{"paper_id":"c454fcec-45d9-446e-bfcc-2204575896da","body_text":"Polycystic ovarian syndrome (PCOS) is a prevalent gynecological disorder that affects\nwomen of reproductive age (15-45 years) and is characterized by a combination of\nhormonal and metabolic manifestations.  \n 1 ,  2 \n  The exact cause of this syndrome remains unclear; however, PCOS is primarily\nassociated with hyperandrogenism and ovulatory dysfunction. Its incidence varies by\nregion and is influenced by lifestyle factors, including diet and physical activity\nhabits. The pathophysiology of PCOS, resulting from hyperandrogenism and insulin\nresistance, can lead to hirsutism, acne, and menstrual cycle irregularities,\nanovulation, endometrial cancer, ovarian enlargement, infertility, type 2 diabetes,\nother cardiovascular diseases.  \n 3 ,  4 \n  Environmental and genetic factors contribute to PCOS development in ovaries.\nMetabolic disorders, including insulin resistance, hypertension, dyslipidemia, and\ncentral obesity, are more common in obese women with PCOS. Furthermore, scientific\nevidence indicates that women with PCOS have higher levels of inflammation-related\nbiomarkers in both their serum and ovarian tissues than women without the condition.  \n 5 ,  6 \n  PCOS causes an imbalance in progesterone and estrogen (sex hormones) levels,\nwhich leads to disturbances in the menstrual cycle. Therefore, oral contraceptives\nare often prescribed for women with PCOS.  \n 7 \n  This condition can be diagnosed by ovarian ultrasonography. The ovary can be\naffected by immune-mediated processes, and these responses can be organ-specific,\naffecting only the ovary, or systemic autoimmune diseases. One autoimmune\nconformation of the ovary, anti-FSH antibody interferes with the function of FSH,\nand FSH’s specific beta-epitope is predominant in endometriosis and\npolycystic ovary syndrome.  \n 8 \n  The pituitary gland abides by slow and fast GnRH pulses. During the early\nfollicular stage, increasing FSH concentrations stimulate estradiol (E2) production.\nElevated E2 selectively suppresses FSH secretion and sustains a high-frequency\npattern of GnRH pulses into the late follicular stage.  \n 9 \n  A hallmark of PCOS is an increase in GnRH levels, leading to elevated\nandrogen levels. The ovary stimulates this oversecretion and produces high LH levels\nfrom the adenohypophysis. This elevation in LH secretion is observed in\napproximately 60% of women with PCOS, and oversecretion of LH prevents oocyte\nmaturation. The main function of LH hormone is to stimulate ovulation and zygote\nimplantation in the uterus, as well as to regulate the menstrual cycle.  \n 10 ,  11 \n  LH receptors on ovarian theca cells regulate steroid production, whereas FSH\nreceptors on granulosa cells regulate follicle development and steroid hormone\nhandling. Evidence from limited studies indicates a possible association between\nthese antibodies and premature ovarian failure in women.  \n 12 \n  In infertile women anti-FSH antibodies were found at high concentrations.\nBecause of their similarities in structure, function, and site of secretion,\nantibodies against both LH and FSH are the same. Pretreatment with oral\ncontraceptives is used to balance the LH/FSH ratio before ovulation, to regulate\novarian induction.  \n 13 \n  There was very little previous studies on the role of anti-FSH and anti-LH\nantibodies in PCOS occurrence so the study was designed to compare the levels of\nthese antibodies and its correlation with imbalanced FSH and LH levels and\nassociation with BMI in PCOS patients.\n\nA study was carried out in Baghdad, about 65 women with PCOS and 56 healthy women\nwere included in the study. The women’s ages ranged between 18 and 45\nyears in patients and controls, about four participants were on treatment for\nPCOS like metformin, vitamin D, and oral contraceptives during sample\ncollection, but they continued to meet the diagnostic criteria for PCOS. The\ndoses of medications were not standardized among participants. The samples were\ncollected using a clinical convenience sampling technique from the Medical City\nHospital, Baghdad, and private women’s infertility clinics. Written\nconsent was obtained from each patient and control before the study started. The\nstudy was approved by the Ethics Committee of the College of Science, University\nof Baghdad (Ref.: CSEC/1124/0100, November 2024 to Jan 2025). Due to the strict\neligibility criteria and logistic challenges in recruiting completely healthy\nindividuals, the sample size of the control group was smaller than the PCOS\ngroup. However, to minimize potential confounding factors, controls were closely\nmatched with patients based on age. BMI was measured using the equation weight\n(kg)/(length).m  2 . The cut off value was 25 as BMI < 25 is normal\nweight, BMI 25-30 is overweight and BMI >30 act as obese. The main symptoms\nthat occur in patients with PCOS are irregular periods, acne, alopecia,\nhirsutism, oily skin, and depression. Hyperandrogenism is a key diagnostic\ncriterion of PCOS. To verify the adequacy of the final sample size (65 cases and\n56 controls), a post-hoc power analysis was conducted using G*Power software.\nBased on current sample size, the study achieved a statistical power (1-β)\nof over 80% (>0.80) at a significant level (α) of 0.05, confirming that\nthe sample size is statistically sufficient to detect significant differences\nbetween the groups.\nThe sample size was calculated by Raosoft; the margin of error is 5%, the\nconfidence level was 95% and response of distribution was 50%.\nThe diagnosis of PCOS was established based on the Revised Rotterdam Consensus\ncriteria, requiring the presence of at least two of the following components:\nOligo- or anovulation, clinical and/or biochemical hyperandrogenism, and PCOS\nmorphology on ultrasound. In this study, the diagnostic criteria are classified\nas follows: Clinical criteria (based on patients’ symptoms, physical\nexamination, and medical history), laboratory criteria (based on blood tests,\nhormonal levels, and immunological assays, including antibody detection), and\nimaging criteria (based on radiological and imaging techniques such as\nultrasound). Specifically, the operational definition included assessing\nirregular periods or chronic amenorrhea for ovulatory dysfunction, and\nevaluating hirsutism, acne, or alopecia alongside serum androgen levels for\nhyperandrogenism.\nAnti-mullerian hormone (AMH) was assessed as part of the laboratory diagnostic\ncriteria to support the evaluation of ovarian function. Additionally, while the\nLH/FSH ratio is not officially recognized as a primary diagnostic criterion\nwithin the Rotterdam guidelines, LH and FSH may serve as useful indicator for\nPCOS. An increase in the LH/FSH ratio to 2 or 3 in women with PCOS, the typical\nratio being 1:1 of LH/FSH, means that the levels of both FSH and LH in the\nbloodstream can be compared.  \n 14 ,  15 \n  Therefore, both AMH levels and the LH/FSH ratio were utilized as\nsupplementary pathophysiological markers rather than primary diagnostic tools.\nThe phenotypes were not specific in this study, as the analysis focused on\noverall diagnosis rather than phenotype classification.\nPregnant or breastfeeding women or those who had hypertension, diabetes, or other\nchronic diseases were excluded from the study. Also, individuals with thyroid\ndysfunction, hyperprolactinemia, and other endocrine conditions that could\ninfluence hormonal or immunological parameters were excluded from the study to\nminimize confounding factors. Only confirmed cases of PCOS were included in the\nstudy.\nUsing the AFIAS-6 apparatus, hormones (Follicle stimulating hormone, luteinizing\nhormone) were measured in the serum of women with PCOS and healthy women during\nthe early follicular phase.\nAll parameters were determined using the serum from women with PCOS and healthy\nwomen (controls) during the early follicular phase. Fertility hormones (anti-FSH\nantibody and anti-LH antibody) were measured by ELISA using immunological kits\n(Cat No: YLA0050HU, Shanghai YL Biotech Company, China) (Cat No: E3130Hu, BT\nLAB, China). The ELISA plate was precoated with human anti-FSH antibodies. Upon\nadding 50 μl of the sample to the wells, any anti-FSH Ab was bound to the\nimmobilized antibodies. Biotin-labeled anti-FSH Ab was dispensed into all wells\nexcept for the standard wells, which were precoated. Biotin-labeled anti-FSH\nantibodies immobilized on the plate. Streptavidin-HRP was added to all wells,\nand the plate was kept in the dark. The mixture was incubated at 37°C for\n60 min. After incubation, unbound streptavidin-HRP was washed away using washing\nbuffer. The washing step was repeated five times, with a one-minute wait for\neach wash. Substrate solution (A and B) was then added, and the resulting color\nindicated the concentration of anti-FSH Ab. The enzymatic reaction was stopped\nby adding an acidic stop solution, and absorbance was measured at 450 nm using a\nmicroplate reader. The same detection steps used for anti-FSH Ab were applied to\nthe anti-LH Ab. According to manufacturers' specifications, the intra-assay and\ninter-assay coefficients of variation (CV) for both anti-LH and anti-FSH ELISA\nkits were <8% and <10%, respectively, confirming the precision and\nreproducibility of the immunological assays.\nData analysis was performed using SPSS version 2019. The normality of continuous\nvariables was formally evaluated using the Shapiro-Wilk test prior to\nstatistical analyses. An independent t-test was used to compare means between\npatients and controls, statistical significance was set at p-value < 0.05.\nWhile the chi-square test was used to compare percentages between groups. Simple\nstatistical analyses were sufficient as the data did not require more complex\nmethods. The chi-square test was used to compare the categorical\nvariables/proportions between groups in this study. A post hoc power analysis\nwas performed using the observed differences in Anti-LH antibody levels between\nPCOS patients and controls. With 65 patients and 56 controls, the statistical\npower was >99% at a significance level of 0.05, indicating that the sample\nsize was adequate to detect significant differences between groups.\n\nThe baseline demographic characteristics of the studied population are summarized in\n Table 1 . The mean age of PCOS patients\nwas 26.80 ± 0.78 years, which was statistically comparable to that of the\nhealthy control group (27.64 ± 1.91 years; p = 0.660), demonstrating successful\nage-matching. Regarding marital status, a significantly higher percentage of PCOS\npatients were married (75.38%, n = 49) compared to the controls (64.29%, n = 36),\nwhereas single participants constituted (24.62%, n = 16) of patients (35.71% n = 13)\nand of controls (p = 0.0001).\nNote: Data are represented as mean ± SE for continuous\nvariables, and as counts and percentage (%) for categorical\nvariables. Statistical significance was set at p < 0.05.\nRegarding fertility distribution, (58.46%, n = 29) of patients had children, while\n(41.54%, n = 20) did not. In the control group (64.29%, n = 23) had children and\n(35.71%, n = 13) did not. Chi-square analysis revealed that this slight difference\nin the prevalence of having children between the two groups was not statistically\nsignificant (p = 0.830), indicating a homogenous distribution of baseline\nparity.\nThere was a non-significant increase in the follicle-stimulating hormone (FSH), and\nluteinizing hormone (LH) in PCOS (8.12 ± 0.74 mIU/ml), (7.61 ± 0.73\nmIU/ml) respectively, as compared to control (7.35 ± 0.62 mIU/ml), (5.62 ±\n0.56 mIU/ml) respectively (p value > 0.05). However, LH levels showed a\nnon-significant increase in patients when compared to controls (p > 0.05). The\npresent study found a highly significant decrease in antibodies against LH hormone\nin patient (45.29 ± 3.04 ng/ml) were found to be less than control (69.16\n± 10.90 ng/ml). Serum anti-LH antibodies were detected in PCOS patients,\nshowing a negative correlation with LH levels. The anti-FSH antibody levels showed a\nnon-significant increase in both patient and control groups. Antibodies against FSH\nin patient (36.22 ± 6.73 ng/ml) was more than the control (34.99 ± 9.78\nng/ml). The LH/FSH ratio revealed a non-significant increase in the patients (1.161\n± 0.37), and control groups (0.780 ± 0.22), as shown in   Table 2 . The LH/FSH ratio was more than 2 was\nin patients 5(7.6%), while in control zero. The ratio was less than 2, in patient 60\n(92%) higher than in control where it was 56 (100%).\nNote: Data are presented as mean ± standard error (SE).\nt: Independent sample t-test statistic; p: Significance value. NS =\nNon-significant (p > 0.05).\nThe statistical sugnificance level was set at (p > 0.05)\nmIU/ml = milli-International Unit per milliliter.\nNg = nanogram per milliliter.\nThe BMI of the patients and controls was as follows: in obese and overweight women\nthe anti-LH antibody decreased in the patient (43.80)%, and control groups (69.90)%,\nwhile the anti- FSH antibody levels increased in the patients (42.60)% compared to\nthe control group (39.70)%, and the LH/FSH ratio showed an increase in the patient\n(1.381), and control groups (0.80)%. In normal weight women, the anti-LH antibody\nwas lower in the patients (47.586) ng/ml, than in controls (68.22)%, and the\nanti-FSH antibody also as anti-LH antibody, in patients (26.48)% increase, compared\nto control (28.80)%, while LH/FSH ratio represented a higher level in the patient\n(0.80)% than in the control (0.80)% as shown in  \nTable 3 . There was a non-significant correlation between the BMI and\n(Anti-FSH, Anti-LH antibodies, and LH/FSH ratio), as shown in   Table 4 .\nNote: Data are presented as prevalence percentages (%) calculated\nindependently within each stratified body mass index (BMI) subgroup\nrelative to the total number of subjects in each respective group\n(case and controls). Due to the descriptive nature of this\nexploratory stratification and the small sample size within these\nspecific individual subgroups, these values represent baseline\nclinical trends.\nNote: r: Pearson correlation coefficient. NS = non-significant (p\n> 0.05). The statistical significance level for all correlation\nanalyses was maintained at p < 0.05.\n\nPolycystic ovarian syndrome can be defined as a hormonal, metabolic, and endocrine\ndisorder, related to irregular androgen production from the ovaries. An increase in\nadrenal androgen levels, combined with obesity, promotes the peripheral conversion\nto estrogen.  \n 16 ,  17 \n  In addition to type 2 diabetes, women with PCOS often exhibit insulin\nresistance, impaired glucose tolerance, and obesity.  \n 18 \n  It was normal to increase the level of LH hormone, and the ratio of LH/FSH;\nit may be between 1-2 in healthy women but increases to reach as high as 2 or 3,\nwhich will lead to anovulation. The gonadotrophin- axis was distributed in PCOS\npatients, so LH levels increased and FSH levels were within the normal range,\nleading to the expression of the normal LH/FSH ratio. This result is not concordant\nwith that reported by Malini and Roy.  \n 19 \n  The results of this study showed an increase in the LH/FSH ratio in female\nwith PCOS, which increased above 1, except in some patients who were, taking an oral\ncontraceptive that balanced the LH/FSH level before the ovulation, which may have\naffected the results. Despite the potential influence of oral contraceptives on the\nLH/FSH ratio, a small proportion (7.5%) of the patients still exhibited values above\n2, which is a hallmark feature of PCOS. This finding further supports the notion\nthat, in addition to endocrine and metabolic disturbances, immune-mediated\nmechanisms, such as the presence of anti-LH Ab and anti-FSH Ab, may contribute to\novarian dysfunction and reproductive impairment in affected women. In addition,\nfindings indicated an elevation in LH concentrations, along with either normal or\nreduced FSH concentrations, in individuals with PCOS, as revealed by previous\nresearch.  \n 20 \n  The medications that some patients with PCOS were taking were vitamin D3,\noral contraceptives, and metformin when samples were collected, but they continued\nto meet the diagnostic criteria for PCOS. The role of metformin and vitamin D in\nPCOS was to decrease the FSH level in women during post-menopause and decrease LH\nlevels in women with PCOS.  \n 21 \n  Some patients (approximately 4 participants) had been taking medications,\nsuch as metformin and vitamin D, which were prescribed by their physician at the\ntime of sample collection. This treatment is commonly used in PCOS to improve\nhormonal balance by lowering LH levels and increasing FSH levels, thereby supporting\novulation and enhancing menstrual regularity. Additionally, metformin and vitamin D\nhave a positive effect on BMI, and their combination is particularly effective in\nmanaging ovulatory dysfunction in patients with PCOS.  \n 22 \n  Pretreatment with oral contraceptives is used to balance the LH/FSH ratio\nbefore ovulation to regulate ovarian induction.  \n 23 \n  Insulin resistance in women with PCOS is reduced by metformin, thereby\ncounteracting adverse metabolic and hormonal disturbances. This improvement is\naccompanied by enhanced GnRH pulsatility, decreased LH secretion, and reduced\nandrogen production, and an increase in sex hormone-binding globulin (SHBG).\nNormalization of LH levels and the LH/FSH ratio in this manner is considered one of\nthe most prominent therapeutic effects of metformin on fertility in women with PCOS.  \n 24 \n  FSH and LH are inhibited by anti-FSH antibody or anti-LH antibody trapping\nthe hormones in the immune complex, and anti-FSH antibody prevents FSH hormone from\nbinding to its receptor.  \n 25 \n  An earlier study showed that there was a higher level of anti-FSH antibody in\npatients with PCOS than in controls. The exact mechanism remains unclear, and a\nprevious study suggested that the epitope of the beta-chain of FSH attacked by\nanti-FSH antibodies. These autoantibodies made the tissue of ovaries and the hormone\nreceptors as target, which activated the gonadotropin-releasing hormone receptor,\nand disrupted the action of the hypothalamic-pituitary-ovarian axis. The rate of\nanti-FSH antibody in this study, increased in female with PCOS, which is consistent\nwith a previous study showing an elevation in anti-FSH antibody levels in women with\nPCOS. However, the anti-LH antibody level did not show that increase. As well as the\ncurrent study showed a non-significant relationship between (BMI) and (anti-FSH\nantibody, anti-LH antibody, and the LH/FSH ratio). There was a non-significant\ndecrease in anti-LH antibody levels, in obese patients and a non-significant\nincrease in obese control women. The results were the same for the non-obese\n(patients and controls). In obese and non-obese (patients and controls), the\nanti-FSH antibody, LH/FSH ratio, revealed a non-significant increase, as shown in\nearlier research.  \n 26 \n  In earlier study, alterations in reproductive hormones were identified in\nPCOS patients, with FSH levels being lower and LH levels elevated. Approximately,\n64.5% of the women exhibited abnormal hormonal profiles involving FSH, LH, along\nwith an increased LH/FSH ratio. The ratio of LH/FSH showed an inverse association\nwith body mass index. Also, these hormonal changes, particularly the imbalance in\nLH, FSH, and their ratio, were related to clinical manifestations of PCOS, such as\nobesity, insulin resistance, and hyperandrogenism.  \n 27 \n  A previous study reported elevated LH levels in PCOS patients accompanied by\ndetectable anti-LH antibodies. These findings suggest that serum anti-LH Abs could\nserve as a potential marker for PCOS. In line with this, the current study observed\nan inverse relationship between LH and anti-LH Abs, as the results showed elevated\nLH levels accompanied by decreased levels of anti-LH antibodies supporting the\nproposed relationship.  \n 28 \n  As such, this study suggested a group of patients with PCOS and controls\n(healthy women), according to their ages, and if they have children (fertile), or\nnot (infertile), to detect the fertility beside that (secondary infertility), and\n(primary infertility) in women with PCOS was clearly increased. However, the levels\nof anti-LH and anti-FSH antibody in primary infertile patients with PCOS were higher\nthan those in secondary infertile patients with PCOS and controls. To review past\nresearch, infertility was defined as primary infertility (when couples never had\nchildren for more than 12 months). Secondary infertility (when couples never had\nchildren after the last child they had, for more than 12 months), and the female was\ndiagnosed with PCOS without taking any treatment. BMI is a numerical value that\nestimates a person’s body fat based on their weight and height; BMI in obese\nand overweight women is above 25, whereas that in low fat women is below 25. In an\nearlier study the women with a BMI less than 25 (normal weight) had fewer menstrual\ncycle irregularities than obese and overweight women.  \n 29\nNon-significant differences appeared among anti-LH antibodies, anti-FSH antibodies,\nand the LH/FSH ratio with BMI in this study.\nSome patients (approximately 4 participants) were on medications (e.g., metformin and\ncontraceptives) during sample collection; nevertheless, they continued to meet the\ndiagnostic criteria for PCOS as confirmed by a specialist. Based on the present\nfindings, several recommendations for future research can be proposed: assess\nadditional immunological markers and apply a more detailed BMI classification.\n\nThis study suggests that immune-mediated mechanisms, including alterations in\nanti-FSH antibody profiles, may play a role alongside endocrine and metabolic\nfactors in the pathophysiology of PCOS. Although an elevated LH/FSH ratio > 2 was\nclinically observed in a small subset of patients, the distribution of fertility\nstatus and baseline parity did not demonstrate statistically significant variations\nbetween the studied groups. Incorporating immunological markers alongside standard\nhormonal screening could improve descriptive patients’ assessment. Future\nstudies with larger sample sizes and broader longitudinal cohorts are required to\nestablish clear causal pathways and guide targeted interventions.\n\nThis study was approved by the Ethics Committee of the College of Science, University\nof Baghdad (Ref.: CSEC/1124/0100, November 2024). All procedures involving human\nparticipants were conducted in accordance with the ethical standards of the\nDeclaration of Helsinki. Written consent was obtained from each patient and control\nbefore the study started.","source_license":"CC-BY-4.0","license_restricted":false}