{"paper_id":"a06758b0-52d9-4b91-8410-055afac48853","body_text":"Infertility has been always addressed as one of\nthe important, serious and costly health issues in\ndifferent societies ( 1 ,  2 ). According to the previous\nstudies performed in different countries, about\n10-15% of couples suffering from infertility regard\nthis disability as the worst experience in their life\n( 3 - 5 ). This problem might cause marital conflicts,\nsocial injuries, divorce and other psycho-social\nproblems ( 2 ,  6 ). At present, in Iran, about 3 million couples suffer from infertility. In 7% of infertile\ncouple population, the cause of infertility is\nunknown.\nThe relationship between metabolism and reproduction\nhas been always considered as controversial\nissues in the field of female endocrinology.\nInsulin, amino acids and low molecular\nweight IGF-binding protein-1 (IGFBP-1) have\nbeen introduced as effective signals in alterations\nof body fat and body mass index (BMI), but these\nalterations have been recently attributed to leptin\nlevel ( 7 ). Leptin, in addition to regulating body\nweight, plays an important role in regulating the\nfunctions of endocrine, reproductive and immune\nsystems through suppressing food intake and increasing\nenergy consumption. Deficiency of leptin\nor its receptors, in addition to causing obesity,\nleads to disturbing reproductive cycle, hormonal\nimbalance, as well as disorders of immune system,\nhematopoietic system and bone metabolism ( 8 ).\nThese observations have indicated the important\nrole of leptin in several physiologic processes and\nthe relationship between abnormal leptin levels\nand many disorders ( 9 ).\nLeptin affects menstrual cycles, directly and indirectly.\nIt have been reported that leptin directly\naffects ovaries and hypothalamic-pituitary axis.\nFurthermore, its effect on follicle stimulating hormone\n(FSH)-dependent estradiol production in animals\nand its role in preventing starvation-induced\ndelay in ovulation in mice show indirect effect of\nleptin on the luteinizing hormone (LH) concentration\n( 10 ).\nLeptin is an adipocyte hormone acting as a link\nbetween adipose tissue and reproductive system.\nIt is also considered as a type 1 cytokine, due to\nits role in cell growth and maturation ( 11 ). Recent\nstudies have reported that leptin is produced by\nboth granulosa and cumulus cells of ovarian follicles\n( 10 ,  11 ).\nLeptin is used in the treatment of hypoleptinemia\ndue to energy deprivation state, leptin deficiency\nand obesity-related hyperleptinemia. Due\nto leptin resistance in some obese individuals, leptin\ntreatmentis used in patients with complete or\nrelative leptin deficiency including patients with\nhypothalamic amenorrhea and lipoatrophy, but coadministration\nof this hormone with leptin sensitizers\nhas been resulted in better outcomes in the\ntreatment of obesity ( 8 ).\nSince it has been proved that there is a definite\nrelationship between infertility and menstrual irregularities\nin women with abnormal obese (OB)\ngene expression and peritoneal fluid is also known\nas an active biologic environment that is essential\nfor regulation of ovarian function, ovulation, zygote\nimplantation, and follicle collection ( 9 ,  11 ),\nany change in the concentration of substances in\nthis environment is likely to affect ovarian function.\nFurthermore it can be postulated that the origin\nof some substances like leptin might be from\nfollicular fluid.\nThe aim of this study was to compare serum and\nfollicular fluid leptin concentrations among explained\ninfertile women, unexplained infertile women and\nfertile women in order to find the role of leptin level\nin infertility. In the present study, without applying\nany invasive method more than the treatment process,\nthree groups were compared in regard to serum\nand follicular fluid leptin concentration using blood\nsample and follicular fluid of assisted reproductive\ntechnology (ART) candidates.\n\nIn this case-control study, 90 women using convenient\nsampling method with regard to the inclusion\ncriteria referred to the Infertility Center of\nAfzalipour Hospital, Kerman, Iran. After ensuring,\nall personal information remained confidential and\nthere was no intervention with their treatment process.\nThis study approved by Ethical Committee of\nKerman University of Medical Sciences and done\nfrom September 2010 to March 2013 and all participants\nprovided an informed consent.\nInclusion criteria were as follow (the presence\nof all 7 criteria was necessary): i. Age between\n20 to 40 years, ii. BMI between 20 to 25 kg/m 2 ,\niii. Normal levels of FSH, LH (on days 2 and 3 of\nmenstrual cycle), prolactin, testosterone and progesterone\n(between days of 19 and 21of menstrual\ncycle), iv. Normal semen fluid analysis and pelvic\nultrasonography, v. Absence of any underlying\ncomplex disorders like diabetes, obesity, cardiovascular\ndisease, any type of metabolic diseases\nand malignancies, vi. Negative result of autoantibody\ntest and vii. No use of anti-inflammatory\nmedicines. It should be mentioned that only infertile\nparticipants underwent laparoscopy and hysterosalpingography (HSG). First the information\nregarding demographic and clinical characteristics\nwere collected and infertility investigations, including\novulation state, cervical and uterine factors,\nand patency of fallopian tubes, were then performed\nin couples referred for  in vitro  fertilization\n(IVF). All partners had normal sperm analysis. If\ninfertility was due to male factor, the case was excluded\nfrom our study because they had no history\nof pregnancy and were not considered as unexplained\ninfertility.\nSubjects were divided into three groups of explained\ninfertile group, unexplained infertile group\nand fertile group. The explained infertile group\n(n=30) contained women with one or more infertility\nfactors. This group was divided into the four\nsubgroups including cervical, endometrial, tubal\nand peritoneal factors based on medical history and\nfindings of physical exam, pelvic ultrasonography,\nHSG or laparoscopy. The unexplained infertile group\n(n=30) contained women with unknown causes of infertility.\nThe normal fertile group (n=30), as control\ngroup, contained normal fertile women referred for\noocyte donation who had regular menstrual cycles\nwith normal fertility factor.\nIn all groups, the long agonist protocol for controlled\novarian hyperstimulation (COH) was used.\nBriefly, COH was performed by administration of\nhuman menopausal gonadotrophin (HMG, Ferring,\nGermany) after pituitary suppression with\nbuserelin (superfact, Aboureyhan, Iran), starting\nin the midluteal phase of the preceding cycle. The\ndosages of gonadotropins were individualized according\nto serum estradiol (E 2 ) levels and transvaginally\nultrasonic measurements of the follicles.\nWhen at least three follicles reached to diameter of\n16-18 mm, ovulation was induced by the administration\nof 10,000 IU human chorionic gonadotropin\n(hCG) 36 hours before puncture. Average\nnumber of follicles of each case was 12 ( 9 - 16 ).\nIn each patient before oocyte aspiration, a peripheral\nblood sample was taken from antecubital vein\none hour prior to puncture. Blood samples were\ntransferred into sterile tubes. Then under general anesthesia\n(same protocol), a surgeon performed the\nultrasound-guided transvaginal oocyte aspiration using\na 16-17 gouge long needle. The follicular fluid\nsamples were carefully collected from the first aspirated\nfollicle of each ovary, and the follicular fluid\nsamples without visible blood contamination were\nused in this study. The oocyte retrieval was continued\nfor all IVF candidates. Blood and follicular fluid\nsamples were immediately centrifuged at 3000 rpm\nfor 10 minutes and the supernatants were stored at\n-70˚C for further analysis.\nTo measure the serum and follicular fluid leptin\nconcentrations, enzyme-linked immunosorbent assay\n(ELISA) kit (Labor Diagnostica Nord GmbH,\nGermany) was used. All measurements were carried\nout in duplicate. The intra- and inter- assay coefficients\nof variation were less than 3.7 and 6.8%, respectively,\nwith the standard range of 0.5-100 ng/ml.\nTo present descriptive statistics, mean ±standard\ndeviation (SD) was used. In order to compare\nfollicular fluid and serum leptin concentrations\namong three groups, Kruskal Wallis test was used.\nTo compare follicular fluid and serum leptin concentrations\nwithin groups, Mann Whitney test\nwas used. All statistical analyses were performed\nthroughthe Statistical Package for the Social Sciences\n(SPSS, SPSS Inc., Chicago, IL, USA) version\n16 and P value less than 0.05 was considered\nas statistically significant.\n\nIn the present study, 90 women including 30 explained\ninfertile women (group 1), 30 unexplained\ninfertile women (group 2) and 30 fertile women\n(group 3) were studied. Demographic and clinical\ndata regarding subsequent IVF of these groups are\nshown in  table 1 . The age and BMI displayed no\nsignificant differences among three groups. Progesterone,\ntestosterone, FSH and LH levels were\nnot significantly different (P>0.05 for all). The\nnumber of transplanted embryos demonstrated\nno significant difference. The percentage of fertility\nrate and good quality embryos in unexplained\ninfertile group were lower in comparison to explained\nand control groups, but differences were\nnot significant (P>0.05).\nIn explained infertile group, mean follicular fluid\nleptin level (19.92 ± 17.87) was lower than mean\nserum leptin level (33.13 ± 17.31), but the difference\nwas not significant (P=0.11). In unexplained\ninfertile group, mean follicular fluid leptin level\n(48.9 ± 20.20) was significantly (P<0.001) higher\nthan mean serum leptin level (27.83 ± 25.29).\nIn fertile group, mean follicular fluid leptin level\n(25.07 ± 22.36) was lower than mean serum leptin\nlevel (31.27 ± 11.02), but the difference was\nnot significant (P=0.19, Table 2 ). Mean follicular\nfluid leptin levels showed a significant difference\nwithin the groups (P<0.001). Among the groups,\nmean follicular fluid leptin level was higher in\nunexplained infertile women (group 2), fertile\nwomen (group 3) and explained infertile (group\n1), respectively. In regard to mean serum leptin,\nexplained infertile women had higher mean serum\nleptin level (33.13 ± 17.31) compared to unexplained\ninfertile women (27.83 ± 25.29) and fertile\nwomen (31.27 ± 11.02), but the difference was\nnot significant (P=0.070,  Fig.1 ).\nFollicular fluid and serum leptin levels in the\neach subgroup of explained infertile women were\ndetermined and the results were compared with\nunexplained infertile and fertile woman. Follicular\nleptin level in all explained infertility subgroups\nwas significantly lower in comparison to unexplained\nand fertile groups (P<0.001), but serum\nleptin level in the explained infertile subgroups\nshowed no significant difference as compared with\nunexplained infertile and fertile groups.\nDemographic data and clinical characteristics\nNo; Number, BMI; Body mass index and COH; Controlled ovarian hyperstimulation.\nLeptin concentration in serum and follicular fluid of three studied groups\n*; Mann-Whitney test, **; Kruskal-Wallis test,  & ; There is a significant difference compared to unexplained infertile group,  ¥ ; Data are\nshown as mean ± standard deviation and  # ; The unit of measurement is ng/ml. P<0.05 is considered statistically significant.\nComparison of leptin concentration in follicular fluid and serum of three studied groups (n=30/per group). Data are presented as mean ± SD.\n\nIn the current investigation, follicular fluid leptin\nlevel in unexplained infertility group was more\nthan that in explained infertility and normal fertile\ngroups.\nInfertility has been always addressed as one of\nthe important, serious and costly health issues in\ndifferent societies ( 1 ,  2 ). For this, various studies\nhave already been performed to discover the\ncauses of infertility, among them high leptin level\nhas been considered as one of the important and\neffective factors in several studies ( 12 ).\nLeptin has a wide range of functions from acting\nas an anti-obesity factor to an effective factor in\nreproduction, hematopoiesis, angiogenesis and T\nlymphocytes system ( 17 ).\nSome investigators have suggested that leptin\nmight exert a double role in regulation of reproduction.\nThey showed that when leptin level is\nlower than normal, it can exert a negative effect on\nendocrine system, regulating reproduction, while\nwhen leptin level is higher than normal, it negatively\naffects normal function of ovary and fetus\ndevelopment ( 18 ,  19 ).\nThe present study suggested that high level of\nfollicular fluid leptin has a negative effect on reproduction\nin unexplained infertility. Enhanced leptin\nlevel may inhibit aromatase activities and prevent\nthe transformation of androgen to estrogen, leading\nto the elevation of serum androgen and interferers\nwith ovarian follicle growth and ovulation\nby suppressing estrogen production ( 20 ).\nA recent study revealed that serum leptin level is\nsignificantly higher in unexplained infertile women\ncompared to the fertile group and suggested that\nleptin as cytokine-like or hormone affects pathophysiology\nof infertility, but due to study limitation,\nleptin levels in serum and peritoneal fluid\nwere not compared ( 19 ).\nIn a study with Takeuchi and Tsutsumi ( 21 ), serum\nleptin level in unexplained infertility group\nwas higher compared to polycystic ovary syndrome\n(PCOS) group, but difference was not significant, whereas in a study with Demir et al. ( 22 ),\ncomparison of serum leptin levels in unexplained\ninfertile woman and fertile woman demonstrated\nsignificant higher serum leptin levels in unexplained\ninfertile woman. However, in the present\nstudy, unexplained infertile group had lower serum\nleptin level in comparison to the explained\ninfertile and fertile groups, but the difference was\nnot significant. It seems that further studies are required\nto clarify this point.\nGogacz et al. ( 10 ) have reported a significant\nincrease in peritoneal fluid leptin level in endometriosis\nand unexplained infertility. They had no\ncontrol group for comparison and suggested similar\nstudies containing a control group. They also\nsuggested peritoneal fluid leptin might be originated\nfrom follicular fluid. To consider the mentioned\nstudy and association between the increased level\nof peritoneal leptin with endometriosis and unexplained\ninfertility, it can be proposed that leptin\nstimulates toxic factors in peritoneal fluid and also\ndecreases the quality of oocyte in endometriosis.\nIt should be noted that the cause of unexplained\ninfertility needs to be identified with new advances\nin infertility treatment and leptin is likely to be\nconsidered as one of the involved factors.\nAnother study represented that peritoneal fluid\nleptin level was significantly higher in unexplained\ninfertile group compared to patients with PCOS. In\nthe mentioned study, serum leptin level was also\nhigher in unexplained infertile group but not significantly\n( 22 ).\nThe results of our study showed significant higher\nfollicular fluid leptin level in unexplained infertile\nwomen compared to the explained infertile and\nfertile groups, but serum leptin level in this group\nwas insignificantly lower than the explained infertile\nand fertile groups. Fertility rate and number\nof good quality embryos in unexplained infertile\ngroup were lower in comparison to explained and\ncontrol group, but there were no significant differences.\nIt seems that further studies are required to\nestablish effect of high follicular leptin level on infertility.\nIt seems that systemic effects of leptin in\nblood flow differ from its local effects in follicular\nfluid as it was observed in the present study that\nunexplained infertile group had higher follicular\nfluid leptin level and lower serum leptin level in\ncomparison to the other two groups. Whether these\neffects act independently or not is a point deserving\nattention.\nThis relationship presents differently in persons\nwith different BMI; for example, in patients with\nanorexia and low BMI and in obese individuals\nwith high BMI, leptin shows contradictory effects.\nIt can be hypothesized that a specified concentration\nof leptin is required for female productivity\nand both high and low levels of leptin can affect\nfertility. This effect is seen for both systemic leptin\nand follicular and peritoneal leptin ( 18 ).\nIn another study, systemic and central effects of\nleptin on gonadotropins have been less accepted\nand leptin effect on ovary has been considered\nmore. There are more leptin receptors on ovary\nthan in central nervous system (CNS) ( 19 ).\n\nThe obtained results showed that high leptin level\nin follicular fluid affect unexplained infertility.\nTherefore, our finding suggested that high leptin\nlevel of follicular fluid is one of the main factors\ninvolved in infertility. The fact that whether leptin\nacts independently or as an associated factor requires\nmore studies.","source_license":"CC-BY-4.0","license_restricted":false}