{"paper_id":"7a677ca9-338a-4c2c-8872-cb0cf1273af0","body_text":"Infertility can be defined as inability to conceive\ndespite sexual relationship without contraception\nfor 1 year. This problem concerns about 10-15%\nof the individuals in the fertile age ( 1 ).\nTo have a family and a child is well-accepted\nand a desired status all over the world. They are\nimportant for the development of the community\nand continuity of the generation. Couples with an\nenthusiasm of having a child but facing the problem\nof infertility, experience a decrease in communication,\nbelief of health, social esteem and\nself-confidence, and become disappointed about\nexpectations for the future. After the diagnosis of infertility is made, people ignore all areas of their\nlives and concentrate on the matter and the methods\nused in the processes of diagnosis and treatment,\nand particularly for the women, it becomes a\npainful chain of physical and emotional events ( 2 ).\nIn vitro  fertilization (IVF) can be defined as one\nof the assisted reproductive techniques medically\napplied on oocyte, sperm or embryo cells  in vitro \nin order to develop pregnancy ( 3 ,  4 ). Cytokines\nas key modulators of the immune system appear\nto modulate other regulatory systems. They also\ncontribute to regulation of the ovarian cycle ( 5 ). A\nproinflammatory cytokine tumor necrosis factoralpha\n(TNF-α) and C-reactive protein (CRP), one\nof the acute phase reactants, can increase resistin\nexpression ( 6 - 9 ). CRP is a negative regulator of\nfunctions of human leptin ( 10 ). Resistin is synthesized\nmostly by inflammatory cells such as\nmacrophages and correlated with TNF-α ( 9 ). Resistin\nlevels are capable of increasing expression\nof TNF-α via nuclear factor (NF)-κβ-dependent\npathway ( 6 ,  8 ,  11 - 13 ). Leptin, a proinflammatory\nfactor, regulates food intake and energy expenditure\n( 14 ). It is also linked to reproductive functions\n( 15 ). Leptin levels may be used as predictive markers\nof assisted reproductive technology (ART) ( 16 ,\n 17 ). It has been demonstrated that combined exposure\nof human monunuclear cells to high concentrations\nof insulin and leptin for 24 hours  in vitro \nstimulates resistin and TNF-α protein expression\n( 12 ). Leptin level is elevated in cases associated\nwith high levels of TNF-α that increases serum\nleptin concentrations ( 14 ,  18 ).\nStudies on reactive oxygen molecules (ROM)\nduring the course of this process and their relationship\nwith proinflammatory factors have gained\nimportance in recent years ( 19 ,  20 ). Shift of the\nequilibrium between pro-oxidants and anti-oxidants\ntowards pro-oxidants results in oxidative\nstress. Effects of oxidative stress on the stages of\nreproduction like oocyte maturation and follicular\ndevelopment are important from the IVF success\npoint of view ( 21 ). Its importance is emphasized\nby the conditions providing low oxygen during\nIVF application. Since the role of oxidative stress\non infertility has not been fully cleared yet, effects\nof lipid peroxidation upon various stages of IVF\nprocess and the contribution of some cytokines\nand hormones upon the process are noteworthy.\nThe aim of this study was to investigate the profiles\nof some pro-inflammatory factors, cytokines\nand hormones, such as CRP, TNF-α, leptin as well\nas resistin, known to be involved in the process of\ninflammation, to evaluate their relationship with\nlipid hydroperoxides, the markers of early lipid\nperoxidation and to assess their associations with\nfemale infertility.\n\nIn this prospective, non-randomized, controlled\nclinical study, the blood samples from 70 women,\nwho consulted to the IVF Center, Obstetrics and\nGynaecology Department, Cerrahpasa Medical\nFaculty, University of Istanbul, Istanbul, Turkey,\nwith the complaint of infertility were used. They\nalso had the features of being between the ages of\n23 and 40, being married for 3 years, having social\nsecurity for 5 years and having two times of intrauterine\ninsemination before, while they were taken\nfor analysis prior to the beginning of the treatment\nin order to determine the suitability for the participation\ninto the study.\nPatient history and gynaecological exam, routine\nbiochemical tests, ultrasonography, serology, basic\ninfertility tests [spermiogram, hormonal tests and\nhysterosalpingography (HSG)] were performed to\nevaluate the causes of infertility before the treatment\nin order to enlighten the source of the problem.\nCauses of reduced female fertility included decreased\novarian reserve, anovulation, uterine disorders\nother than endometriosis, fertility-sparing\nsurgery with unilateral salpingo-oophorectomy,\nmethylene tetrahydrofolate reductase gene mutation,\nunexplained reasons and presence of more\nthan one factor. Patients with polycystic ovary\nsyndrome (PCOS) were also included in the study\npopulation, with the result that both pregnant and\nnon-pregnant groups had a proportionate distribution\nto eliminate the effects of their possible contribution\nin terms of inflammation.\nA signed written informed consent was obtained\nfrom all participants prior to the study. Procedures\nwere carried out in accordance with Declaration of\nHelsinki. This project was approved by the Ethics\nCommittee and Institutional Board of Cerrahpasa\nMedical Faculty, Istanbul University, Istanbul,\nTurkey.\nTwenty-six women, who had given a birth without\nany medication constituted the control group (group 1). The blood samples from 70 infertile\nwomen were collected during the early follicular\nphase (the 3rd day of the cycle) before the onset\nof the intervention. A total of 26 individuals managed\nto complete the process with the appropriate\nrespond to treatment. Their \"pre-IVF samples\"\n(group 2) out of 70 were included into the study to\nconstitute the paired data with the \"post-IVF samples\"\n(group 3) taken on the 15 th  day of the application\nof embryo transfer from these 26 women.\nThere was no statistically significant difference\nbetween the age and body mass index (BMI) values\nof the control and patient groups (P=0.909,\nP=0.431, respectively).\nAnthropometric measurements and demographic\ncharacteristics of the women participated in the\nstudy were recorded. Blood samples were taken\ninto sterile vacuum operated tubes at 08:00 – 10:00\nam while fasting before the IVF treatment on the\n3rd day of the menstruation (follicular phase) and\non the 15 th  day after the embryo transfer, and were\ncentrifuged in 2000 rpm for 10 minutes. Serum\nsamples were stored at -80˚C until assayed.\nSerum anti-mullerian hormone (AMH), inhibin\nB, follicle stimulating hormone (FSH), luteinizing\nhormone (LH), estradiol (E 2 ), prolactin, and\nthyroid stimulating hormone (TSH) levels were\ndetermined in all women. The levels of TNF-α\n(Human TNF-α Elisa Kit, Assaypro, USA), resistin\n(Human Resistin Elisa Kit, Assaypro USA),\nleptin (Human Leptin Elisa Kit, Assaypro, USA),\nleptin receptor (Human Leptin Receptor Elisa\nKit, BioVendor, EU), and high sensitive -C reactive\nprotein (hs-CRP) (CRP HS ELISA Kit, DRG\nInt, Inc. USA) were determined by commercially\navailable enzyme-linked immunosorbent assay\n(ELISA) kits. Lipid hydroperoxide levels, one of\nthe important markers of oxidative stress, were\ndetermined by a spectrophotometric method [Lipid\nHydroperoxide (LPO) Assay Kit, Cayman\nChem Comp., USA].\nAll samples were assayed using the AssayMax\nHuman TNF-alpha ELISA kit (AssayPro, USA).\nThe intra-assay and inter-assay coefficients of\nvariation (CV) were 5.5 and 7.0%, respectively,\nusing the AssayMax Human Resistin ELISA kit\n(Assaypro, USA). The intra-assay and inter-assay\nCV were 4.0 and 7.2%, respectively, using the\nAssayMax Human Leptin ELISA kit (Assaypro,\nUSA). The intra-assay and inter-assay CV were\n4.0 and 7.7%, respectively, using the Human Leptin\nReceptor ELISA kit (BioVendor Research and\nDiagnostic Products, EU). The intra-assay and\ninter-assay CV were 7.2 and 9.8%, respectively,\nusing the hs-CRP ELISA kit (DRG International,\nInc., USA). The intra-assay and inter-assay CV\nwere 4.1 and 7.5%, respectively, using the Lipid\nHydroperoxide (LPO) Assay kit (Cayman Chemical\nCompany, USA).\nStatistical analyses were performed using the\nStatistical Package for Social Sciences (SPSS,\nSPSS Inc., Chicago, IL, USA) software package.\nData were analyzed using descriptive-analytic\ntests. Parametric variables were represented as\nmean and standard error (SE), and categorical data\nwere represented by number (n) and percentage\n(%). The values for arithmetical mean, standard\ndeviation (SD) and SE were calculated for the\npregnant and non-pregnant women in pre-IVF and\npost-IVF groups as well as for the participants in\ncontrol group. Mann-Whitney U test, paired sample\nt test, Wilcoxon signed-rank test as well as\nPearson correlation analysis were performed. P\nvalues less than 0.05 were considered significantly.\n\nIVF-applied group had a mean age (mean ± SE)\nof 31.2 ± 1.4 years and BMI of 25.4 ± 2.8 kg/m 2 .\nControl group was consisted of healthy women\nwho took no medication, had no illnesses, were\nspontaneously-conceived and volunteered. Mean\nage of this group was 31.4 ± 1.5 years and BMI\nvalue was 24.1 ± 1.1 kg/m 2 . There was no statistically\nsignificant difference between the ages and\nBMI values of the groups.\nEight of 26 women conceived after application\nof IVF treatment (30.8%). Two of them were concluded\nwith medical abortion due to unembryonic/\nempty sac pregnancy. Six of 26 women finished\nthe period with live birth (23.1%).\nIn  table 1 , some demographical and clinical parameters\nin non-pregnant and pregnant women are\nsummarized.\nValues for mean ± SE for the parameters of control,\npre- and post-IVF groups as well as P values\nthat define the statistical differences between the\ngroups are shown in  table 2 .\nThe values of LPO (P=0.000) and leptin receptor (P=0.01) between control and pre-IVF groups showed\nstatistically significant differences.\nThere were statistically significant differences between\nLPO (P=0.000), CRP (P=0.023) and resistin\n(P=0.002) levels obtained in control and post-IVF\ngroups.\nThe differences between pre-IVF and post-IVF levels\nof resistin, LPO (P=0.003, P=0.001, respectively)\nand TNF-α (P=0.020) were statistically significant.\nAs far as the values for the parameters in the pregnancy\n(+) and pregnancy (-) groups of preand post-\nIVF women were considered, TNF-α showed a statistically\nsignificant increase after IVF in pregnancy\n(-) women (P=0.026). Similar post-IVF profiles were\nobserved for the resistin (P=0.028, P=0.016, respectively)\nand LPO (P=0.046, P=0.003, respectively)\nlevels in pregnancy (+) and pregnancy (-) groups.  Table 3  shows the mean ± SE and p values of the nonpregnant\nand pregnant women in pre-IVF and post-\nIVF groups.\nBMI had a positive correlation with leptin (r=0.615,\nP=0.001), a negative correlation with leptin receptor\n(r=-0.505, P=0.008) and a positive correlation with\nCRP (r=0.464, P=0.039). Also, leptin had a negative\ncorrelation with leptin receptor (r=-0.534, P=0.005)\nand a positive correlation with CRP (r=0.639,\nP=0.002) in the control group. In this group, resistin\nhad also positive correlation with age (P≤0.05).\nBefore IVF, statistically significant correlations\nwere observed between BMI and leptin (r=0.486,\nP=0.012) with resistin (r=0.517, P=0.007); between\nleptin and leptin receptor (r=-0.574, P=0.002) with\nresistin (P=0.047) and between TNF-α and LPO\n(P=0.016). After IVF, a significant correlation between\nBMI and leptin (r=0.641, P=0.000) was found.\nStrong correlations were detected for leptin (r=0.599,\nP=0.001) and LPO (r=0.715, P=0.000) between preand\npost-IVF values.\nImportant correlations were determined between\nBMI and leptin before (r=-0.547, P≤0.05) and after\nIVF (r=0.771, P≤0.01) for the women who were in\nthe pregnancy (-) group. Before IVF, also, a statistically\nimportant relationship was observed between\nBMI and resistin (r=0.686, P≤0.01). An inverse association\nwas noted between leptin and leptin receptor\n(r=-0.617, P≤0.01).\nWomen, who could conceive following IVF treatment\nshowed significant correlations between leptin\nand resistin (r=0.874, P≤0.01) before IVF as well as\nleptin and TNF-α (r=0.841, P≤0.01) after IVF. None\nof these correlations were detected in the pregnancy\n(-) group.\nComparison of some demographical and clinical parameters (mean ± SE) in non-pregnant and pregnant women\nSE; Standard error, BMI; Body mass index, AMH; Anti-mullerian hormone, LH; Luteinizing hormone, FSH; Follicule stimulating hormone\nand TSH; Thyroid stimulating hormone.\nThe mean ± SE and P values of the control and IVF groups\nSE; Standard error, IVF;  In vitro  fertilization, TNF-α; Tumor necrosis factor alpha, CRP; C-reactive protein and LPO; Lipid hydroperoxides.\nThe mean ± SE and P values of the non-pregnant and pregnant women in pre-IVF and post-IVF groups\nSE; Standard error, IVF;  In vitro  fertilization, TNF-α; Tumor necrosis factor alpha, CRP; C-reactive protein and LPO; Lipid hydroperoxides.\n\nInfertile couples have to face emotional and\neconomical sides of the problem, because success\nrates of IVF trials have not reached to the desired\nlevel, yet. Immunologic factors may contribute to\nunexplained losses and thus, studies on the matter\nare being accelerated.\nCytokines are polypeptides that occur at the\ncrossroads of immunological pathways. Maternal\ninflammatory response plays an important role in\nthe early stages of pregnancy; however, there is no\nconsensus on the roles of inflammatory parameters\nwithin this period ( 22 ).\nIn this study, mean TNF-α levels were found\nhigher (P≤0.05) in the pregnancy (-) group than\npregnancy (+) group after IVF. This situation reminds\nus a question whether the success rates of\nIVF applications in infertile women can be increased\nby use of TNF-α blockers.\nIt was reported that use of TNF-α inhibitors and\nintravenous immunoglobulins (IVIG) in young infertile\nwomen improved the result of IVF application\nand increased the success rates of IVF. TNF-α/\ninterleukin-10 (IL-10) elevation before pregnancy might relate with the risk of failure in IVF ( 23 - 25 ).\nSerum resistin levels might be a good predictor\nof ovarian response in infertile women during IVF\n( 7 ). In the present study, resistin levels were almost\nthe same in the control and pre-IVF groups\n(12.2 ± 1.1 ng/ml vs. 12.5 ± 1.5 ng/ml). This level\nincreased to 22.2 ± 2.9 ng/ml after IVF. This increase\nsuggested that the profile of this parameter\ncould be important.\nResistin shares several features with proinflammatory\ncytokines in humans and can partially\ncontribute to regulation of inflammation and immunity.\nMacrophages incubated with recombinant\nresistin caused elevated production of TNF-α via\nthe transcription factor NF-κβ dependent pathway\n( 6 ,  13 ). Elevated TNF-α and resistin levels may\ncontribute to increased inflammation, which may\nlead to poor quality oocytes and embryos ( 7 ,  26 ).\nIn the previous study, resistin was reported to increase\nthe expression of TNF-α ( 27 ). In this study,\npost-IVF levels of TNF-α also increased in a parallel\nmanner with resistin. CRP levels were similarly increased\nbut much higher than the levels of TNF-α.\nIn a similar manner, CRP levels of post-IVF\ngroup were statistically higher than those of control\nand pre-IVF groups. In Pre-IVF group, a statistically\nsignificant difference was found between\npregnancy (+) and pregnancy (-) groups (2.8 mg/L\nvs. 3.8 mg/L). It was noted that pre-IVF pregnancy\n(+) group had lower levels of CRP than the other\ngroup.\nProbable effects between leptin and systemic inflammation\nare on-going discussion subject. Studies\non culture cells and mouse models reported that\nhuman CRP prevented binding of leptin to its specific\nreceptor and blocked the signal transduction.\nThus, this parameter may weaken the physiologic\nfunction of leptin that contributes to \"leptin resistance\"\n( 10 ,  28 ,  29 ).\nLeptin, an adipocyte-derived hormone, does not\nonly take role in the regulation of food intake, but\nis also involved in many reproductive functions\nincluding steroideogenic potential of ovary ( 15 ).\nOvary is a target organ for leptin because leptin, its\nmRNA as well as its receptors are found in reproductive\ntissues ( 15 ,  30 ). Since leptin may influence\nfollicular growth as well as oocyte development,\nleptin and leptin receptors were also investigated\nin this study.\nIn general terms, investigation of the effects of\nhormones that were applied in the extent of IVF\ntreatment protocol showed decreases in leptin levels\nof the post-IVF group compared to pre-IVF\nvalues (52.7 ± 6.8 ng/ml vs. 62.6 ± 10.5 ng/ml).\nPre-IVF levels of leptin decreased significantly\nin the pregnancy (+) group compared to the pregnancy\n(-) group (54.6 ng/ml vs. 64.9 ng/ml). Our\nresults were consistent with the report stating that\nelevated leptin may exert adverse impacts on pregnancy\nsuccess ( 15 ). Several investigations reported\nthat high leptin is associated with low pregnancy\nrates in IVF cycles ( 16 ,  31 ). The effect of leptin on\nembryo quality is currently a controversial topic\n( 30 ,  32 ,  33 ). However, it remains elucidated how\nelevated leptin concentrations negatively impact\nIVF outcome ( 31 ).\nCertain studies ( 34 - 36 ) showed that soluble\nleptin receptor levels were inversely correlated\nwith BMI. A similar relationship was found for\nthe control group in this study. Levels of leptin\nreceptor were higher in the post-IVF period (42.7\n± 5.7 ng/ml) compared with the pre-IVF period\n(32.0 ± 2.4 ng/ml), but there was no statistically\nsignificant difference between the levels in pre-\nIVF and post-IVF pregnancy (+) and pregnancy\n(-) groups.\nIn this study a positive correlation was found\nbetween levels of leptin and TNF-α in post-IVF\npregnancy (+) women. This suggested that leptin\nmay have a relationship with some other inflammatory\nparameters.\nThe best investigated adipocytokin up-tillnow\nis resistin. It is claimed that resistin takes\nrole as an acute phase reactant due to its up-regulation\nin patients with severe sepsis and septic\nshock ( 10 ,  28 ,  37 ). However, there is little\nknowledge about its potential association with\nleptin. A finding that may contribute to this subject\nwas obtained in this study. A strong pre-IVF\nrelationship was determined between leptin and\nresistin in pregnancy (+) women following IVF\napplication.\nOn the other hand, in a recently published article,\nleptin and resistin are reported as negative and\npositive outcome predictors, respectively, in women\nundergoing IVF ( 38 ). Our results were consistent\nwith these findings. In pregnancy (+) group,\nas pre-IVF samples showed significant decreases in leptin concentrations, increased resistin levels\nwere observed for post-IVF samples.\nOxidative stress may be used as a predictive\nmarker in controlled ovarian stimulation success\n( 39 ). Although it is not sufficient to measure\nLPO levels alone to interpret oxidative stress,\nthey give some notion about the matter as the\nmarkers of early lipid peroxidation. Significant\ndifferences were found between LPO levels of\nthe control group and pre-IVF as well as post-\nIVF groups. Also, a strong correlation was found\nbetween pre-IVF and post-IVF values of this\nparameter. Levels for this parameter were determined\nlower in pre-IVF pregnancy (+) group\ncompared to pregnancy (-) group.\n\nIn women, who ended up the IVF attempt with\na successful pregnancy, a relationship between\nleptin and resistin is noted beside the association\nof these parameters with TNF-α. Relationships\nbetween leptin and resistin as well as TNF-α are\nexpected, because double-sided effects are being\nobserved among them. Resistin increases TNF-α,\nwhich in turn induces resistin. Leptin stimulates\nboth resistin and TNF-α, that increases serum\nleptin concentrations. Association of TNF-α as\nwell as resistin levels with quality of oocytes\nand embryos, and also influence of leptin upon\nfollicular growth as well as oocyte development\nsupport the leptin-resistin and leptin-TNF-α correlations,\nwhich appear to be effective upon IVF\noutcome. Monitoring the levels of these parameters\nwithin the period, which follows IVF attempt\nmay reveal more significant relationships.","source_license":"CC0","license_restricted":false}