\nent stimulation programs and their interaction with the cause \nof infertility in the study participants, a two-way ANOVA was \nused; it confirmed that only the causes of infertility were \nsignificant ( P=0.03), while the type of stimulation protocol \nfailed to score statistical differences, as shown in Table 4. The \nTable 2. The outcome of the ICSI program based on subfertility causes\nICSI parameter Endometriosis (n=12) Tubal factor (n=22) PCOS (n=26) Unexplained (n=48) P-value\nNo-of retrieved oocyte 11.67±4.80 6.18±2.75 12.69±5.63 7.63±4.67 0.002\nb)\nGerminal vesicles 1.50±1.76 0.36±0.81 0.77±0.93 0.67±1.47 0.379\nM 1 1.50±1.38 0.82±0.87 1.08±1.44 0.63±1.06 0.364\nMature oocyte 8.67±5.43 5.00±3.16 10.85±5.19 6.29±3.93 0.006\nb)\nNo. of two pronuclei 5.67±4.8 4.45±2.88 7.69±4.75 5.00±3.34 0.152\nGood quality embryo 3.83±3.55 4.09±2.77 6.15±3.39 6.15±3.39 0.147\nBad quality embryo 1.83±2.56 0.27±0.46 1.00±1.78 0.54±0.83 0.108\nNo. of transferred embryos 2.50±1.05 2.82±1.33 3.08±0.86 2.58±0.97 0.518\nImplantation rate 12.8±3.4 27.2±5.6 37.0±7.8 8.3±2.5 0.068\nClinical pregnancy rates\na)\n (%) 15.0 44.4 50.0 23.8 0.250\nValues are presented as mean±standard deviation or number. \nICSI, intracytoplasmic sperm injection; PCOS, polycystic ovary syndrome.\na)\nData are presented as percentages and compared by chi-square test. \nb)\nStatistically significant differences between groups.\nTable 3. A multi-variant logistic regression and odds ratio for causes of infertility and their effect on pregnancy rate\nVariable Odds ratio 95% confidence interval P-value\nEndometriosis cases Reference Reference Reference\nCases of PCOS 2.22 0.19 to 25.72 0.52\nTubal factors 1.87 0.15 to 23.40 0.63\nUnexplained infertility 2.05 0.20 to 20.96 0.54\nPCOS, polycystic ovarian syndrome.\nFig. 1. The level of FF 8-OHdG based on the causes of subfertility. \nFF , follicular fluid; 8-OHdG, 8-oxo-2’-deoxyguanosine.\n4.0\n3.5\n3.0\n2.5\n2.0\n1.5\n1.0\n0.5\nFF 8-OHdG\nEndometriosis\nPolycystic ovarian syndrome\nTubal factors\nUnexplained\n\nwww.ogscience.org 435\nWassan Nori, et al. Follicular fluid 8-OHdG in ICSI cycles\nlevel of FF 8-OHdG was significantly higher in non-pregnant \n(2.37±0.75 ng/mL) vs. pregnant (1.58±0.39 ng/mL), P<0.001, \nas shown in Fig. 2. Correlation analysis showed that FF \n8-OHdG was significantly correlated with female age (0.4; \nP=0.006), D2 estradiol levels (-0.311; P=0.02), and good-\nquality embryos (-0.27; P=0.04) (Table 5). The ROC curve  \nestimated an 8-OHdG cutoff value of 1.85 ng/mL linked with \nthe highest sensitivity and specificity (86.7% and 74.4%; \nTable 4. A two-way ANOVA showing the effect of stimulation \nprotocol and cause of infertility on ICSI outcome.\nIntervention F-ratio P-value\nType of protocol 3.29 0.076\nCause of infertility 3.24 0.03\na)\nCause of infertility×type of protocol 0.15 0.928\nOnly subfertility cause was influential.\nANOVA, analysis of variance; ICSI, intracytoplasmic sperm injection. \na)\nStatistically significant value.\nFig. 2.  The level of FF 8-OHdG in non-pregnant vs. pregnant. \n8-OHdG, 8-oxo-2’-deoxyguanosine; FF , follicular fluid.\n4.0\n3.5\n3.0\n2.5\n2.0\n1.5\n1.0\n0.5\n8-OHdG\nNegative Positive\nPregancy rate\nTable 5. Correlation of FF 8-OHdG vs. study parameters\nStudy parameter Coefficient of correlation (r) P-value 95% confidence interval\nAge (yr) 0.4 0.006\na)\n0.12 to 0.57\nBody mass index (kg/m\n2\n) 0.11 0.42 0.16 to 0.37\nFollicle stimulating hormone (mlU/mL) 0.22 0.12 0.46 to 0.05\nLuteinizing hormone (mlU/mL) -0.12 0.38 -0.37 to 0.15\nEstradiol (pg/mL) -0.311 0.02\na)\n-0.53 to -0.04\nNo of oocyte -0.032 0.82 -0.29 to 0.23\nGerminal vesical 0.15 0.29 -0.13 to 0.39\nM1 -0.001 0.99 -0.27 to 0.27\nMature oocyte -0.07 0.59 -0.33 to 0.19\nNo. of two pronuclei -0.20 0.14 -0.45 to 0.06\nGood quality embryo -0.27 0.04\na)\n-0.50 to -0.0004\nBad quality embryo 0.07 0.63 -0.21 to 0.33\nNo. of embryo transferred -0.20 0.13 -0.45 to 0.06\nFertilization rate (%) 0.24 0.08 -0.47 to 0.02\nFF, follicular fluid; 8-OHdG, 8-oxo-2’-deoxyguanosine; M1, metaphase one oocytes.\na)\nStatistically significant value.\nFig. 3. The ROC curve showing FF 8-OHdG critical value in pre -\ndicting clinical pregnancy test. 8-OHdG, 8-oxo-2’-deoxyguanosine; \nAUC, area under the curve; ROC, receiver operating characteristic; \nFF , follicular fluid.\nSensitivity\n0 20 40 60 80\n100-specificity\nAUC=0.829 \nP<0.001\nSensitivity: 86.7 \nSpecificity: 74.4 \nCriterion: ≤1.853\n8-OHdG\n100\n100\n80\n60\n40\n20\n0\n\nwww.ogscience.org436\nVol. 66, No. 5, 2023\nrespectively), an area under the curve (AUC) of 0.83, and \nP=0.001 in predicting clinical pregnancy, as illustrated in Fig. 3.\nDiscussion\nThe analysis highlighted significant differences in the num -\nbers (No.) of retrieved oocytes and M2 and FF 8-OHdG levels \nbased on the cause of infertility. FF 8-OHdG had the highest \nconcentration among the endometriosis groups, followed \nby the PCOS group. The OR for clinical pregnancy was the \nhighest among PCOS cases and lowest in the endometriosis \ngroup. Only subfertility affected ICSI outcomes, whereas the \nstimulation program had no effect. FF 8-OHdG levels were \nsignificantly higher in non-pregnant women than in pregnant \nwomen. This confirmed strong positive correlations with fe -\nmale age and inverse correlations with E2 and good-quality \nembryos. Finally, the ROC estimated an 8-OHdG cutoff value \n<1.85 ng/mL that predicted clinical pregnancies with 86.7% \nsensitivity, 74.4% specificity, and P<0.001.\nNishihara et al. [18] addressed the correlation between FF \nOS markers (8-OHdG) and antioxidant status (total glutathi -\none) with ICSI outcomes and embryo transfer rates. In accor-\ndance with our results, patients with endometriosis had the \nhighest FF for 8-OHdG.\nAnother study investigated IVF outcomes in two sub -\ngroups: with and without endometriosis. They confirmed an \ninverse correlation of FF 8-OHdG with good-quality embryos; \nhowever, they found no difference in 8-OHdG levels either in \nthe FF or in the serum of pregnant vs. non-pregnant women. \nFurthermore, only FF 8-OHdG levels were significantly higher \nin the endometriotic group, whereas serum levels showed a \ntrend toward 8-OHdG elevation [19].\nSeveral studies have reported that women with endometri-\nosis have poorer outcomes. Affected women have different \nstem cell compositions and proliferation, hormone sensitivity, \ninvasiveness, and immunological modulation [18].\nFurthermore, the endometrium of affected patients resists \nselective progesterone activity, which regulates decidualiza -\ntion and modifies local inflammatory reactions throughout \nimplantation. This explains the poor outcome among the en-\ndometriosis group, as shown by our results showing higher \nFF 8-OHdG levels and the lowest OR for clinical pregnancy \n[18,19]. \nThe OR for clinical pregnancy was the highest for the PCOS \nsubgroup. Sova et al. [20] reported that patients with PCOS \nhad considerably lower serum 8-OHdG levels than age- and \nBMI-matched healthy controls. These levels were further \nreduced by metformin therapy. The authors assumed that \n8-OHdG is not only a result of oxidative DNA damage but \nalso possesses ROS-suppressing capabilities in many in vitro \nexperiments, implying that it may play a role in preventing \nOS and fine-tuning the reactivity to OS [20].\nFabjan et al. [21] reported that the FF (8-OHdG) concen -\ntration was significantly lower in patients with PCOS. They \nsuggested that it was a good predictor of oocyte fertiliza -\ntion and maturation. The authors explained that high ROS \nlevels would stimulate more antioxidant enzymes, reducing \noxidative stress. Consequently, this prevents the interaction \nbetween ROS and DNA and reduces 8-OHdG formation. \nMany researchers have confirmed that the levels of several \nantioxidant enzymes are significantly higher in women with \nPCOS [22]. \nMany female diseases, such as endometriosis, tubal block -\nage, and polycystic ovary syndrome, are associated with free \nradicals that can damage DNA. Excess ROS may harm the \nendometrium.\nPrevious studies suggested that biomarkers related to OS \nfall into the following categories: 8-OHdG is classified as a \nsensitive measure of DNA damage and OS, which underpins \noocyte damage and negatively impacts oocyte quality, fertil -\nization, embryo grading, and endometrial adequacy [19-21]. \nDifferent causes of infertility result in different OS levels, mir-\nrored by the 8-OHdG levels [1,4,7]. The effect of OS markers \non infertility is only a small fraction of the puzzle; FF-OHdG \nis not the only marker that indicates OS in infertile couples. \nOther markers of OS marker their role in OS damage leading \nto infertility requires further research. No single OS marker \nhas been recommended, nor how anti-oxidants contribute to \neffective ICSI results has been elucidated [6,8,11].\nFew studies have discussed the potential association be -\ntween oxidative stress, type of stimulation program, and IVF \noutcomes. Our data showed that infertility causes affected \nICSI outcomes, while the stimulation program failed to have \na significant effect. Tuli ć et al. [23] investigated the differ -\nences in the serum levels of OS parameters before and after \nagonist and antagonist stimulation. Patients without OS \nexhibited better IVF outcomes after stimulation. However, in \nline with our results, the ovarian stimulation program was \nnot linked to any change in OS parameters or ICSI outcomes; \n\nwww.ogscience.org 437\nWassan Nori, et al. Follicular fluid 8-OHdG in ICSI cycles\nthey showed an insignificant difference in the rates of bio -\nchemical pregnancies, abortions, and live births for both sub-\ngroups.\nIn contrast, Thaker et al. [24] showed that the long-agonist \nprogram had a higher No. of retrieved ova in comparison \nwith the gonadotropin-releasing hormone antagonist pro -\ngram. However, the number of women with a positive out -\ncome was conversely low in the long-agonist regimen. The \nauthors attributed this to a higher no of retrieved follicles, \nwhich may have contributed to implantation failure. For an -\ntagonist and short agonist programs, pregnancy rates were \nmatched [24,25].\nThe analysis confirmed a significantly high FF 8-OHdG level \namong non-pregnant women, in line with an earlier study \nthat linked OS biomarkers to failed pregnancies [26].\nAnother study showed that high FF 8-OHdG levels in wom-\nen with low fertilization rates were linked to poor-quality \nblastocytes. The authors recommended FF 8-OHdG and total \nglutathione as reliable biomarkers for successful fertilization \nin assisted reproduction [18]. Many studies have discussed \nthe complex correlation between ROS and antioxidant status \nin the FF. ROS are unwanted byproducts of biological oxida -\ntion. Low levels of ROS may serve as a physiological signaling \npathway in the embryo [27]. However, once this delicate bal-\nance in FF between OS and antioxidant defense mechanisms \nis disturbed by the overproduction of free radicals and/or un-\nderproduction of antioxidants, the increase in free radicals in \nFF consequently causes poor oocyte quality and low fertiliza-\ntion rates [28,29]. Nishihara et al. [18] reported contradictory \nresults; they showed an insignificant correlation between \nOS and antioxidant levels in positive pregnancies. Our data \nhighlighted the highest OS among the endometriosis groups \nrepresented by FF 8-OHdG levels, followed by the PCOS, \ntubal, and unexplained groups. However, tubal factors had \nthe lowest OR for becoming pregnant [18]. \nNo single OS biomarker has been recommended, nor has \nthe mechanism by which antioxidants contribute to success -\nful ICSI outcomes been elucidated; therefore, the authors \nrecommended individual estimation of OS markers and anti-\noxidants simultaneously in the FF [30].        \nVárnagy et al. [19] showed that FF 8-OHdG levels were \nnegatively correlated with No. of a good-quality embryo in \na patient undergoing a stimulation program; however, they \nfound no correlation between FF 8-OHdG levels and mature \noocyte No. Likewise, Seino et al. [31] declared that granulosa \ncells 8-OHdG negatively correlated with ova, embryo quality, \nand fertilization rates. Another study confirmed that the FF \n8-OHdG levels were significantly higher in patients with high-\ngrade oocyte degeneration [32]. \nThe influence of maternal age on egg quality and fertiliza -\ntion is a well-known phenomenon in female fertility and was \npositively linked to the follicular oxidative state in the cur -\nrent study. Maternal age inversely affects the success of the \nICSI cycle. Da Broi et al. [33] found that women older than \n38 years experience a decrease in growing follicles, oocyte \nquality, and changes in the quality of the surrounding cells, \nunderlining the importance of FF. Furthermore, female aging \nis associated with increased ROS production, decreased anti-\noxidant production, and oocyte competency [19]. \nAdvanced maternal age is linked to increased oocyte and \nembryo aneuploidies, and OS triggers aneuploidy in animal \nmodels. Our results indicated a strong positive correlation \nbetween FF 8-OHdG levels and maternal age. This ominous \nalliance increases chromosomal abnormalities in the offspring \n[8,32-35].\nThe inverse correlations of FF 8-OHdG with E2 and good-\nquality embryos were in agreement with earlier research; \nserum E2 levels were correlated with FF OS biomarkers that \ninversely affect ovarian responses [26]. \nIt has been suggested that the immunomodulatory action \nof estrogen limits OS and simultaneously upregulates en -\ndogenous anti-oxidants [36]. Furthermore, immature ovarian \nfollicles are E2 dependent. Women with reduced E2 levels \nexperience embryonic arrest, which explains their inverse cor-\nrelation with good-quality embryos [37]. \nThe cutoff value of FF 8-OHdG (<1.85 ng/mL) predicted \nclinical pregnancy with a sensitivity and specificity of 86.7% \nand 74.4%, respectively (AUC, 0.83 and P=0.001), making \nit a valuable marker for clinical use. Many markers for OS \nin the FF have been extensively examined because of their \nclose correlation with fertilization and pregnancy potential \nin IVF patients. In addition, 8-OHdG is regarded not only as \na byproduct of oxidative DNA damage but also for its ROS-\nsuppressing ability, which implies its potential applications in \nmitigating and tuning the OS response [20].        \nAlthough the OR for becoming pregnant was higher in \nPCOS cases and lowest in endometriosis cases, the difference \nwas not statistically significant. Furthermore, the implanta -\ntion and clinical pregnancy rates did not differ significantly \nbetween the groups. Therefore, one may assume that FF \n\nwww.ogscience.org438\nVol. 66, No. 5, 2023\n8-OHdG correlates with clinical pregnancy rates rather than \nthe causes of infertility, which is worthy of further research. \nStudy strengths: this study addressed the correlation be -\ntween the causes of infertility and the effects of different \nstimulation protocols. Furthermore, we used FF 8-OHdG \nrather than serum, a more sensitive marker of reproductive \npotential [18].\nWhat is unique about FF 8-OHdG, besides its correlation \nwith infertility, is that earlier studies discussed an additional \nprognostic value; its levels were reduced following metfor -\nmin therapy in PCOS cases, which opens therapeutic and \nprognostic avenues, especially in PCOS cases [20]. OS can be \ntreated, and optimizing couples’ conditions before embark -\ning on ICSI cycles can alleviate many financial, psychological, \nand health complications related to failed IVF trials [1].\nThe strong positive correlation between FF 8-OHdG and \nmaternal age implies that reducing OS will improve the ICSI \noutcome and prevent increased chromosomal abnormalities \nin the fetus. Additionally, FF 8-OHdG has been validated as a \nquantitative marker of ROS DNA damage in male sperm [38]. \nStudy limitations: smoking is a recognized confounder of \n8-OHdG levels. However, the effects of passive smoking on \nfemale partners have not been addressed. A high level of \n8-OHdG mirrors the underlying oxidative DNA damage and \nmay signify a decline in the DNA repair rate [39,40]. Because \nonly ICSI cycles were studied, this may represent a source of \nbias in the general effect of FF 8-OHdG on ART outcomes. In \naddition, the subgroup analysis included a small number of \nendometriosis cases, which may have impacted the results; \ntherefore, the current results should be interpreted with cau-\ntion.\nAlthough many pieces of the puzzle on how to optimize \nICSI outcomes are missing, it seems that the crosstalk be -\ntween infertility causes and OS biomarkers plays a decisive \nrole in improving the outcome. FF 8-OHdG, an OS marker, \nwas the highest among the endometriosis cases and was \ninversely related to estradiol levels and the number of good-\nquality embryos. It distinguished women with clinical preg -\nnancies with high sensitivity and specificity. Further studies \nare warranted to determine its prognostic and therapeutic \napplications in ICSI treatment.\nConflict of interest\nThe corresponding author states no conflict of interest on \nbehalf of all authors.\nEthics approval\nIt was issued by the Ethical Committee of Mustansiriyah \nUniversity/Medical College. Reference No. 167 on April 23, \n2019.\nPatient consent \nAll couples gave informed written and verbal consent.\nFunding information\nNone.\nReferences\n  1. Niederberger C, Pellicer A, Cohen J, Gardner DK, Pal -\nermo GD, O’Neill CL, et al. Forty years of IVF. Fertil Steril \n2018;110:185-324.e5. \n  2. Isikoglu M, Avci A, Kendirci Ceviren A, Aydınuraz B, Ata \nB. Conventional IVF revisited: is ICSI better for non-male \nfactor infertility? Randomized controlled double blind \nstudy. J Gynecol Obstet Hum Reprod 2021;50:101990. \n  3. Varni JW, Limbers CA, Burwinkle TM. How young can \nchildren reliably and validly self-report their health-relat -\ned quality of life?: an analysis of 8,591 children across \nage subgroups with the PedsQL 4.0 generic core scales. \nHealth Qual Life Outcomes 2007;5:1. \n  4. Yang J, Li Y, Li S, Zhang Y, Feng R, Huang R, et al. 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