Comparison of the effects of recombinant hCG and GnRH agonist on follicular fluid pentraxin 3, NF-kB and redox balance

Comparison of the effects of recombinant hCG and GnRH agonist on follicular fluid pentraxin 3, NF-kB and redox balance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of the effects of recombinant hCG and GnRH agonist on follicular fluid pentraxin 3, NF-kB and redox balance Fatma Tanılır, Sevil Çiçek, Pınar Kırıcı This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7180678/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives To compare the effects of ovulation triggering with recombinant human chorionic gonadotropin (hCG) or GnRH agonist on follicular fluid (FF) long pentraxin 3 (PTX3), nuclear factor kappaB (NF-kB), and redox balance markers in infertile patients. Materials and Methods A total of 40 patients under the age of 35 who were planned for assisted conception were included in the study. Participants were grouped according to their infertility etiology as PCOS (n = 13), uni- or bilateral endometrioma (n = 9), male factor (n = 11), and recurrent implantation failure (RIF, n = 7).The selection of which patient would be triggered with hCG or agonist was done randomly. PTX3 mRNA expression and NF-kB protein analysis were performed in FF samples collected from metaphase II mature oocytes. Follicular unit redox balance was determined by total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI). Results In the rhCG-triggered group, FF-NF-kB protein concentration was significantly higher than in the GnRH agonist-triggered group. Similarly, while FF-TAS levels were higher in the rhCG group, FF-TOS and OSI values ​​were significantly higher than in the agonist group.FF-PTX3 mRNA expression in the rhCG-triggered group was significantly higher than the GnRH agonist group. A 5-fold up-regulation in the relative expression of FF-PTX3 mRNA was detected in the hCG group compared to the GnRH agonist group (1.02 ± 0.26 vs. 5.39 ± 0.29, p < 0.001). When PTX3 mRNA expression was evaluated according to subgroups, significant downregulation was detected in the PCOS group compared to the other three groups. The relative FF-PTX3 mRNA expression of the endometrioma group was higher than all groups. Relative gene expression levels of male factor and RIF groups were similar. Conclusions rhCG induces ovulation more effectively than GnRHa agonists by providing a clearer balance between proinflammatory cytokines and redox balance markers. Follicular fluid long pTX3 NF-kB Redox balance GnRH hCG Figures Figure 1 Figure 2 Figure 3 Introduction Germ cells undergo complex morphological and metabolic changes until they develop into mature oocytes. This process, called folliculogenesis, begins with the reactivation of arrested follicles in the intrauterine period and continues until ovulation. The interaction of follicular unit elements formed in the oocyte and granulosa cells ensures the ovulation of a mature oocyte that has completed its developmental capacity. Mural granulosa cells on the inner surface of the follicle and cumulus granulosa cells (CCs) around the oocyte are critical for the development of a mature (MII) oocyte ( 1 ). In the pre-ovulation period, the accumulation of hyaluronic acid-rich matrix in the extracellular space and the radial opening of CCs is called cumulus expansion ( 2 , 3 ). In natural cycles, CC expansion is mandatory for ovulation, resumption of meiosis, and successful fertilization ( 4 ). In vivo, CC expansion also plays a role in the accumulation of sperm around the COC in the ampullary region ( 3 ). The ovarian follicle is the basic functional unit composed of somatic and germ cell components responsible for oocyte development ( 5 ).Normal follicular development and ovulation are achieved with adequate inflammatory and oxidative stress ( 6 , 7 ).Cumulus cells themselves and the ocytes keep cumulus cell differentiation and expansion under tight control ( 1 ).Since the fertilization potential of oocytes with inadequate cumulus expansion is limited, evolutionarily cumulus cell expansion is maintained by more than one gene ( 8 , 9 ). Hyaluronic acid synthase 2, gremlin1, nuclear factor kappa beta (NF-kB), and long pentraxin 3 (PTX3) are the main genes responsible for cumulus expansion. Oocytes expressing these genes have a higher fertilization potential than those that do not ( 5 , 8 ).Nuclear factor-κappaB (NF-κB) and long pentraxin 3 (PTX3) work together to provide the physiological inflammatory conditions necessary for many cellular functions ( 5 , 10 , 11 ).NF-kB, the main cellular regulator of the inflammatory cascade, is phosphorylated by different cytokines and enables the expression of immune response genes ( 6 , 7 ).In mice and humans, NF-kB and long Ptx3 mRNA are expressed only by cumulus cells from the follicular unit elements ( 3 , 5 ).Long PTX3 and NF-kBare involved in cumulus matrix stability and expansion ( 10 , 12 ). PTX3, whose expression increases secondary to inflammation, prevents tissue damage by regulating apoptosis on the basis of inflammation ( 13 ). Follicular development and ovulation are significantly affected by cumulus cells expressing sufficient amounts of NF-kB and PTX3 ( 3 , 5 ). In natural cycles, successful ovulation is possible when pituitary LH balances the release of inflammatory molecules and reactive oxygen species in the somatic compartment of the follicular unit. Follicular fluid (FF) located in the antral space plays a role in oocyte-granulosa cell communication. FF, which carries nutrients from the CCs to the ocyte, is like a living medium consisting of oocyte and follicle cell-derived hormones, enzymes, and electrolytes. While oocytes from mice null for some proinflammatory cytokines do not fertilize in vivo, the same oocytes fertilize in vitro ( 3 ).This finding suggests that natural cycles and assisted reproductive cycles may be different in terms of inflammatory cytokines and redox balance markers. Controlled ovarian stimulation protocols used to achieve multifollicular development in assisted conception is a process that causes supraphysiological changes in the morphology, number and functions of follicular unit elements.Drugs used to trigger ovulation mimic the physiological LH peak and cause cumulus expansion. The most common drugs used to induce ovulation in IVF/ICSI cycles are recombinant human chorionic gonadotropin (rHCG) and gonadotropin-releasing hormone (GnRHa) agonists. It has been reported that hCG injection into mice causes a significant increase in inflammatory cytokines expression( 3 ). Unlike natural cycles, there are no data on how ovulation-inducing agents used in assisted reproductive techniques affect the synthesis and release of follicular fluid pro-inflammatory cytokines and reactive oxygen species (ROS).This study was aimed to analyze the follicular fluid PTX3, NF-kB and redox balance markers of infertile patients whose ovulation was triggered with recombinant hCG or GnRH agonist.The effects of rHCG and GnRHa on follicular fluid oxidative stress indices were evaluated by total oxidant status (TOS), total antioxidant status (TAS), and oxidative stress index (OSI). Materials and Methods A total of 40 patients under the age of 35 who were planned for assisted conception due to different infertility etiologies were included in the study. According to infertility etiologies, participants were grouped as PCOS (n = 13), uni-or bilateral endometrioma (n = 9), male factor (n = 11) and recurrent implantation failure (n = 7).Failure of two or more IVF/ICSI attempts was considered RIF ( 14 ). Those who met at least two of the Rotterdam criteria of ovulatory dysfunction, hyperandrogenemia and polycystic ovarian morphology were considered PCOS ( 15 ). The diagnosis of endometrioma was made by transvaginal ultrasonography and its presence was confirmed for at least two cycles. Patients with severe oligoasthenoteratatospermia or azoospermia in at least two separate semen analyzes ( 16 ) were defined as male factor infertility. Participants in each group underwent controlled ovarian stimulation with a flexible GnRH antagonist protocol. Recombinant FSH treatment (Gonal-f, Merck-Serono, Bari, Italy) was started on the second or third day of the cycle, with the dose adjusted according to the patient's age, BMI, and AFC values. When the dominant follicle reached ≥ 14 mm, the pituitary was suppressed with 0.25 mg/day cetrotide (Merck-Serono, France). When two or more follicles had a diameter of at least 18 mm, ovulation was triggered in patients with either 0.2 mg triptorelin (Gonapeptyl; Ferring, Istanbul, Turkey) or 250 micrograms/0.5 mL recombinant hCG (Ovitrelle; Merck-Serono, Bari, Italy).Ovulation was triggered by hCG in 23 patients and by GnRH agonist in 17 patients. The selection of which patient would be triggered with hCG or agonist was done randomly, unless they were at high risk of OHSS. Agonist trigger was used for those at high risk for OHSS. Oocytes were collected under ultrasound guidance 35–36 hours after the ovulation trigger. Follicular fluidsampling Follicular fluid samples that did not show massive blood contamination during oocyte retrieval were placed in Eppendorf tubes (Isolab Laborgeräte GmbH, Germany) containing RNA stabilization buffer (Qiagen, Hilden, Germany) and centrifuged at 5000 g for 10 min. The supernatant was then removed and stored at -20°C until RNA extraction. Only follicular fluids containing mature MII oocytes that had shed their first polar bodies were evaluated. FFs of oocytes in the GV or MI stages were excluded from the evaluation.ICSI was performed on all the MII oocytes.The relationship between PTX3, NF-kB, redox markers and fertilization rate was recorded. Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) FF samples of MII oocytes collected from patients administered hCG or agonist triggers were subjected to qRT-PCR to detect long PTX3 mRNA expression.Glyceraldehyde-3-Phosphate Dehydrogenase was taken as the reference gene. Total RNA isolation of cumulus cells was performed with PureLink Total RNA Mini Kit (Invitrogen). RNA was reverse transcribed using the High Capacity cDNA RT Kit (Applied Biosystems, Foster City, CA). Blirt Amplyfyme SYBR Green Master Mix was used in the real-time PCR section. Primer sequences for qRT-PCR were as follows: PTX3: Forward 5'-TGGACAACGAAATAG ACATGG-3', Reverse 5' CTCTCATCTGCGAGTTCTCC-3', GAPDH: Forward 5' GAAGATGGTGATGGGATTTC-3', Reverse 5'-GAAGGTGAAGGTCGGAGTC- 3'. ELISA analysis of NF-kB After thawing the frozen FF samples, the follicular fluid NF-kB concentration was measured using the commercially available human NF-kB kit (Sunred Biotechnology, Shanghai, China) and the quantitative sandwich enzyme immunoassay principle in accordance with the kit procedures. The intra-assay and inter-assay coefficients of variation of the kit were < 10% and < 12%, respectively.The assay range was 0.15–40 ng/mL, and the sensitivity was 0.146 ng/mL.A Bio-Tek ELx50 device (BioTek Instruments, USA)was used for plate washing, and a Bio-Tek ELx800 device (BioTek Instruments, USA)was used for absorbance readings. The concentrations corresponding to absorbance were calculated by the standard curve table formula. Spectrophotometric analysis of follicular fluid oxidant and antioxidant status The follicular fluid TAS and TOS concentrations were determined using a spectrophotometric method with commercially available kits according to the manufacturer's instructions (Rel Assay, Mega Medicine Industry & Trade Co., Gaziantep, Turkey).The oxidative stress index (OSI) was calculated as the percentage of TOS to TAS. Statistical Analysis The normality of the data distribution was assessed using the Shapiro-Wilk test. Variables showing normal distribution were reported as mean ± standard deviation, while those not showing normal distribution were reported as median (1st quartile–3rd quartile). The analysis of variables between groups was performed using Student's t-test or the Mann-Whitney U test, depending on the normality of the distribution. The relationships between the variables were evaluated using Spearman’s correlation analyses. All statistical analyses were performed using IBM SPSS version 27.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 10.4.2 software (GraphPad Software, San Diego, California, USA). The P significance level was set at < 0.05. To create the correlation matrix showing the correlation analysis, R Studio version 4.3.3 and the “Metan” package were used( 17 ). Results As shown in Table 1 , the average age, BMI, and infertility duration of the agonist and hCG trigger groups were similar. The mean endometrial thickness measured on the day of ovulation triggering was similar in both groups. The total amount of gonadotropin consumed, total number of oocytes, and serum estradiol levels measured on the ovulation trigger day were similar in both groups. In the endometrioma group, cysts were located bilaterally in three cases and unilaterally in six cases.Median endometrioma diameter was recorded as 3.8 cm (range 3–5 cm). FF-PTX3 mRNA expression in the rhCG-triggered group was significantly higher than the GnRH agonist trigger group. A 5-fold up-regulation in the relative expression of FF-PTX3 mRNA was detected in the hCG group compared to the GnRH agonist trigger group (1.02 ± 0.26 vs. 5.39 ± 0.29, p < 0.001). When PTX3 mRNA expression was evaluated according to subgroups, significant downregulation was detected in the PCOS group compared to the other three groups. The relative FF-PTX3 mRNA expression of the endometrioma group was higher than all groups. Relative gene expression levels of male factor and RIF groups were similar (Fig. 1 ). In the rhCG-triggered group, the FF-NF-κB protein concentration was significantly higher than that in the GnRH agonist-triggered group (p < 0.001). Similarly, FF-TAS levels in the rhCG group were significantly higher than those in the agonist group (p = 0.001). The FF-TOS and OSI values ​​of the rhCG group were significantly lower than those of the agonist group (p = 0.010 and p < 0.001, respectively) (Fig. 2 ). FF-TAS levels were positively correlated with FF-NF-κB (r = 0.506, p = 0.014) and FF-PTX3 levels (r = 0.530, p = 0.009). FF-TOS levels were negatively correlated with FF-NF-kB (r=-0.480, p = 0.020) and FF-PTX3 (r=-0.440, p = 0.036). FF-OSI levels were negatively correlated with FF-NF-kB (r=-0.497, p = 0.016) and FF-PTX3 (r=-0.443, p = 0.034). (Fig. 3 ). Table 1 Demographic characteristics of both the participant groups. hCG trigger GnRH agonist trigger P-values N, % 23 (57.5) 17 ( 42.5) Age (years) 30.91 ± 2.47 29.8 ± 2.79 0.190 a BMI (kg/m 2 ) 23.18 ± 3.08 22.99 ± 3.24 0.855 a Infertility duration (years) 3 (2.5-4) 3.5 ( 3 – 4 ) 0.085 b LH (mIU/mL) 4.59 ± 1.57 4.77 ± 1.11 0.696 a FSH (mIU/mL) 5.87 ± 1.93 5.57 ± 1.23 0.579 a Endometrial thickness (mm) 9.25 ± 0.48 9.48 ± 0.68 0.232 a Total rFSH dose (IU) 2380.58 ± 625.31 2241.03 ± 846.47 0.552 a Estradiol on the day of hCG (pg/mL) 1566.03 ± 599.4 1599.41 ± 714.7 0.873 a Number of oocytes collected (n) 7.13 ± 1.84 7.65 ± 1.97 0.399 a FF-PTX3 (mRNA) 5.39 ± 0.29 1.02 ± 0.26 < 0.001 a NF-kB (ng/mL) 7.63 ± 2.93 3.14 ± 1.35 < 0.001 a TAS (mmol Trolox Eq/L) 1.70 ± 0.37 1.33 ± 0.25 0.001 a TOS (µmol H 2 O 2 Eq/L) 29.6 ± 9.04 36.99 ± 7.65 0.010 a OSI (arbitrary unit) 1.90 ± 0.87 2.95 ± 0.80 < 0.001 a a Independent sample t-test, b Mann-Whitney U test. BMI:body mass index, FSH: follicle-stimulating hormone, LH: luteinizing hormone,hCG: human chorionic gonadotrophin NF-kB: nuclear factor-κB, PTX3: long pentraxin 3, TAS:total antioxidant status, TOS: total oxidant status, OSI: oxidative stress index. We did not observe any significant morphological changes in the microscopic examination of COCs in the hCG group with high FF-PTX3 and NF-kB expression and the agonist trigger group with low FF-PTX3 and NF-kB levels. CCs in both groups had bright cytoplasm and radial projections. Low PTX3 did not cause loss of transparency in CCs. The presence of a 2PN zygote was considered fertilization. 2PN zygote rates of hCG and agonist trigger groups were similar regardless of PTX3 expression. The 2 PN zygote rate was recorded as 80.5% in the hCG group and 74% in the agonist trigger group. Discussion In natural cycles, the transformation of a primordial germ cell into a mature follicle is a complex and coordinated process regulated by pituitary gonadotropins, local growth factors, and cytokines ( 18 ). Gonadotropins, which are the main managers of the process of producing an oocyte that has completed its developmental and steroidogenic capacity, manage this process by regulating the synthesis of many intermediary molecules within the follicular unit ( 19 ). With the combined effect of pulsatile FSH and LH, proliferation, neo-vascularization and a hyaluronan-rich extracellular matrix begins to form in the follicular unit. The developing follicle undergoes metamorphosis both morphologically and functionally and becomes an endocrine organ ( 20 ). Cumulus granulosa cells, which are the basic elements of the follicular unit along with the oocyte, play an important role in the expulsion of the mature follicle out of the follicle by providing the necessary nutrients during the process of the oocyte becoming mature, and by expanding during ovulation. Proinflammatory cytokines, whose synthesis and release are regulated by LH and the oocyte, are critical molecules that play a role in oocyte maturation and cumulus expansion by regulating local inflammation ( 3 , 18 ). Although the role of inflammatory molecules synthesized in cumulus and mural granulosa cells, which are elements of the follicular unit, and contribute to ovulation is well known in natural cycles, there is no clinical study on how these molecules change in controlled ovarian stimulation cycles. This study is important as it is the first study to analyze how recombinant hCG or GnRH agonist used to stimulate the final stage of follicular maturation in patients undergoing IVF/ICSI with an antagonist protocol affects FF-PTX3, NF-kB and redox balance. The main finding of the study was that FF-PTX3 mRNA and NF-kB protein concentrations were significantly higher in the rhCG group compared to the GnRH agonist group. Conversely, FF-PTX3 and NF-kB concentrations were significantly downregulated in the GnRH agonist group compared to the hCG group. It is a known fact that the LH/hCG peak increases cumulus expansion in both natural and stimulated cycles ( 3 , 12 , 21 ). LH/hCG stimulation increases the release of growth factors, such as amphiregulin, epiregulin, betacellulin, PGE2, and EGF, and causes cumulus expansion ( 22 – 25 ). The synthesis and release of these molecules are regulated by cumulus cell genes such as hyaluronic acid synthase 2, gremlin1, and long PTX3 ( 3 , 25 ). The present study provides evidence that rhCG, used to trigger ovulation in stimulated cycles, contributes to ovulation by stimulating PTX3 and NF-kB levels in the follicular unit. Consistent with our results, PTX3 mRNA expression was shown to start 2 h after hCG injection in mice and peak at 6 h ( 3 ).Since we collected oocytes 36 h after rhCG administration, the changes in FF-PTX3 and NF-kB levels reflect inflammatory cytokine changes during this time period. Successful oocyte collection within 36 h and a sufficient number of MII oocytes obtained are evidence that hCG injection stimulates FF-PTX3 and NF-kB levels to ensure late-stage follicle maturation. We do not know the mechanisms by which rhCG induces FF-PTX3 and NF-kB production. Unlike mural granulosa cells, cumulus cells do not contain LH receptors. In addition, mural granulosa cells do not express PTX3 ( 3 ). Therefore, we can suggest that hCG increases FF-PTX3 and NF-kB expression by activating oocyte-derived paracrine molecules such as GDF-9 rather than directly by cumulus cells ( 3 , 25 ). However, our last statement is an interpretation that requires clinical and laboratory confirmation. We do not clearly know the mechanism underlying the low PTX3 and NF-κB levels in the GnRH agonist group. However, despite the low PTX3 and NF-κB levels in the GnRH agonist group, the fact that similar amounts of MII oocytes were obtained with the hCG group suggests that NF-κB and PTX3 are not the only inflammatory molecules that determine final stage oocyte maturation. Indeed, cumulus expansion is a process controlled by other genes such as hyaluronic acid synthase 2 and gremlin1, apart from NF-κB and PTX3. Since we analyzed only two molecules, we cannot make clear comments about the other molecules involved in cumulus expansion. Most likely, GnRH agonists may provide final oocyte maturation by increasing the expression of genes other than PTX3 and NF-κB. This question can be answered by analyzing all cumulus expansion genes in cycles triggered by hCG or agonists. Although reactive oxygen species (ROS) are byproducts of intracellular metabolism, they have been directly linked to folliculogenesis ( 26 ).Although oocyte development is driven by reduction-oxidation reactions ( 26 , 27 ), there are no clear clinical data on how controlled ovarian stimulation affects the intrafollicular redox balance. The present study is clinically important because it shows that rhCG and GnRH agonists used to trigger ovulation in stimulated cycles alter the intrafollicular redox balance.The fact that FF-TAS levels were higher in the rhCG group compared to the agonist group may be evidence that rhCG enables cumulus expansion with its antioxidant effect. In line with this, the fact that oxidant indicators TOS and OSI of the patients in the rhCG group were lower than those in the agonist group may be an indication that rhCG contributes to ovulation by activating the redox balance in an antioxidant way. The fact that TAS levels were low and TOS and OSI levels were high in the GnRH agonist group suggests that these drugs negatively affect the redox balance. However, the fact that the number of eggs collected was similar to the rhCG group suggests that the oxidative redox balance did not significantly affect the egg collection rates. However, Dumollard et al. ( 28 ) showed that the intrafollicular redox balance is altered during mouse oogenesis and is critical for early oocyte development and oocyte-embryo transition. It has also been shown that oogenesis and early embryogenesis are negatively affected when the redox balance is disrupted ( 29 ). The discrepancy between our results and the experimental data may be due to the different species of participants in the study groups. Mechanical collection of oocytes in stimulated cycles may also be another reason for the difference between experimental studies and human studies. A clear conclusion can be reached by analyzing the follicular fluid redox balance markers in patients undergoing natural and spontaneous cycles. Conclusion The similarity of COCs morphologies in the hCG and agonist trigger groups suggests that low PTX3 and NF-kB expression do not cause a morphological change, and no interpretation can be made regarding gene expression based on microscopic images. In addition, the similar fertilization rates of both trigger groups allow the choice of agonist or hCG trigger to be left to the IVF practitioner. Since the participant profiles in the rhCG and agonist groups were similar, we can attribute the differences in FF-PTX3 and NF-kB expression between the groups to the applied trigger agent.Although the redox balance was in the antioxidative direction in the rhCG group and in the oxidative direction in the agonist group, similar egg collection rates suggest that mechanical egg collection is not affected by the type of ovulation-inducing drug. It will be possible to find answers to questions that we could not clarify with more comprehensive studies examining more participants and all cumulus expansion genes. Declarations Funding : This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by Local Ethics Committee (protocol code 10-22 and date of approval 27.07.2023 ). Authors had received the informed consent from the study participants. Informed Consent Statement: Informed consent was obtained from all the participants involved in this study. Conflict Interests: The authors report there are no conflict interests to declare. Author Contributions: Conceptualization, F.T.C..; Software,F.T.C..; Formal analysis, P.K., and P.K.; Investigation,S.E..; Resources, S.E.; Data curation, Z.K.; Visualization, Z.K.; Project administration, F.T.C. All authors have read and agreed to the published version of the manuscript. References Sayutti N, Abu MA, Ahmad MF. PCOS and Role of Cumulus Gene Expression in Assessing Oocytes Quality. Front Endocrinol (Lausanne). 2022;13:843867. Fülöp C, Kamath RV, Li Y, Otto JM, Salustri A, Olsen BR, Glant TT, Hascall VC. Coding sequence, exon-intron structure and chromosomal localization of murine TNF-stimulated gene 6 that is specifically expressed by expanding cumulus cell-oocyte complexes. Gene. 1997;202:95–102. 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Nat Rev Mol Cell Biol. 2014;15:411–21. Petrova B, Liu K, Tian C, Kitaoka M, Freinkman E, Yang J, Orr-Weaver TL. Dynamic redox balance directs the oocyte-to-embryo transition via developmentally controlled reactive cysteine changes. Proc Natl Acad Sci U S A. 2018;115:E7978–86. Dumollard R, Ward Z, Carroll J, Duchen MR. Regulation of redox metabolism in the mouse oocyte and embryo. Development. 2007;134:455–65. Urner F, Sakkas D. Involvement of the pentose phosphate pathway and redox regulation in fertilization in the mouse. Mol Reprod Dev. 2005;70:494–503. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7180678","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496574571,"identity":"2a6dc44a-db88-4103-a56f-8a63b7c639c7","order_by":0,"name":"Fatma Tanılır","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYBAC9gYog42dsYHhI5jH2HgAnxaeA8xAMgGohZmxgXFmA4MEUEsDcVoYgDQzL1gLAwN+Leznj274+cMuj4+Zue2z7Q6bOt32w0BbamyicWrhSWa72ZOQXAx0WPPs3DNpEmZnEoFajqXlNuDQYs+QzHaDJ4E5sQ2ohTm37bCE2QGgFsaGwzi18PA/Zrv5J6EeosUSpOX8QwJaJJLZbvMkHIZoYQRpuUHIFonHZrdl0o6DtTD2tqVJbrsBtCUBj194+BOf3XxjU504v739McPPNht+s/PpDx98qLHBqQUHSCBN+SgYBaNgFIwCNAAA+6Jc3z8xU2EAAAAASUVORK5CYII=","orcid":"","institution":"private clınıc diyarbakır","correspondingAuthor":true,"prefix":"","firstName":"Fatma","middleName":"","lastName":"Tanılır","suffix":""},{"id":496574572,"identity":"a93fb6f4-5697-47b9-b0b0-c38874623d0f","order_by":1,"name":"Sevil Çiçek","email":"","orcid":"","institution":"Medeniyet Üniversitesi Göztepe Eğitim ve Araştırma Hastanesi","correspondingAuthor":false,"prefix":"","firstName":"Sevil","middleName":"","lastName":"Çiçek","suffix":""},{"id":496574573,"identity":"e181e538-d356-4782-b3a3-5788d22ab4fb","order_by":2,"name":"Pınar Kırıcı","email":"","orcid":"","institution":"Malatya Turgut Özal Üniversitesi","correspondingAuthor":false,"prefix":"","firstName":"Pınar","middleName":"","lastName":"Kırıcı","suffix":""}],"badges":[],"createdAt":"2025-07-21 20:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7180678/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7180678/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88642926,"identity":"923a3378-4199-42db-affc-6c6a27aac309","added_by":"auto","created_at":"2025-08-08 16:12:45","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95840,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical presentation of the fold change in FF-PTX3 mRNA expression in MII oocytes of the rhCG and GnRH agonist-triggering groups. FF-PTX3 mRNA expression of the rhCG group was approximately five fold higher than the expression level agonist goup \u003cem\u003e(​Data are expressed as the mean ± SD). An independent sample t-test was used for comparison.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7180678/v1/d1370dacdda293389b7db1cb.jpeg"},{"id":88642933,"identity":"00183ff8-bfe3-4e8e-b660-4af838a50e76","added_by":"auto","created_at":"2025-08-08 16:12:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":286336,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical presentation of the comparative analysis of follicular fluid NF-kB (A), TAS (B), TOS (C), and OSI (D) levels between the rhCG and GnRH agonist triggering groups.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7180678/v1/ad2ed2c32ed1fc6172539a89.jpeg"},{"id":88642929,"identity":"ec17a5a6-7b39-4f37-823b-bf02617d7f72","added_by":"auto","created_at":"2025-08-08 16:12:45","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43167,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical presentation of the correlation matrix illustrates the significant correlations between the relative expression of PTX-3 in follicular fluid and other variables. Blue hues denote positive correlations, while red hues indicate negative correlations.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7180678/v1/dc75e46d5d24b2377fce5475.jpeg"},{"id":94852500,"identity":"937438cc-dbb4-4fe2-b233-a3968b345e2c","added_by":"auto","created_at":"2025-10-31 11:24:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":977251,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7180678/v1/5fbc6463-81a9-4ecc-b7e9-e8595711f4ac.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of the effects of recombinant hCG and GnRH agonist on follicular fluid pentraxin 3, NF-kB and redox balance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGerm cells undergo complex morphological and metabolic changes until they develop into mature oocytes. This process, called folliculogenesis, begins with the reactivation of arrested follicles in the intrauterine period and continues until ovulation. The interaction of follicular unit elements formed in the oocyte and granulosa cells ensures the ovulation of a mature oocyte that has completed its developmental capacity. Mural granulosa cells on the inner surface of the follicle and cumulus granulosa cells (CCs) around the oocyte are critical for the development of a mature (MII) oocyte (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In the pre-ovulation period, the accumulation of hyaluronic acid-rich matrix in the extracellular space and the radial opening of CCs is called cumulus expansion (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In natural cycles, CC expansion is mandatory for ovulation, resumption of meiosis, and successful fertilization (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In vivo, CC expansion also plays a role in the accumulation of sperm around the COC in the ampullary region (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe ovarian follicle is the basic functional unit composed of somatic and germ cell components responsible for oocyte development (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).Normal follicular development and ovulation are achieved with adequate inflammatory and oxidative stress (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).Cumulus cells themselves and the ocytes keep cumulus cell differentiation and expansion under tight control (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).Since the fertilization potential of oocytes with inadequate cumulus expansion is limited, evolutionarily cumulus cell expansion is maintained by more than one gene (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Hyaluronic acid synthase 2, gremlin1, nuclear factor kappa beta (NF-kB), and long pentraxin 3 (PTX3) are the main genes responsible for cumulus expansion. Oocytes expressing these genes have a higher fertilization potential than those that do not (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).Nuclear factor-κappaB (NF-κB) and long pentraxin 3 (PTX3) work together to provide the physiological inflammatory conditions necessary for many cellular functions (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).NF-kB, the main cellular regulator of the inflammatory cascade, is phosphorylated by different cytokines and enables the expression of immune response genes (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).In mice and humans, NF-kB and long Ptx3 mRNA are expressed only by cumulus cells from the follicular unit elements (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).Long PTX3 and NF-kBare involved in cumulus matrix stability and expansion (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). PTX3, whose expression increases secondary to inflammation, prevents tissue damage by regulating apoptosis on the basis of inflammation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Follicular development and ovulation are significantly affected by cumulus cells expressing sufficient amounts of NF-kB and PTX3 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn natural cycles, successful ovulation is possible when pituitary LH balances the release of inflammatory molecules and reactive oxygen species in the somatic compartment of the follicular unit. Follicular fluid (FF) located in the antral space plays a role in oocyte-granulosa cell communication. FF, which carries nutrients from the CCs to the ocyte, is like a living medium consisting of oocyte and follicle cell-derived hormones, enzymes, and electrolytes. While oocytes from mice null for some proinflammatory cytokines do not fertilize in vivo, the same oocytes fertilize in vitro (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).This finding suggests that natural cycles and assisted reproductive cycles may be different in terms of inflammatory cytokines and redox balance markers. Controlled ovarian stimulation protocols used to achieve multifollicular development in assisted conception is a process that causes supraphysiological changes in the morphology, number and functions of follicular unit elements.Drugs used to trigger ovulation mimic the physiological LH peak and cause cumulus expansion. The most common drugs used to induce ovulation in IVF/ICSI cycles are recombinant human chorionic gonadotropin (rHCG) and gonadotropin-releasing hormone (GnRHa) agonists. It has been reported that hCG injection into mice causes a significant increase in inflammatory cytokines expression(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Unlike natural cycles, there are no data on how ovulation-inducing agents used in assisted reproductive techniques affect the synthesis and release of follicular fluid pro-inflammatory cytokines and reactive oxygen species (ROS).This study was aimed to analyze the follicular fluid PTX3, NF-kB and redox balance markers of infertile patients whose ovulation was triggered with recombinant hCG or GnRH agonist.The effects of rHCG and GnRHa on follicular fluid oxidative stress indices were evaluated by total oxidant status (TOS), total antioxidant status (TAS), and oxidative stress index (OSI).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eA total of 40 patients under the age of 35 who were planned for assisted conception due to different infertility etiologies were included in the study. According to infertility etiologies, participants were grouped as PCOS (n\u0026thinsp;=\u0026thinsp;13), uni-or bilateral endometrioma (n\u0026thinsp;=\u0026thinsp;9), male factor (n\u0026thinsp;=\u0026thinsp;11) and recurrent implantation failure (n\u0026thinsp;=\u0026thinsp;7).Failure of two or more IVF/ICSI attempts was considered RIF (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Those who met at least two of the Rotterdam criteria of ovulatory dysfunction, hyperandrogenemia and polycystic ovarian morphology were considered PCOS (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The diagnosis of endometrioma was made by transvaginal ultrasonography and its presence was confirmed for at least two cycles. Patients with severe oligoasthenoteratatospermia or azoospermia in at least two separate semen analyzes (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) were defined as male factor infertility.\u003c/p\u003e\u003cp\u003eParticipants in each group underwent controlled ovarian stimulation with a flexible GnRH antagonist protocol. Recombinant FSH treatment (Gonal-f, Merck-Serono, Bari, Italy) was started on the second or third day of the cycle, with the dose adjusted according to the patient's age, BMI, and AFC values. When the dominant follicle reached\u0026thinsp;\u0026ge;\u0026thinsp;14 mm, the pituitary was suppressed with 0.25 mg/day cetrotide (Merck-Serono, France). When two or more follicles had a diameter of at least 18 mm, ovulation was triggered in patients with either 0.2 mg triptorelin (Gonapeptyl; Ferring, Istanbul, Turkey) or 250 micrograms/0.5 mL recombinant hCG (Ovitrelle; Merck-Serono, Bari, Italy).Ovulation was triggered by hCG in 23 patients and by GnRH agonist in 17 patients. The selection of which patient would be triggered with hCG or agonist was done randomly, unless they were at high risk of OHSS. Agonist trigger was used for those at high risk for OHSS. Oocytes were collected under ultrasound guidance 35\u0026ndash;36 hours after the ovulation trigger.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFollicular fluidsampling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollicular fluid samples that did not show massive blood contamination during oocyte retrieval were placed in Eppendorf tubes (Isolab Laborger\u0026auml;te GmbH, Germany) containing RNA stabilization buffer (Qiagen, Hilden, Germany) and centrifuged at 5000 g for 10 min. The supernatant was then removed and stored at -20\u0026deg;C until RNA extraction. Only follicular fluids containing mature MII oocytes that had shed their first polar bodies were evaluated. FFs of oocytes in the GV or MI stages were excluded from the evaluation.ICSI was performed on all the MII oocytes.The relationship between PTX3, NF-kB, redox markers and fertilization rate was recorded.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFF samples of MII oocytes collected from patients administered hCG or agonist triggers were subjected to qRT-PCR to detect long PTX3 mRNA expression.Glyceraldehyde-3-Phosphate Dehydrogenase was taken as the reference gene. Total RNA isolation of cumulus cells was performed with PureLink Total RNA Mini Kit (Invitrogen). RNA was reverse transcribed using the High Capacity cDNA RT Kit (Applied Biosystems, Foster City, CA). Blirt Amplyfyme SYBR Green Master Mix was used in the real-time PCR section. Primer sequences for qRT-PCR were as follows: PTX3: Forward 5'-TGGACAACGAAATAG ACATGG-3', Reverse 5' CTCTCATCTGCGAGTTCTCC-3', GAPDH: Forward 5' GAAGATGGTGATGGGATTTC-3', Reverse 5'-GAAGGTGAAGGTCGGAGTC- 3'.\u003c/p\u003e\u003cp\u003e\u003cb\u003eELISA analysis of NF-kB\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter thawing the frozen FF samples, the follicular fluid NF-kB concentration was measured using the commercially available human NF-kB kit (Sunred Biotechnology, Shanghai, China) and the quantitative sandwich enzyme immunoassay principle in accordance with the kit procedures. The intra-assay and inter-assay coefficients of variation of the kit were \u0026lt;\u0026thinsp;10% and \u0026lt;\u0026thinsp;12%, respectively.The assay range was 0.15\u0026ndash;40 ng/mL, and the sensitivity was 0.146 ng/mL.A Bio-Tek ELx50 device (BioTek Instruments, USA)was used for plate washing, and a Bio-Tek ELx800 device (BioTek Instruments, USA)was used for absorbance readings. The concentrations corresponding to absorbance were calculated by the standard curve table formula.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpectrophotometric analysis of follicular fluid oxidant and antioxidant status\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe follicular fluid TAS and TOS concentrations were determined using a spectrophotometric method with commercially available kits according to the manufacturer's instructions (Rel Assay, Mega Medicine Industry \u0026amp; Trade Co., Gaziantep, Turkey).The oxidative stress index (OSI) was calculated as the percentage of TOS to TAS.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe normality of the data distribution was assessed using the Shapiro-Wilk test. Variables showing normal distribution were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, while those not showing normal distribution were reported as median (1st quartile\u0026ndash;3rd quartile). The analysis of variables between groups was performed using Student's t-test or the Mann-Whitney U test, depending on the normality of the distribution. The relationships between the variables were evaluated using Spearman\u0026rsquo;s correlation analyses. All statistical analyses were performed using IBM SPSS version 27.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 10.4.2 software (GraphPad Software, San Diego, California, USA). The P significance level was set at \u0026lt;\u0026thinsp;0.05. To create the correlation matrix showing the correlation analysis, R Studio version 4.3.3 and the \u0026ldquo;Metan\u0026rdquo; package were used(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the average age, BMI, and infertility duration of the agonist and hCG trigger groups were similar. The mean endometrial thickness measured on the day of ovulation triggering was similar in both groups. The total amount of gonadotropin consumed, total number of oocytes, and serum estradiol levels measured on the ovulation trigger day were similar in both groups. In the endometrioma group, cysts were located bilaterally in three cases and unilaterally in six cases.Median endometrioma diameter was recorded as 3.8 cm (range 3\u0026ndash;5 cm).\u003c/p\u003e\u003cp\u003eFF-PTX3 mRNA expression in the rhCG-triggered group was significantly higher than the GnRH agonist trigger group. A 5-fold up-regulation in the relative expression of FF-PTX3 mRNA was detected in the hCG group compared to the GnRH agonist trigger group (1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 vs. 5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When PTX3 mRNA expression was evaluated according to subgroups, significant downregulation was detected in the PCOS group compared to the other three groups. The relative FF-PTX3 mRNA expression of the endometrioma group was higher than all groups. Relative gene expression levels of male factor and RIF groups were similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the rhCG-triggered group, the FF-NF-κB protein concentration was significantly higher than that in the GnRH agonist-triggered group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, FF-TAS levels in the rhCG group were significantly higher than those in the agonist group (p\u0026thinsp;=\u0026thinsp;0.001). The FF-TOS and OSI values ​​of the rhCG group were significantly lower than those of the agonist group (p\u0026thinsp;=\u0026thinsp;0.010 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). FF-TAS levels were positively correlated with FF-NF-κB (r\u0026thinsp;=\u0026thinsp;0.506, p\u0026thinsp;=\u0026thinsp;0.014) and FF-PTX3 levels (r\u0026thinsp;=\u0026thinsp;0.530, p\u0026thinsp;=\u0026thinsp;0.009). FF-TOS levels were negatively correlated with FF-NF-kB (r=-0.480, p\u0026thinsp;=\u0026thinsp;0.020) and FF-PTX3 (r=-0.440, p\u0026thinsp;=\u0026thinsp;0.036). FF-OSI levels were negatively correlated with FF-NF-kB (r=-0.497, p\u0026thinsp;=\u0026thinsp;0.016) and FF-PTX3 (r=-0.443, p\u0026thinsp;=\u0026thinsp;0.034). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographic characteristics of both the participant groups.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehCG trigger\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGnRH agonist trigger\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP-values\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN, %\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23 (57.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17 ( 42.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.190\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.855\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInfertility duration (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 (2.5-4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.5 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.085\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLH (mIU/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.696 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFSH (mIU/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.579 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEndometrial thickness (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.232\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal rFSH dose (IU)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2380.58\u0026thinsp;\u0026plusmn;\u0026thinsp;625.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2241.03\u0026thinsp;\u0026plusmn;\u0026thinsp;846.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.552\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEstradiol on the day of hCG (pg/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1566.03\u0026thinsp;\u0026plusmn;\u0026thinsp;599.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1599.41\u0026thinsp;\u0026plusmn;\u0026thinsp;714.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.873 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of oocytes collected (n)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.399 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFF-PTX3 (mRNA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNF-kB (ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.63\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTAS (mmol Trolox Eq/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTOS (\u0026micro;mol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Eq/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36.99\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.010\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOSI (arbitrary unit)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003eIndependent sample t-test, \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003eMann-Whitney U test. BMI:body mass index, FSH: follicle-stimulating hormone, LH: luteinizing hormone,hCG: human chorionic gonadotrophin NF-kB: nuclear factor-κB, PTX3: long pentraxin 3, TAS:total antioxidant status, TOS: total oxidant status, OSI: oxidative stress index.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWe did not observe any significant morphological changes in the microscopic examination of COCs in the hCG group with high FF-PTX3 and NF-kB expression and the agonist trigger group with low FF-PTX3 and NF-kB levels. CCs in both groups had bright cytoplasm and radial projections. Low PTX3 did not cause loss of transparency in CCs. The presence of a 2PN zygote was considered fertilization. 2PN zygote rates of hCG and agonist trigger groups were similar regardless of PTX3 expression. The 2 PN zygote rate was recorded as 80.5% in the hCG group and 74% in the agonist trigger group.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn natural cycles, the transformation of a primordial germ cell into a mature follicle is a complex and coordinated process regulated by pituitary gonadotropins, local growth factors, and cytokines (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Gonadotropins, which are the main managers of the process of producing an oocyte that has completed its developmental and steroidogenic capacity, manage this process by regulating the synthesis of many intermediary molecules within the follicular unit (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). With the combined effect of pulsatile FSH and LH, proliferation, neo-vascularization and a hyaluronan-rich extracellular matrix begins to form in the follicular unit. The developing follicle undergoes metamorphosis both morphologically and functionally and becomes an endocrine organ (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Cumulus granulosa cells, which are the basic elements of the follicular unit along with the oocyte, play an important role in the expulsion of the mature follicle out of the follicle by providing the necessary nutrients during the process of the oocyte becoming mature, and by expanding during ovulation. Proinflammatory cytokines, whose synthesis and release are regulated by LH and the oocyte, are critical molecules that play a role in oocyte maturation and cumulus expansion by regulating local inflammation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough the role of inflammatory molecules synthesized in cumulus and mural granulosa cells, which are elements of the follicular unit, and contribute to ovulation is well known in natural cycles, there is no clinical study on how these molecules change in controlled ovarian stimulation cycles. This study is important as it is the first study to analyze how recombinant hCG or GnRH agonist used to stimulate the final stage of follicular maturation in patients undergoing IVF/ICSI with an antagonist protocol affects FF-PTX3, NF-kB and redox balance. The main finding of the study was that FF-PTX3 mRNA and NF-kB protein concentrations were significantly higher in the rhCG group compared to the GnRH agonist group. Conversely, FF-PTX3 and NF-kB concentrations were significantly downregulated in the GnRH agonist group compared to the hCG group. It is a known fact that the LH/hCG peak increases cumulus expansion in both natural and stimulated cycles (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). LH/hCG stimulation increases the release of growth factors, such as amphiregulin, epiregulin, betacellulin, PGE2, and EGF, and causes cumulus expansion (\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The synthesis and release of these molecules are regulated by cumulus cell genes such as hyaluronic acid synthase 2, gremlin1, and long PTX3 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The present study provides evidence that rhCG, used to trigger ovulation in stimulated cycles, contributes to ovulation by stimulating PTX3 and NF-kB levels in the follicular unit. Consistent with our results, PTX3 mRNA expression was shown to start 2 h after hCG injection in mice and peak at 6 h (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).Since we collected oocytes 36 h after rhCG administration, the changes in FF-PTX3 and NF-kB levels reflect inflammatory cytokine changes during this time period. Successful oocyte collection within 36 h and a sufficient number of MII oocytes obtained are evidence that hCG injection stimulates FF-PTX3 and NF-kB levels to ensure late-stage follicle maturation. We do not know the mechanisms by which rhCG induces FF-PTX3 and NF-kB production. Unlike mural granulosa cells, cumulus cells do not contain LH receptors. In addition, mural granulosa cells do not express PTX3 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Therefore, we can suggest that hCG increases FF-PTX3 and NF-kB expression by activating oocyte-derived paracrine molecules such as GDF-9 rather than directly by cumulus cells (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, our last statement is an interpretation that requires clinical and laboratory confirmation.\u003c/p\u003e\u003cp\u003eWe do not clearly know the mechanism underlying the low PTX3 and NF-κB levels in the GnRH agonist group. However, despite the low PTX3 and NF-κB levels in the GnRH agonist group, the fact that similar amounts of MII oocytes were obtained with the hCG group suggests that NF-κB and PTX3 are not the only inflammatory molecules that determine final stage oocyte maturation. Indeed, cumulus expansion is a process controlled by other genes such as hyaluronic acid synthase 2 and gremlin1, apart from NF-κB and PTX3. Since we analyzed only two molecules, we cannot make clear comments about the other molecules involved in cumulus expansion. Most likely, GnRH agonists may provide final oocyte maturation by increasing the expression of genes other than PTX3 and NF-κB. This question can be answered by analyzing all cumulus expansion genes in cycles triggered by hCG or agonists.\u003c/p\u003e\u003cp\u003eAlthough reactive oxygen species (ROS) are byproducts of intracellular metabolism, they have been directly linked to folliculogenesis (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).Although oocyte development is driven by reduction-oxidation reactions (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), there are no clear clinical data on how controlled ovarian stimulation affects the intrafollicular redox balance. The present study is clinically important because it shows that rhCG and GnRH agonists used to trigger ovulation in stimulated cycles alter the intrafollicular redox balance.The fact that FF-TAS levels were higher in the rhCG group compared to the agonist group may be evidence that rhCG enables cumulus expansion with its antioxidant effect. In line with this, the fact that oxidant indicators TOS and OSI of the patients in the rhCG group were lower than those in the agonist group may be an indication that rhCG contributes to ovulation by activating the redox balance in an antioxidant way. The fact that TAS levels were low and TOS and OSI levels were high in the GnRH agonist group suggests that these drugs negatively affect the redox balance. However, the fact that the number of eggs collected was similar to the rhCG group suggests that the oxidative redox balance did not significantly affect the egg collection rates. However, Dumollard et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) showed that the intrafollicular redox balance is altered during mouse oogenesis and is critical for early oocyte development and oocyte-embryo transition. It has also been shown that oogenesis and early embryogenesis are negatively affected when the redox balance is disrupted (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The discrepancy between our results and the experimental data may be due to the different species of participants in the study groups. Mechanical collection of oocytes in stimulated cycles may also be another reason for the difference between experimental studies and human studies. A clear conclusion can be reached by analyzing the follicular fluid redox balance markers in patients undergoing natural and spontaneous cycles.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe similarity of COCs morphologies in the hCG and agonist trigger groups suggests that low PTX3 and NF-kB expression do not cause a morphological change, and no interpretation can be made regarding gene expression based on microscopic images. In addition, the similar fertilization rates of both trigger groups allow the choice of agonist or hCG trigger to be left to the IVF practitioner. Since the participant profiles in the rhCG and agonist groups were similar, we can attribute the differences in FF-PTX3 and NF-kB expression between the groups to the applied trigger agent.Although the redox balance was in the antioxidative direction in the rhCG group and in the oxidative direction in the agonist group, similar egg collection rates suggest that mechanical egg collection is not affected by the type of ovulation-inducing drug. It will be possible to find answers to questions that we could not clarify with more comprehensive studies examining more participants and all cumulus expansion genes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e The study was conducted in accordance with the Declaration of Helsinki, and approved by Local Ethics Committee (protocol code 10-22 and date of approval 27.07.2023\u003cstrong\u003e).\u003c/strong\u003eAuthors had received the informed consent from the study participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Informed consent was obtained from all the participants involved in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict Interests:\u003c/strong\u003e The authors report there are no conflict \u0026nbsp;interests to declare.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, F.T.C..; Software,F.T.C..; Formal analysis, P.K., and P.K.; Investigation,S.E..; Resources, S.E.; Data curation, Z.K.; Visualization, Z.K.; Project administration, F.T.C. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSayutti N, Abu MA, Ahmad MF. PCOS and Role of Cumulus Gene Expression in Assessing Oocytes Quality. Front Endocrinol (Lausanne). 2022;13:843867.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eF\u0026uuml;l\u0026ouml;p C, Kamath RV, Li Y, Otto JM, Salustri A, Olsen BR, Glant TT, Hascall VC. Coding sequence, exon-intron structure and chromosomal localization of murine TNF-stimulated gene 6 that is specifically expressed by expanding cumulus cell-oocyte complexes. Gene. 1997;202:95\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSalustri A, Garlanda C, Hirsch E, De Acetis M, Maccagno A, Bottazzi B, Doni A, Bastone A, Mantovani G, Beck Peccoz P, Salvatori G, Mahoney DJ, Day AJ, Siracusa G, Romani L, Mantovani A. 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Mol Reprod Dev. 2005;70:494\u0026ndash;503.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Follicular fluid, long pTX3, NF-kB, Redox balance, GnRH, hCG","lastPublishedDoi":"10.21203/rs.3.rs-7180678/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7180678/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e\u003cp\u003eTo compare the effects of ovulation triggering with recombinant human chorionic gonadotropin (hCG) or GnRH agonist on follicular fluid (FF) long pentraxin 3 (PTX3), nuclear factor kappaB (NF-kB), and redox balance markers in infertile patients.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e\u003cp\u003eA total of 40 patients under the age of 35 who were planned for assisted conception were included in the study. Participants were grouped according to their infertility etiology as PCOS (n\u0026thinsp;=\u0026thinsp;13), uni- or bilateral endometrioma (n\u0026thinsp;=\u0026thinsp;9), male factor (n\u0026thinsp;=\u0026thinsp;11), and recurrent implantation failure (RIF, n\u0026thinsp;=\u0026thinsp;7).The selection of which patient would be triggered with hCG or agonist was done randomly. PTX3 mRNA expression and NF-kB protein analysis were performed in FF samples collected from metaphase II mature oocytes. Follicular unit redox balance was determined by total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn the rhCG-triggered group, FF-NF-kB protein concentration was significantly higher than in the GnRH agonist-triggered group. Similarly, while FF-TAS levels were higher in the rhCG group, FF-TOS and OSI values ​​were significantly higher than in the agonist group.FF-PTX3 mRNA expression in the rhCG-triggered group was significantly higher than the GnRH agonist group. A 5-fold up-regulation in the relative expression of FF-PTX3 mRNA was detected in the hCG group compared to the GnRH agonist group (1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 vs. 5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When PTX3 mRNA expression was evaluated according to subgroups, significant downregulation was detected in the PCOS group compared to the other three groups. The relative FF-PTX3 mRNA expression of the endometrioma group was higher than all groups. Relative gene expression levels of male factor and RIF groups were similar.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003erhCG induces ovulation more effectively than GnRHa agonists by providing a clearer balance between proinflammatory cytokines and redox balance markers.\u003c/p\u003e","manuscriptTitle":"Comparison of the effects of recombinant hCG and GnRH agonist on follicular fluid pentraxin 3, NF-kB and redox balance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 16:12:40","doi":"10.21203/rs.3.rs-7180678/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9f1c8d1-d3d8-4331-b273-8e1b25e12b03","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-31T11:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-08 16:12:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7180678","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7180678","identity":"rs-7180678","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}