{"paper_id":"931f8169-d915-49dc-9100-f4ad1fbe137b","body_text":"Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorder among\nwomen of reproductive age, affecting approximately 11 to 13% of the global female\npopulation  ( 1 , 2 ) . It is a heterogeneous condition characterized by\nthree core diagnostic features – hyperandrogenism, chronic anovulation, and\npolycystic ovarian morphology – often accompanied by metabolic abnormalities such as\ninsulin resistance (IR) and obesity  ( 3 ) . Although the pathophysiology of PCOS is not fullyunderstood,\nit is thought to involve a combination of genetic predisposition, environmental\nfactors, oxidative stress, chronic low-grade inflammation, and metabolic\ndysregulation  ( 3 , 4 ) . Insulin resistance, which plays a central role in\nthe etiology of the disease, is present in approximately 50 to 80% of patients and\ncontributes to hyperandrogenemia and reproductive dysfunction through multiple\nmechanisms  ( 5 , 6 ) . However, the exact mechanisms underlying IR in\nPCOS remain unclear, warranting further research  ( 4 ) . It has long been established that alterations in\nglucose transporters (GLUT) are involved in the development of IR. The primary\nmechanism is insulin-stimulated cellular glucose uptake, which is facilitated by the\ntranslocation of GLUT4-containing vesicles from the cytoplasm to the plasma membrane\n ( 7 ) . Insulin-regulated\naminopeptidase (IRAP), a cellular protein featuring both intracellular and\nextracellular functional sites, is essential for this translocation process and\nplays a critical role in insulin-mediated glucose uptake via GLUT-4 in skeletal\nmuscle and adipose tissue. During this process, its extracellular domain is cleaved\nby metalloproteases and released into the bloodstream  ( 8 ) . Given these functions, circulating IRAP levels\nhave been proposed as a potential biomarker for IR, as demonstrated in previous\nstudies in patients with type 2 diabetes  ( 8 , 9 ) . However, a review\nof the existing literature indicates that this association has not yet been\ninvestigated in women with PCOS.\nFor these reasons, this study aimed to measure serum IRAP levels in women diagnosed\nwith PCOS and to investigate their potential contribution of these levels to the\ndevelopment of IR, which plays a central role in the pathophysiology of PCOS.\n\nThis case-control study was conducted between May and December 2021 at the Department\nof Obstetrics and Research Hospital. Ethical approval was obtained from the Ethics\nCommittee of the Faculty of Medicine, Sakarya University (date: May 7, 2021,\napproval no: 90). The study was conducted in accordance with the principles of the\nDeclaration of Helsinki, and written informed consent was obtained from all\nvolunteers.\nThe study included 40 patients diagnosed with PCOS according to the Rotterdam\ncriteria (2003). The control group consisted of 40 healthy women of similar age and\nbody mass index (BMI) who presented to the clinic for routine gynecological\nexaminations. The inclusion criteria for all participants were an age range of 18 to\n40 years and a BMI between 20 to 35 kg/m 2 . For the control group, the\nabsence of any significant gynecological pathology was required; only minor\ncomplaints such as dysmenorrhea or premenstrual syndrome were permitted.\nThe exclusion criteria for all participants were as follows: a history of smoking;\ncurrent pregnancy or lactation; previous ovarian surgery; a diagnosis of\nendometrioma or endometriosis; thyroid disorders or abnormal prolactin levels; use\nof hormonal therapy (e.g., oral contraceptives) within the past 6 months; use of\nmedications known to affect carbohydrate metabolism (e.g., insulin, cortisol); a\nhistory of chronic disease (e.g., epilepsy, renal failure, heart disease); and a\nhistory of cancer.\nClinical data and laboratory results of the participants were retrospectively\ncollected from medical records. The following parameters were recorded: age, height,\nweight, intermenstrual interval, and the presence of oligo-anovulation and\nhirsutism. Body mass index was calculated using the formula: BMI = weight (kg) /\n[height (m)] 2 .\nThe diagnosis of PCOS was established according to the Rotterdam criteria, which\nrequires the presence of at least two of the following three features:\nOligo-ovulation or amenorrhea, defined as a menstrual cycle exceeding 35 days\nor the absence of menses for 3 consecutive months, respectively.\nClinical hyperandrogenism, primarily evidenced by hirsutism. This was\nassessed using the modified Ferriman-Gallwey (mFG) scoring system, where\nnine androgen-sensitive body areas were assigned a score from zero (no\nterminal hair growth) to 4 (extensive hair growth). A total mFG score of\n≥ 8 was considered diagnostic  ( 11 ) .\nBiochemical hyperandrogenism, defined as an elevated serum level of any of\nthe following androgens: total testosterone (reference range: 0.04 to 4.18\nng/dL), dehydroepiandrosterone sulfate (DHEAS) (reference range: 10 to 248\nµg/dL), or 17-OH-progesterone (reference range: 0.2 to 1.0 ng/mL)\n ( 12 - 14 ) . All participants\nunderwent routine gynecological and transvaginal ultrasonographic\nexaminations during the early follicular phase (days 2 to 4) of a\nspontaneous menstrual cycle. For the diagnosis of PCOS, polycystic ovarian\nmorphology was defined according to the Rotterdam criteria as the presence\nof ≥ 20 follicles (2 to 9 mm in diameter) per ovary and/or an ovarian\nvolume > 10 mL  ( 15 ) .\nThe control group consisted of healthy women with regular menstrual cycles\n(26 to 32 days), normal ovarian morphology, and no evidence of clinical or\nbiochemical hyperandrogenism.\nBlood samples were obtained from the brachial vein during the early follicular\nphase (cycle days 2 to 4) following an 8-hour overnight fast. A total of 16 mL\nof venous blood was collected from each participant into two tubes in the early\nmorning. From one tube, the following serum analyses were performed:\nanti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH),\nluteinizing hormone (LH), thyroid-stimulating hormone (TSH), total testosterone,\nfree testosterone, DHEAS, 17-hydroxyprogesterone (17-OHP), fasting glucose, and\nfasting insulin. Serum AMH levels were quantified using a chemiluminescent\nimmunoassay (Access AMH kits, Beckman Coulter Access 2 analyzer). This assay had\na measurement range of 0.02 to 24 ng/mL, with an intra-assay coefficient of\nvariation (CV) of 1.7% and an inter-assay CV of 3.1%. FSH, LH, TSH, total\ntestosterone, insulin, and DHEAS levels were measured by chemiluminescent\nimmunoassay on an Abbott Architect i2000 analyzer (Abbott Diagnostics). The\ninter-assay CVs were as follows: ≤ 10% for FSH, ≤ 7% for LH,\n≤ 10% for TSH, ≤ 10% for total testosterone, ≤ 7% for\ninsulin, and ≤ 10% for DHEAS. Fasting glucose levels were determined via\nthe hexokinase method on a Beckman Coulter AU5800 analyzer. The assay\ndemonstrated an intra-assay CV of 0.7%, a total CV of 0.9%, and a measurement\nrange of 10 to 800 mg/dL. Serum 17-OHP and free testosterone levels were\nmeasured using radioimmunoassays (RIA) with Diasource kits. For the 17-OHP\nassay, the intra-assay CV was 6.8%, the inter-assay CV was 8.7%, and the\nmeasurement range was 0.17 to 14 ng/mL. For the free testosterone assay, the\nintra-assay CV was 5.7%, the inter-assay CV was 7.3%, and the measurement range\nwas 0.3 to 90 pg/mL. Insulin resistance was evaluated using the homeostasis\nmodel assessment of insulin resistance (HOMA-IR), calculated as [fasting insulin\n(µU/mL) × fasting glucose (mg/dL)] / 405. A HOMA-IR value ≥\n2.5 was defined as indicative of IR.\nThe blood samples taken with the second tube blood sample were centrifuged at\n4,000 rpm for 10 minutes, then the serums were separated and put into a deep\nfreezer to be stored at -80°C until the day of analysis. On the study day, all\nsamples were thawed in the same month and Human ICE protease-activating factor\n(IRAP) levels were measured using ELISA kits (MyBioSource, San Diego, USA,\nCatalog no: MBS260922). The results were calculated using the Biotek ELX800\n(USA) ELISA reader, while the intra-measurement CV was reported as < 8% and\nthe inter-measurement coefficient of variation as < 12%.\nDescriptive statistics are presented as mean ± standard deviation for\nnormally distributed continuous variables and median (first-third quartiles) for\nnon-normally distributed variables. Comparisons between groups for parametric\nand non-parametric data were performed using the Student’s\n t -test and the Mann-Whitney U test, respectively. Correlations\nbetween continuous variables were assessed using Pearson’s correlation\ncoefficient. The discriminatory power of IRAP for diagnosing PCOS was evaluated\nusing Receiver Operating Characteristic (ROC) curve analysis. A p-value of less\nthan 0.05 was considered statistically significant. All analyses were conducted\nusing IBM Statitical Package for the Social Sciences (SPSS) statistics, Version\n20.0.\n\nThe clinical and laboratory findings of the participants in the patient and control\ngroups included in this study are shown in  Table 1 . Serum LH, total testosterone, AMH, DHEAS, fasting\nglucose, insulin, and HOMA-IR values were significantly higher in the PCOS group\nthan the control group. In contrast, serum IRAP levels were significantly lower in\nthe PCOS group compared to the control group. Laboratory data comparing PCOS\npatients with and without IR are shown in  Table 2 . Accordingly, serum IRAP levels were significantly\nlower in the insulin-resistant group. The correlation of serum IRAP levels with\nbiochemical variables is presented in  Table\n3 . IRAP levels show a significant negative correlation with\ninsulin, HbA1c, fasting blood glucose levels, and HOMA-IR.\nComparison of the clinical and laboratory characteristics of the women with\npolycystic ovary syndrome and the control group\nMann-Whitney U Test,\nStudent t-test.\nResults expressed as mean ± standard deviation.\nPOCUS: polycystic ovary syndrome; BMI: body mass index; LH: luteinizing\nhormone; AMH: Anti-Müllerian hormone; FSH: follicle-stimulating\nhormone; TSH: thyroid-stimulating hormone; HDL: high-density\nlipoprotein; LDL: low-density lipoprotein; DHEAS: dehydroepiandrosterone\nsulfate; F-G score: Ferriman-Gallwey score; HOMA-IR: homeostatic model\nassessment for insulin resistance; HbA1c: glycated hemoglobin; IRAP:\ninsulin-regulated aminopeptidase.\nComparison of laboratory tests in insulin resistant and non-insulin resistant\npolycystic ovary syndrome patients according to the homeostatic model\nassessment for insulin resistance cut-off of 2.5\nStudent t test.\nPOCUS: polycystic ovary syndrome; HOMA-IR: homeostatic model assessment\nfor insulin resistance; HbA1c: glycated hemoglobin; IRAP:\ninsulin-regulated aminopeptidase.\nCorrelation of serum insulin-regulated aminopeptidase levels with biochemical\nvariables in the polycystic ovary syndrome group\nr: Pearson correlation coefficient.\nHOMA-IR: homeostatic model assessment for insulin resistance; HbA1c:\nglycated hemoglobin; SDHEA: serum dehydroepiandrosterone sulfate.\nThe ROC curve for IRAP concentrations in the PCOS group is shown in  Figure 1 . The area under the curve was\n83.1% (95% confidence interval: 0.744-0.918). The optimal cut-off value was 0.655\nng/mL, and the rates below this value were 82.5% and 70.0% for sensitivity and\nspecificity, respectively (p=0.001).\nReceiver Operating Characteristic analysis of the discrimination of\ninsulin-regulated aminopeptidase values of polycystic ovary syndrome\ncases.\n\nOur results demonstrate that serum IRAP levels are significantly lower in the PCOS\ngroup than in the control group. Notably, a further reduction in IRAP levels was\nobserved in PCOS patients with IR relative to those without, suggesting a\nsignificant influence of IR on IRAP expression. These findings suggest that IRAP may\nbe associated not only with IR but also with hormonal disorders. Significant\nnegative correlations were also observed between serum IRAP levels and fasting\ninsulin, fasting glucose, HbA1c, and HOMA-IR. These correlations suggest that\ndecreased IRAP levels are linked to IR and dysregulated glucose metabolism,\npositioning IRAP as a potential biomarker for IR. This finding aligns with existing\nliterature. Furthermore, weak negative correlations were identified between IRAP\nlevels and serum DHEAS, free testosterone, and total testosterone concentrations in\nPCOS patients. These findings suggest that IRAP may be associated not only with IR\nbut also with hormonal disorders. The interactions between insulin, GLUT-4, and its\nclosely associated protein, IRAP, are integral to regulating cellular glucose\nuptake, maintaining whole-body glucose homeostasis, and ensuring metabolic\nequilibrium. Disruptions in this intricate process can lead to IR. A review of the\nliterature indicates that previous investigations into GLUT-4 and its role in IR in\nPCOS patients have relied on invasive methods, such as biopsies of adipocyte and\nendometrial tissue  ( 16 - 18 ) . As a less invasive alternative\nto these approaches, we hypothesized that measuring serum IRAP levels could serve as\na surrogate marker for GLUT-4 translocation to the plasma membrane and, by\nextension, provide a reflection of IR in PCOS patients. This premise formed the\nrationale for the present study.\nA review of the literature indicates that numerous studies have investigated the\nrelationship between IR and IRAP. Research has demonstrated reduced IRAP\ntranslocation in both diabetic rats 19 and patients with type 2 diabetes mellitus\n(T2DM)  ( 20 , 21 ) . These foundational studies, which utilized\ninvasive biopsy methods to analyze adipocyte  ( 19 , 21 )  and skeletal\nmuscle tissue  ( 20 ) , established a\nclear link between impaired IRAP activity and IR. Subsequent clinical investigations\nhave employed a more practical, non-invasive approach by measuring serum IRAP\nlevels. These studies consistently report decreased circulating IRAP levels in\nindividuals with T2DM  ( 8 , 9 )  and in pregnant women with\ngestational diabetes  ( 22 ) .\nCollectively, these findings from both tissue-based and serum-based analyses suggest\nthat reduced IRAP levels may serve as a useful indicator of IR.\nIn our study, we found that the serum IRAP levels were significantly lower in the\ninsulin-resistant group. It is well-established that insulin stimulation triggers\nthe translocation of IRAP and GLUT4 to the plasma membrane as a complex.\nSubsequently, the extracellular domain of IRAP is cleaved by metalloproteases and\nreleased into the circulation  ( 8 , 23 ) . In states of IR, this\nphysiological process is disrupted, leading to reduced cleavage and secretion of\nIRAP and, consequently, lower circulating levels. The findings of the present study\nare consistent with this mechanism and align with previous reports in the\nliterature.\nThe PCOS group exhibited significantly higher levels of IR markers, including fasting\ninsulin, fasting glucose, HbA1c, and HOMA-IR, relative to healthy controls.\nConsistent with this metabolic profile, serum IRAP levels showed a significant\nnegative correlation with these indices. This inverse association provides important\nevidence linking lower IRAP levels to the development of IR in PCOS.\nAs a result of our study, we found that serum IRAP levels were significantly lower in\nthe insulin-resistant group. Moreover, IRAP levels demonstrated strong and\nstatistically significant negative correlations with metabolic parameters, including\nfasting glucose, insulin, HOMA-IR, and HbA1c, indicating that higher IR and poorer\nglycemic control are associated with lower IRAP levels in PCOS patients. In\ncontrast, IRAP levels showed weak and statistically non-significant correlations\nwith hormonal parameters, including free and total testosterone and DHEA-S. Overall,\nthese findings suggest that IRAP is more closely associated with metabolic\ndisturbances than with androgen levels in PCOS, implying that circulating IRAP\nlevels may primarily reflect IR, while their potential relationship with other\nhormonal parameters warrants further investigation in future studies.\nThe main limitations of this study include its case-control design, the relatively\nsmall sample size, the limited number of participants in the PCOS subgroups, and the\nuse of the HOMA-IR index rather than the hyperinsulinemic-euglycemic clamp, which is\nconsidered the gold-standard method for assessing IR. An additional limitation is\nthat the ACTH stimulation test was not performed to definitively rule out\nnon-classical congenital adrenal hyperplasia (in patients with elevated 17-OH\nprogesterone levels. Although clinical evaluation and DHEAS levels did not indicate\nnon-classical congenital adrenal hyperplasia, future studies should incorporate this\ntest to provide a more definitive differential diagnosis.\n\nThis study provides the first evidence linking reduced serum insulin-regulated\naminopeptidase levels to polycystic ovary syndrome. Notably, insulin-regulated\naminopeptidase levels were significantly lower in polycystic ovary syndrome patients\nboth with and without insulin resistance compared to controls. The significant\nnegative correlations observed between insulin-regulated aminopeptidase levels and\nindices of both insulin resistance and hyperandrogenemia indicate that\ninsulin-regulated aminopeptidase may be involved in the core metabolic and endocrine\ndisturbances characterizing the pathogenesis of polycystic ovary syndrome.","source_license":"CC-BY-4.0","license_restricted":false}