Relation of Follicular Fluid Soluble Receptor for Advanced Glycation End-Products Concentration and Anti Mullerian Hormone in Polycystic Ovary Syndrome and Non-PCOS Women Referring to In Vitro Fertilization Center: Case-Control Study.

Polycystic ovary syndrome (PCOS) stands as one of the most prevalent endocrine disorders affecting 4 to 20% of women in their reproductive years ( 1 ). The precise underlying causes of PCOS remain elusive, although various hypotheses have emerged attributing its etiology to environmental influences, dietary patterns, and genetic predispositions. Recent studies have provided growing evidence that reactive molecules, namely advanced glycation end products (AGEs), play a role in the etiology of PCOS ( 2 ). Findings suggest that dietary AGEs have a significant impact on the reproductive and metabolic dysfunctions relevant to PCOS ( 3 ). AGEs are generated through the non-enzymatic glycation process, which involves the interaction between reducing sugars and proteins, lipids, or nucleic acids. Furthermore, endogenous AGE formation can occur under normal metabolic conditions or in abnormal situations such as diabetes, renal disease, or other inflammatory disorders. Women with PCOS exhibit increased levels of serum AGEs, which are associated with insulin resistance. Consequently, a substantial deposition of AGEs in the ovarian tissue occurs, leading to anovulation and hyperandrogenism ( 4 ). Inflammatory signaling pathways, as well as oxidative stress signaling pathways ( 5 , 6 ) are triggered by this interaction between AGEs and receptors for advanced glycation end-products (RAGE), resulting in tissue destruction. Soluble RAGE (sRAGE) represents the cleaved form of the membrane-bound RAGE receptor, generated through proteolytic cleavage ( 7 ). These sRAGE receptors circulate in the bloodstream and can be found in various bodily fluids, including follicular fluid (FF). They play a paramount role as the neutralizers of the effects engendered by AGEs ( 8 ). By preventing the binding of AGEs to RAGE, sRAGE effectively mitigates the pro-inflammatory actions of AGEs ( 9 ). Consequently, sRAGE has been utilized as a biological indicator in various pathologies, such as diabetes and atherosclerosis ( 7 ). It is noteworthy that studies have shown significantly lower levels of sRAGE in the FF of women with PCOS compared to those without PCOS ( 2 ). The anti-mullerian hormone (AMH) is a significant hormone involved in the process of folliculogenesis. It exerts an inhibitory effect by suppressing the differentiation of granulosa cells, thereby preventing the maturation and atresia of follicles in the early stages of development ( 10 ). Considering PCOS, it is widely recognized that women with this condition exhibit abnormally high levels of AMH in both serum and ovarian tissues ( 11 ). Such elevation in AMH levels is associated with abnormal folliculogenesis and anovulation, further compounded by the presence of elevated levels of AGEs. The quantity of ovarian follicles serves as an indicator of women’s reproductive age and their potential for reproduction ( 12 ). The level of AMH, considered the most reliable indirect measure of ovarian reserve ( 1 ), holds various clinical implications. Its implication ranges from diagnosing conditions such as women with PCOS and diminished ovarian reserve to predicting the success rates of infertility treatments and estimating the time until menopause ( 13 ). The rate at which AMH levels decrease was proposed as a predictive factor for the time until menopause, regardless of the baseline AMH value and chronological age ( 14 ), as well as independent of metabolic and menopausal risk factors ( 15 ). In addition to age and genetic factors, which are considered key predictors of ovarian reserve, lifestyle and environmental influences seem to play a significant role in modifying follicular recruitment and/or follicular atresia ( 16 ). While some epidemiological studies found associations between certain dietary factors and the timing of menopause, the findings have been inconsistent across studies ( 17 ). However, these findings indirectly suggest the potential impact of nutrition and dietary intake on ovarian reserve. Notably, reducing the consumption of diets containing AGEs demonstrated favorable effects on metabolic and hormonal profiles, as well as ovarian function, particularly in women with PCOS ( 18 ). We posit the hypothesis that the concentration of soluble receptors for sRAGE in FF can serve as an indicator of ovarian reserve in women undergoing in vitro fertilization (IVF). This can be observed through the FF concentration of AMH and the number of retrieved oocytes and embryos. This study aims to explore whether there is an association between FF sRAGE levels and markers of ovarian reserve in both Iranian women with PCOS and those without PCOS. We hypothesise that the concentration of sRAGE is lower in women with PCOS, independent of age. Additionally, we believe that age may influence the concentration of this receptor and ovarian reserve.

Table 1 presents the demographic and clinical characteristics of the 43 participants who completed the study. The participants included 17 individuals with PCOS as cases and 26 non-PCOS individuals as controls. The mean age of the participants was 35 years with a SD of 3.98, while the mean body mass index (BMI) was 25.99 kg/m² with an SD of 3.98. There was a statistically significant difference in age between the two groups, with the mean age of PCOS participants being 32.4118 years, which was lower than that of the control group (38.2692) (P=0.000). Nevertheless, no statistically significant difference was observed in the mean (SD) BMI between the two groups. The mean (SD) concentration of FF sRAGE also did not show a statistically significant difference between the two groups, as indicated in this table. Demographic and clinical characteristics of the patients’ FF sRAGE in the PCOS and control group Data analysis using the t test. *; Significance at P<0.05, BMI; Body mass index, FF; Folicular fluid, sRAGE; Soluble receptor for advanced glycation end product, PCOS; Polycystic ovary syndrome. Distribution of women in PCOS group (n=17 women)/non-PCOS group (n=26 women). After categorizing the participants into age groups (3040 years), it turned out that in PCOS women younger than 30 years, the serum AMH concentration was significantly higher compared to nonPCOS women (P=0.00). However, there were no noticeable differences in the numbers of oocytes and embryos between the two groups. Concerning the age group of >30<40 years, PCOS women exhibited significantly higher levels of AMH concentration, as well as higher numbers of oocytes and embryos compared to non-PCOS women (P=0.00, P=0.016, P=0.044, respectively). Among those aged >40 years, PCOS women displayed significantly higher serum AMH concentration and numbers of oocytes compared to non-PCOS women (P=0.003, P=0.00, Table 2 ). Regarding the correlation analysis without age matching, a significant and positive relationship was observed between FF sRAG and serum AMH concentration in PCOS women (P=0.012, r=0.596) as detailed in Table 3. When analyzing the age group of 30<40 years among PCOS women, there was no significant relationship between serum sRAGE concentration and AMH concentration, as well as oocyte and embryo numbers. Transitioning to the age group of women over 40 years with PCOS, a significant and positive correlation was noted between serum AMH and FF sRAGE (P=0.001, r=1). Furthermore, in the same age group, a significant and inverse relationship was observed between FF sRAG and oocyte numbers (P<0.001, r=- 1 ), as well as between FF sRAG and embryo numbers (P<0.001, r=-1). In conclusion, it can be inferred that across all age categories of non-PCOS women, no correlation was found between sRAGE and serum AMH concentration, embryo numbers, and oocyte numbers. Tables 4 and 5 present the correlation analysis depicting the relationships between sRAGE and three variables within the PCOS group, stratified by age classification, and the correlation analysis between sRAGE and three variables within the non-PCOS group, also categorized by age classification, respectively. Comparison of FF sRAGE (ng/mL), serum AMH (ng/mL), oocytes and embryo numbers in the PCOS and control group based on the age adjustment Data analysis using the t test. FF; Folicular fluid, sRAGE; Soluble receptor for advanced glycation end product, AMH; Anti mullerian hormone, PCOS; Polycystic ovary syndrome, and * ; Significance at P<0.05. Correlation table between sRAGE and three variables in two groups without age classification Data analysis using the Pearson correlation coefficient. sRAGE; Soluble receptor for advanced glycation end product, AMH; Anti mullerian hormone, PCOS; Polycystic ovary syndrome, and *; Significance at P<0.05. Correlation table between sRAGE and three variables in the PCOS group with age classification Data analysis using the Pearson correlation coefficient. sRAGE; Soluble receptor for advanced glycation end product, PCOS; Polycystic ovary syndrome, AMH; Anti mullerian hormone, and *; Significance at P<0.05. Correlation table between sRAGE and three variables in the nonPCOS group with age classification Data analysis using the Pearson correlation coefficient. sRAGE; Soluble receptor for advanced glycation end product, PCOS; Polycystic ovary syndrome, and AMH; Anti mullerian hormone. Significance at P<0.05.

This study aimed to find the relation between FF (sRAGE) concentration and AMH in PCOS and nonPCOS women. Several studies have shown that a higher concentration of sRAGE indicates less inflammation and greater health in people. Consequently, it is anticipated that higher concentrations of the decoy receptor sRAGE could enhance granulosa cell function within the follicular milieu, leading to increased production and secretion of AMH ( 20 ). There is growing recognition that the buildup of AGEs within the ovarian follicle could potentially initiate premature ovarian aging, particularly impacting infertile women with reduced ovarian reserve ( 21 ). The potential presence of AGE accumulation in the ovary could explain reduced vascularization efficiency and activation of oxidative stress pathways through interactions with cellular RAGE receptors ( 22 ). The results of the present study indicate a positive correlation between FF sRAGE and serum AMH protein in women with PCOS. Ovarian reserve testing is widely used in clinical practice to predict how the ovaries will respond to controlled ovarian hyperstimulation (COH) and improve the accuracy of predicting the female partner’s age alone ( 23 ). However, it is important to note that environmental factors, such as obesity and nutritional status, can influence AMH levels and potentially affect its accuracy as a marker for ovarian reserve ( 24 ). To the best of the authors’ knowledge, women with PCOS had a higher intake of AGEs compared to nonPCOS women. Notwithstanding this finding, the concentration of sRAGE was similar between the two groups ( 25 ). Regarding this observation, Jinno et al. ( 26 ) investigated the levels of toxic AGEs in the blood and FF of 157 women undergoing IVF to assess the association between AGE accumulation and IVF outcomes. The results demonstrated a negative correlation between the accumulation of AGEs in the FF and serum and factors such as follicular growth, fertilization, and embryonic development. Remarkably, even in younger individuals (40 years old), elevated serum AGE levels were indicative of ovarian dysfunction and reduced fertility. While Jinno et al.’s ( 26 ) study did not measure the level of sRAGE, it is reasonable to hypothesize that the presence of sRAGE, functioning as a decoy receptor by binding to AGEs in serum and FF, may show inverse correlations. Therefore, it can be concluded that sRAGE has the potential to act as a valuable biological marker for evaluating the ovarian follicular environment. Given the inverse relationship between the concentration of sRAGE and the concentration of AGEs metabolite, and the direct relationship between the concentration of this receptor and ovarian reserve observed in the current study, it can be inferred that an increase in sRAGE concentration may lead to higher success rates in IVF procedures. The potential accumulation of AGEs within the ovary may contribute to impaired vascularization and activation of oxidative stress response through their interaction with cellular receptors known as RAGE. In the same way as FF sRAGE, AMH represents ovarian health and may act as a biological marker of follicular health. In this study, we presented novel findings demonstrating a correlation between follicular sRAGE and AMH concentrations in reproductive-aged women with PCOS, specifically in those under 30 and over 40 years of age. This suggests a potential association between sRAGE and the reproductive environment, which may be influenced by reproductive age. While the precise functionality of sRAGE within the FF remains uncertain, it is plausible that its presence may be indicative of the function of the AGE-RAGE system in ovarian follicles. This receptor has the potential to serve as a viable biomarker for evaluating the nutritional status of individuals. A study conducted in 2014 by Irani and Merhi ( 27 ) suggested that in women with PCOS, vitamin D3 supplementation could potentially mitigate the inflammatory effects of AGEs by increasing circulating levels of sRAGE. The observed normalization of serum AMH following replacement therapy with vitamin D3 implies an enhancement in folliculogenesis. This finding suggests that the quantity of AGEs ingested may indeed have an impact on AMH levels. Despite the potential of a high concentration of sRAGE to serve as an indicator of favorable fertility status, there exists an age-related limitation. Our study reveals that as age increases, particularly in women with PCOS who are over 40 years old, elevated levels of this receptor may suggest unfavorable reproductive conditions. The current findings demonstrate an inverse relationship between sRAGE concentration and the number of embryos and oocytes, while a direct relationship is observed with AMH levels. Elevated AMH levels typically indicate a higher number of immature eggs in PCOS women. However, intriguingly, at a younger age of around 30 years, a higher concentration of this receptor appears to be indicative of favorable fertility status in women. Conversely, in older age groups, the results show a reversed pattern, where only AMH exhibits a direct relationship with sRAGE. Notably, a recent study by Bonetti et al. ( 28 ) reported that increasing intrafollicular sRAGE concentration was predictive of poor-quality embryos. It is well established that the number of follicles per ovary and ovarian volume exhibits a declining pattern as individuals age. Utilizing these markers, along with agespecific thresholds, has proven to be more effective in diagnosing PCOS compared to using a single threshold ( 29 ). Furthermore, research indicates that AMH levels exhibit a gradual decrease as women age. However, it is important to note that the rate of AMH decline may vary among women of reproductive age ( 30 ). Interestingly, recent studies indicated that the depletion of the ovarian pool occurs more gradually in women with PCOS compared to those without PCOS. Research findings suggest that the rate of decline in AMH levels accelerates once individuals exceed the age of 40 ( 31 ). However, it has been observed that this decline occurs at a slower pace in women with PCOS compared to those with normal ovulation, suggesting that PCOS women may experience sustained fertility. Although our previous finding did not show a correlation between age and sRAGE, it is feasible that this relationship may alter at specific ages ( 25 ). Prakash et al. ( 32 ) demonstrated a decrease in sRAGE levels with age, which exhibited an inverse association with BMI and fat-free mass (FFM) in healthy individuals. Interestingly, a study conducted on healthy centenarians revealed higher plasma levels of sRAGE compared to young and healthy individuals, supporting the notion of sRAGE serving as a marker for healthy aging and longevity. It would be beneficial to elucidate the individual contributions of each factor to the decline in sRAGE and explore potential associations with age-related risk factors for diseases. Scavello et al. ( 33 ) revealed two types of sRAGE and suggested that in a healthy population, the cRAGE isoform serves as a biomarker for aging, whereas sRAGE represents a more reliable marker for obesity and insulin resistance. Consequently, the levels of sRAGE isoforms may be differentially associated with risk factors for age-related diseases. The association between BMI and PCOS, as well as BMI and AMH, shows inconsistency ( 34 ). Through a meta-analysis, Moslehi et al. ( 35 ) revealed that obese women have significantly lower levels of markers associated with ovarian reserve, i.e. AMH and FSH, in comparison to non-obese ones. In addition, there was a negative correlation observed between BMI and AMH in all populations studied, as well as with FSH in subgroups of fertile women without PCOS. This suggests that the relationship between AMH and PCOS is influenced by BMI ( 34 ). The present study found no significant association between AMH and BMI in either of the groups examined, and there was no noteworthy difference in BMI between women with PCOS and the control group. As a result, we did not make any adjustments to the agespecific AMH cut-off values for BMI.

Utilization of age-specific cut-off values for AMH, employing robust advanced statistical methods, can effectively evaluate the value of AMH in discriminating PCOS patients. Moreover, such cut-off values could also be employed as an initial diagnostic tool for PCOS. Notably, while sRAGE partially reflects conditions associated with inflammation and fertility status, the beneficial effects on AMH levels and IVF appear to be dependent on the age of the individuals involved. Certainly, the limited sample size is a serious limitation. With larger sample sizes in future tests, we can confidently discuss the results.

A total of 43 women of reproductive age were recruited in this case-control study to examine the influence of FF sRAGE concentrations on AMH, the number of retrieved oocytes, and embryos at Erfan and Arash Hospitals in Tehran, Iran, between June 2022 to September 2022. The control group consisted of 26 women whose husbands were diagnosed with infertility, while the case group included 17 women with PCOS. The inclusion criteria for PCOS women were based on the 2003 Rotterdam criteria, requiring the presence of two or more significant symptoms of the syndrome (oligo‐anovulation, hyperandrogenism and polycystic ovaries (≥ 12 follicles measuring 2‐9 mm in diameter and/or an ovarian volume > 10 mL in at least one ovary) ( 19 ). PCOS diagnosis was confirmed by medical practitioners affiliated with the Tehran University of Medical Sciences. Before the sampling process, demographic information such as age and weight, as well as data on food frequency and physical activity from a questionnaire, were collected and recorded by the examiners. Exclusion criteria included a history of diabetes, chronic kidney disease, any chronic metabolic disorders, endometriosis, use of medications affecting glucose and lipid metabolism, alcohol consumption, and smoking. Both hospitals followed similar standards for pituitary desensitization in both the control and study groups, employing gonadotropin-releasing hormone (GnRH) antagonists. The protocols allowed for flexibility, with subcutaneous injections of gonadotropins (GONAL-F 150 IU/d, Merck, Germany) administered from the third to the fifth day of menstruation to stimulate follicular growth. Note that the size of follicles was monitored using transvaginal ultrasounds after 5-7 days. Once at least two follicles reached a mean diameter of 15-17 mm, two injections of recombinant human chorionic gonadotropin (HCG) (OVITRELLE 6500 IU/d, Merck, Germany) were admin istered. Following thirty-four hours, transvaginal ultrasound-guided oocyte retrieval was carried out. Oocytes were collected through transvaginal, ultrasound-guided puncture within 34-36 hours after hormonal treatment. FF was obtained from the first large aspirated follicle that was free of blood and subsequently centrifuged at 15,000 g for 5 minutes. The resulting supernatant was immediately frozen at -80°C until further use. Aiming at measuring FF sRAGEs and serum AMH concentration, commercially available ELISA kits ZELLBIO ZB-10027C-H9648 (GmbH) from ZELLBIO in Hamburg, Germany were employed. Data were initially tested for normal distribution using the Kolmogorove-Smirnov test. Descriptive statistics, including the mean and standard deviation (SD), were employed to summarize normally distributed variables. To compare the PCOS group with the control group, an independent sample t test was taken into account. Pearson correlation analysis was conducted to examine the relationship between serum sRAGE and AMH in three steps: i. In the whole population to detect the possible relationship regardless of PCOS/non-PCOS, ii. Only in PCOS women, and iii. Only in non-PCOS women. To adjust the effect of possible confounding variables, such as age, on the relationship between AMH and sRAGE, correlation analysis was performed as both crude (without adjusting) and age stratification. The statistical software SPSS (version 22.0 for Windows; SPSS Inc., IBM, USA) was used for all statistical analyses. Statistical significance was determined at a P<0.05. This study received approval from the Ethics Committee of the Tehran University of Medical Sciences, Tehran, Iran (IR.TUMS.MEDICINE. REC.1401.066). All participants provided signed informed consent prior to the study.