Alteration of the follicular fluid amino acid profile reveals the important roles of several amino acids in embryo quality in patients with polycystic ovary syndrome.

The demographic and clinical details of the patients were presented in Supplementary Table 1. It was discovered that individuals with PCOS displayed markedly elevated levels of AMH, LH, and testosterone, while exhibiting lower levels of FSH in comparison to the control group (Fig.  1 A-D). There were no glaring disparities in crucial metabolic indicators like age, BMI, PRL, and E2 levels. Interestingly, the PCOS group demonstrated a substantially higher number of antral follicles and mature oocytes (MII) when juxtaposed with the control group (Fig.  1 E-F). The control group had higher rates of fertilization on day 1, higher proportions of high-quality embryos on day 3, and higher rates of blastocyst formation on day 6 (Fig.  1 G-I, Supplementary Table 1). Total testosterone and FAI were significantly higher in follicular fluid in PCOS patients(Fig.  1 J-K, Supplementary Table 1). Fig. 1 Comparison of clinical characteristics of participants in PCOS and Control group. Comparison of clinical characteristics were assessed using t-test and the Mann-Whitney U test for normally and non-normally dispersed parameters, respectively. PCOS = polycystic ovarian syndrome, BMI = body mass index, AMH = Anti-Müllerian hormone, FSH = Follicle- stimulating hormone, LH = Luteinizing hormone, T = Testosterone. FF = Follicular Fluid, SHBG = Sex Hormone-Binding Globulin, FAI = Free androgen index. p  < 0.05 was taken as statistically significant Comparison of clinical characteristics of participants in PCOS and Control group. Comparison of clinical characteristics were assessed using t-test and the Mann-Whitney U test for normally and non-normally dispersed parameters, respectively. PCOS = polycystic ovarian syndrome, BMI = body mass index, AMH = Anti-Müllerian hormone, FSH = Follicle- stimulating hormone, LH = Luteinizing hormone, T = Testosterone. FF = Follicular Fluid, SHBG = Sex Hormone-Binding Globulin, FAI = Free androgen index. p  < 0.05 was taken as statistically significant During the analysis of the follicular fluid samples, we were able to measure a total of 30 amino acids. The concentration values can be seen in Supplementary Table S2, with glutamine, alanine, and glutamic acid being the most abundant. These amino acids have important physiological roles in the development of oocytes. The P-values for the quantitative results of the amino acids, determined through statistical analysis, are shown in Fig.  2 . The concentrations of 11 amino acids were significantly reduced in the PCOS group. These were glutamine ( p  = 0.025), taurine ( p  = 0.017), phenylalanine ( p  = 0.006), arginine ( p  = 0.002), histidine ( p  = 0.001), serine ( p  = 0.001), Tryptophan ( p  = 0.037), citrulline ( p  = 0.05), lysine ( p  = 0.012), sarcosine ( p  = 0.028) and 1-Methylhistidine ( p  = 0.006) (Fig.  2 A-K). Seventy-nine PCOS women, 28 subjects with reduced insulinsensitivity (IR-PCOS) and 51 subjects with normal insulin-sensitivity (IS-PCOS) were included in the study. The amino acids in the follicular fluid of the subgroups of PCOS women displaying insulin resistance (IR-PCOS) and insulin sensitivity (IS-PCOS) are shown in Supplementary Table S3. Taurine ( p  = 0.01), alanine ( p  = 0.003), Tryptophan ( p  = 0.02), arginine ( p  = 0.02), 1-Methionine ( p  = 0.004), citrulline ( p  = 0.001), sarcosine ( p  = 0.03), α-aminoadipic acid ( p  = 0.01) were significantly decreased in insulin sensitivity PCOS. Meanwhile, in Supplementary Table 4, the control and PCOS groups were divided into normal BMI and high BMI groups according to BMI less than or equal to 24 (normal BMI) and BMI greater than 24 (high BMI), respectively. We found no difference in amino acids between normal BMI and high BMI in the control group. While, in PCOS group, there were downregulation of taurine ( p  = 0.032), alanine ( p  = 0.004), Tryptophan ( p  = 0.03), methionine ( p  = 0.001), citrulline ( p  = 0.003) and α-Aminoadipic acid ( p  = 0.007) in high BMI group. Fig. 2 Differential amino acid profiles in the follicular fluid of PCOS and control patients. Statistical significance was determined by Mann-Whitney U test. Con = control, PCOS = polycystic ovarian syndrome. p  < 0.05 was taken as statistically significant Differential amino acid profiles in the follicular fluid of PCOS and control patients. Statistical significance was determined by Mann-Whitney U test. Con = control, PCOS = polycystic ovarian syndrome. p  < 0.05 was taken as statistically significant Patients were divided into two groups based on the quality of embryos present on day 3. The high-quality embryo group had a proportion of ≥ 50%, while the low-quality embryo group had a proportion of < 50%. Based on these parameters, the control participants were further divided into two subgroups: CON-Top ( n  = 42) and CON-Poor ( n  = 22), while the PCOS patients were divided into PCOS-Top ( n  = 37) and PCOS-Poor ( n  = 42). The control group had a higher rate of high-quality embryos. The differences in amino acid levels were further analyzed in the Top-quality and Poor-quality subgroups in PCOS and control groups. In the PCOS group, taurine ( p  = 0.019), aspartic acid ( p  = 0.02) and threonine ( p  = 0.018) levels were higher in the top quality-subgroup (Fig.  3 A-C). The significant changes in amino acid profile continued to be observed in the control group, alanine ( p  = 0.002), glutamic acid ( p  = 0.001), tyrosine ( p  = 0.036), Tryptophan ( p  = 0.039), glycine ( p  = 0.008), ornithine ( p  = 0.022), threonine ( p  = 0.027) and methionine ( p  = 0.003) were lower in the poor quality subgroup as were presented in Fig.  3 D and L and Supplementary Table 5. Threonine, the only metabolite, was identified as a risk factor for both women with regular ovarian function and those with polycystic ovary syndrome (PCOS). Fig. 3 Differential amino acid profiles in the follicular fluid of the subgroup of control and PCOS patients. A A total of 143 participants were separated into two subgroups based on their proportions of good embryos on day 3. Top quality embryo group was defined as patients with proportions of good embryos on day 3 ≥ 50% and poor quality embryo group was defined as patients with proportions of good embryos on day 3 < 50%. B The box and scatter plots show the concentration of differential metabolites in different subgroups. Only the metabolites with significant differences between CON-Top and CON-Poor or PCOS-Top and PCOS-Poor were displayed. p  < 0.05 was taken as statistically significant Differential amino acid profiles in the follicular fluid of the subgroup of control and PCOS patients. A A total of 143 participants were separated into two subgroups based on their proportions of good embryos on day 3. Top quality embryo group was defined as patients with proportions of good embryos on day 3 ≥ 50% and poor quality embryo group was defined as patients with proportions of good embryos on day 3 < 50%. B The box and scatter plots show the concentration of differential metabolites in different subgroups. Only the metabolites with significant differences between CON-Top and CON-Poor or PCOS-Top and PCOS-Poor were displayed. p  < 0.05 was taken as statistically significant We discovered 11 different metabolites that showed a connection with embryo quality in the group of patients with regular ovarian function. These metabolites were α-Aminoadipic acid, citrulline, methionine, threonine, histidine, ornithine, glycine, Tryptophan, tyrosine, glutamic acid, and alanine. These embryo quality-related amino acids were associated with a 0.735-fold (95%CI 0.612–0.857), 0.715-fold (95%CI 0.589–0.84), 0.729-fold (95%CI 0.579–0.88), 0.679-fold (95%CI 0.539–0.82), 0.654-fold (95%CI 0.511–0.798), 0.675-fold (95%CI 0.532–0.818), 0.703-fold (95%CI 0.563–0.844), 0.658-fold (95%CI 0.513–0.803), 0.661-fold (95%CI 0.511–0.81), 0.745-fold (95%CI 0.622–0.869), 0.739-fold (95%CI 0.614–0.865) higher risk of poor embryo quality in CON group per SD increase, respectively (Fig.  4 A). As for PCOS patients, taurine, aspartic acid and threonine were associated with a 64.6% (95%CI 0.524–0.768), 66.4% (95%CI 0.541–0.787) and 65.5% (95%CI 0.532–0.778) higher risk of poor embryo quality per SD increase (Fig.  4 B). Fig. 4 OR (95% CIs) of good quality embryos risk per one standard deviation increase of differential metabolites after adjusting for confounding factors and the ROC curve of metabolites. A OR (95% CIs) of good quality embryos risk per one standard deviation increase of control group differential metabolites and the ROC curve of metabolites. B OR (95% CIs) of good quality embryos risk per one standard deviation increase of PCOS group differential metabolites and the ROC curve of metabolites. The bold fonts mean the p.value of OR of relevant metabolites is < 0.05. OR = odd ratio, CI = confidence interval. The sensitivity is demonstrated on the y-axis and 1-the specificity on the x-axis OR (95% CIs) of good quality embryos risk per one standard deviation increase of differential metabolites after adjusting for confounding factors and the ROC curve of metabolites. A OR (95% CIs) of good quality embryos risk per one standard deviation increase of control group differential metabolites and the ROC curve of metabolites. B OR (95% CIs) of good quality embryos risk per one standard deviation increase of PCOS group differential metabolites and the ROC curve of metabolites. The bold fonts mean the p.value of OR of relevant metabolites is < 0.05. OR = odd ratio, CI = confidence interval. The sensitivity is demonstrated on the y-axis and 1-the specificity on the x-axis In the control participants, as depicted in Fig.  5 A, the majority of amino acids were positively correlated with basal serum AMH (6/30), number of MII oocytes (13/30), and age (5/30). Glutamic acid and threonine were positively correlated with basal serum E2 levels, while glutamic acid and threonine were negatively correlated with top-quality embryo rate. Meanwhile, alanine, histidine, methionine, citrulline, and α-aminoadipic acid were shown a significantly negative correlation with the rate of top-quality embryos. Phenylalanine, arginine, serine, lysine, and leucine showed a significantly negative correlation with levels of endocrine indicator basal serum PRL (Fig.  5 ). Fig. 5 Spearman correlation coefficients of amino acid profiles with clinical characteristics in the control group. The orange ovals show a positive relationship between the relevant metabolite and clinical characteristic. The blue ovals show an inverse relationship between the relevant metabolite and clinical characteristic. Only the p .values of the correlation < 0.05 are colored Spearman correlation coefficients of amino acid profiles with clinical characteristics in the control group. The orange ovals show a positive relationship between the relevant metabolite and clinical characteristic. The blue ovals show an inverse relationship between the relevant metabolite and clinical characteristic. Only the p .values of the correlation < 0.05 are colored In the PCOS patients, nine metabolites such as asparagine, alanine, aspartic acid, arginine, ornithine, serine, methionine, sarcosine and α-aminobutyric acid had a positive correlation with the number of mature oocytes retrieved. Furthermore, we observed a positive relationship between taurine and glutamic acid, arginine and proline and basal serum PRL level. Sarcosine and the rate of fertilization were positively correlated. A negative relationship was observed between asparagine, aspartic acid and threonine and the rate of top-quality embryos. Alanine, aspartic acid, glycine, histidine, serine, proline, methionine and sarcosine were shown negative association with endocrine indicators, such as basal serum FSH, LH and E2. Tryptophan and α-Aminoadipic acid were shown positive association with metabolic indicators, such as BMI (Fig.  6 ). Fig. 6 Spearman correlation coefficients of amino acid profiles with clinical characteristics in the PCOS group. The orange ovals show a positive relationship between the relevant metabolite and clinical characteristic. The blue ovals show an inverse relationship between the relevant metabolite and clinical characteristic. Only the p .values of the correlation < 0.05 are colored Spearman correlation coefficients of amino acid profiles with clinical characteristics in the PCOS group. The orange ovals show a positive relationship between the relevant metabolite and clinical characteristic. The blue ovals show an inverse relationship between the relevant metabolite and clinical characteristic. Only the p .values of the correlation < 0.05 are colored In order to better understand the connection between metabolites related to embryo quality and clinical characteristics, we conducted an analysis of KEGG pathways between PCOS and non-PCOS groups. By utilizing topological analysis with a cutoff value of 0.1, we identified a number of metabolic pathways that may play a key role in PCOS. A total of 11 metabolic pathways exceeded this cutoff value (Supplementary Table 6). Among these pathways, arginine biosynthesis showed the lowest significance level ( p  < 0.05), but three other pathways - alanine, aspartate and glutamate metabolism, histidine metabolism, and arginine and proline metabolism - were also identified as potential targets (Fig.  7 ). Fig. 7 Pathway analysis based on different metabolism of PCOS and control groups. The pathway enrichment analysis is a quantitative analysis using the concentration values of the compounds compared to the list of the compounds used with over-representation analysis. Based on enrichment (y-axis) and topology analysis (x-axis), this figure illustrates pathways that are significantly changed. The higher impact values represent the relative importance of the pathway, and the size of the circles illustrates the impact of the pathway (the larger circle represents greater pathway enrichment). The color of the circles indicates the significance (the more intense the red color, the lower the p value is) Pathway analysis based on different metabolism of PCOS and control groups. The pathway enrichment analysis is a quantitative analysis using the concentration values of the compounds compared to the list of the compounds used with over-representation analysis. Based on enrichment (y-axis) and topology analysis (x-axis), this figure illustrates pathways that are significantly changed. The higher impact values represent the relative importance of the pathway, and the size of the circles illustrates the impact of the pathway (the larger circle represents greater pathway enrichment). The color of the circles indicates the significance (the more intense the red color, the lower the p value is)

A total of 143 women, including 79 PCOS patients and 64 control participants, were recruited for this study, all of which underwent in vitro fertilization (IVF) treatment at the Zhongshan Hospital of Fudan University from September 2020 to September 2022. The diagnosis of PCOS was according to the 2003 Rotterdam criteria [ 18 ]. All the PCOS patients presented with oligo-ovulation and polycystic ovaries. An age- and weight-matched control group of females was included in the study. The control participants were infertile for tubal obstruction or male factor. All of the control women had regular menstrual cycles, normal ovarian morphology, and normal follicular development which was confirmed by follicular monitoring via ultrasound. The first exclusion criterion was the use of oral contraceptives or medications that may affect hormonal and metabolic levels within the last 6 months. To avoid the effects of some diseases on FF amino acid, we also excluded patients with metabolic disorders, known active infections or inflammatory diseases, autoimmune diseases, endometriosis, or diminished ovarian reserve. The research was approved by the Ethical Committee of Zhongshan Hospital, Fudan University, Shanghai, China on December 30, 2022 (Permission number: B2022-625R) and all participants provided written consent to take part. The physician evaluates the initial infertility diagnosis according to the definition set by the Society for Assisted Reproductive Technology (SART) [ 19 ]. All patients in our study received the appropriate controlled ovarian stimulation protocol with exogenous follicle-stimulating hormone (FSH) at individualized doses and according to their own situation. Individual doses of rFSH ranging from 150 to 250 IU per day were used for stimulation, depending on the maturity of the follicles. The starting dose was determined based on BMI and age. A maximum Daily dose of 300 IU was given to those with a previously determined low response. We supplemented the stimulation with rLH or hMG individually according to the patient’s age or response. From the 6th day of the cycle, we monitored the follicular maturity using ultrasound every other day. Gonadotropin was administered individually according to the size of the follicles. When the diameter of one or two follicles was ≥ 18 mm or those of three or more follicles were ≥ 17 mm, human chorionic gonadotropin (hCG) and/or the gonadotropin-releasing hormone (GnRH) agonist was administered to induce ovulation. Since the risk of OHSS in PCOS patients is higher than other infertile patients, the dosages of ovulation induction drugs are related to the incidence of OHSS. Therefore, the application of low-dose hCG (3000IU) in the late follicular stage for PCOS patients can safely and effectively maintain the growth and development of follicles, and does not affect the outcome of IVF/ICSI-FET treatment. After 36 h, oocytes were extracted by transvaginal ultrasound guidance. In cases of severe male infertility, technicians combine oocytes in metaphase-II (MII) with freshly retrieved sperm using intracytoplasmic injection technique (ICSI) [ 20 ]. The other patients used conventional IVF. Patients with severe oligoasthenospermia and testicle-derived sperm in the male partner were excluded from the study. The successful fertilization is confirmed within 16 to 18 Hours by the presence of a 2PN oocyte with two pronuclei. Expert evaluators then judge the quality of the resulting embryos on days 2 and 3 after fertilization, following the SART guidelines [ 19 ]. Any embryo deemed suitable for transfer must meet the following criteria: a) an absence of Multinucleated blastomeres, b) 7 to 9 blastomeres on day 3, and c) less than 20% anucleated fragments. Any other embryos are considered of low quality. On day 5 or 6, blastocysts were evaluated and graded according to the Gardner score. The Gardner system evaluates blastocysts based on three sequential morphological criteria. First, the expansion grade (1–6) is assigned based on the blastocoel cavity size and hatching stage: Grade 1–2 indicate early cavity formation (less than or equal to half the embryo volume), Grade 3 signifies a full blastocyst, Grade 4 denotes expansion with a thinning zona, Grade 5 shows hatching, and Grade 6 represents a fully hatched blastocyst. Higher expansion grades (especially 3–6) indicate better maturity. Second, the quality of the inner cell mass (ICM), destined to become the fetus, is graded A-C: ‘A’ (many tightly packed cells), ‘B’ (fewer, loosely grouped cells), or ‘C’ (very few/poorly defined cells). Third, the trophectoderm (TE), which forms the placenta, is similarly graded A-C: ‘A’ (many cohesive epithelial cells), ‘B’ (fewer, less organized cells), or ‘C’ (very few, large, or degenerate cells). The final grade combines these assessments (e.g., 4AA, 5BA, 3BB), where high expansion (≥ 3) combined with A or B grades for ICM and TE indicates the best quality and highest implantation potential. The rate of mature oocytes (MII rate) is calculated by dividing the number of MII oocytes by the all retrieved oocytes. The rate of high-quality embryos is found by dividing the number of high-quality embryos by the number of 2PN zygotes per woman. Each woman contributes one IVF cycle (as long as it is their first) to the study. Specialists inspect IVF/ICSI embryos under a microscope every morning (from day 1 to day 6) to track fertilization, cleavage, and embryo quality. The dominant follicles of the patient were examined via transvaginal ultrasound-guided aspiration to collect the FF. After oocyte separation, the aspirated FF which wasn’t contaminated by visible blood from the first one to two mature follicles with a diameter of 17–20 mm was collected. After centrifugation at 2500 rpm 10 min, supernatants were stored at −80◦C for further testing. Amino acids concentrations were determined by high-performance liquid chromatography (ExionLC AD, USA)-tandem mass spectrometry (AB SCIEX Triple Quad 6500+, USA) (HPLC-MS/MS) according to the instructions. In brief, 120 µl of internal standard working solution was added to 10 µl of sample solution and mixed. The solution was derivated with 50 µl of n-butyl alcohol at 60℃for 30 min. 1 µl derivatization sample were injected into the HPLC. Chromatographic separations were performed on an Phenomenex C18 column (2.1*100 mm, 2.6 μm particle size) at 40℃. The mobile phase consisted of water including 0.1% formic acid (solvent A) and methanol including 0.1% formic acid (solvent B). The solvent was delivered to the Kinetex C18 column column at a flow rate of 0.4 ml/min. Ion source is electrospray ion source, the positive ion detection mode was used and the scanning mode in the Multiple reaction monitoring mode. The data were obtained using Analyst software version 1.6.3(AB Sciex). The metabolite XIC plot shows the ion current chromatogram of the metabolite standard extracted in LC-MS/MS (Supplementary Fig. 1). Targeted mass spectrometry detection was performed at Calibra Scientific, Inc., Key Laboratory of Digital Technology of Zhejiang Province, China. The levels of total testosterone in FF of the patients were measured with chemiluminescent assays (Diagnostic Products Corporation, USA). The concentrations of sex hormone-binding globulin (SHBG) in FF were measured with commercial enzyme-linked immunosorbent assay kits (Adlitteram Diagnostic Laboratories, USA). Free androgen index (FAI) was calculated as testosterone (nmol/l) divided by SHBG (nmol/l) × 100. SPSS (Chicago, Illinois, USA) was utilized for statistical analysis. The t-test and Mann-Whitney U test were used to compare amino acid profiles for normally and non-normally dispersed parameters. All the comparisons in this study were corrected by Bonferroni. The Spearman correlation was used to calculate correlations. We utilized logistic regression to determine the odds ratio (ORs) and 95% confidence interval (95% CI) for the impact of follicular fluid amino acid concentrations on the embryo quality after adjusting for confounding factors. The set of variables considered necessary for adjusting to estimate unbiased associations between follicular fluid amino acid levels and embryo quality included: maternal age (in years), body mass index (BMI), serum sex hormone levels and basal antral follicle count (AFC). Among them, age (23–29, 30–34, and 35–44 y), BMI ( 24 kg/m 2 ) were adjusted as categorical variables in logistic regression models, while others were treated as continuous variables.

Our findings may offer insight into how amino acid imbalances in follicular fluid could contribute to the development of PCOS. In our study, we followed infertile couples undergoing IVF treatment and found that exposure to certain amino acids, such as threonine, was inversely associated with embryo quality. Moreover, we observed significant relationships between arginine, histidine, and serine in the PCOS group. In the control group, a combination of ten follicular fluid amino acids was linked to a lower rate of high-quality embryos. Overall, lower concentrations of amino acids in follicular fluid were linked to poorer embryo quality during IVF. However, further research is necessary to validate these results and determine the potential impact on pregnancy and live birth outcomes.

Our study showed that the number of oocytes obtained by PCOS women and the number of MII oocytes increased, and the fertilization rate and blastocyst rate decreased, indicating a decreased ability of oocyte development. This result is consistent with previous research results [ 8 , 21 , 22 ]. Studies by Wissing et al. have shown that embryos in women with hyperandrogenic PCOS develop more slowly in early life compared to women without PCOS [ 23 ]. However, some studies have also found no difference in oocyte quality between PCOS and control groups during ICSI cycles [ 24 , 25 ]. The reason for this could be the larger number of oocytes found in women with PCOS compared to those without. Another challenge in examining oocyte quality in PCOS patients is the significant variation in oocyte parameters, influenced by factors like laboratory conditions and type of treatment used for stimulation. As PCOS is a diverse condition, our study focused on comparing oocyte quality in non-obese PCOS patients with high levels of testosterone. Our findings suggest that it is elevated androgens, rather than obesity, that have a major impact on the altered metabolic effects of PCOS. Our study aimed to quantify the levels of 30 amino acids in the follicular fluid of patients undergoing in vitro fertilization (IVF) and analyze the differences in amino acid profiles between PCOS patients and those with regular ovarian function. By comparing the FF amino acid concentration of PCOS patients with the control group, it was found that the concentrations of several amino acids such as glutamine, taurine, phenylalanine, arginine, histidine, serine, tryptophan, citrulline, 1-methylhistidine, lysine and sarcosine had significantly changed, which was consistent with previous findings in plasma [ 26 , 27 ]. Among them, arginine, histidine and serine were mostly altered. Other studies report higher concentrations of branched-chain amino acids (leucine, isoleucine, valine) and aromatic amino acids (phenylalanine, tyrosine) in PCOS follicular fluid compared to controls, correlating with insulin resistance and hyperandrogenism [ 28 , 29 ]. BCAAs and glutamate/glutamine ratio may serve as diagnostic markers for PCOS-related ovarian dysfunction [ 30 ]. Glutamine and alanine levels are often increased, potentially linked to altered ovarian microenvironment and oxidative stress [ 31 ]. PCOS FF shows decreased glycine, which are associated with impaired one-carbon metabolism and follicular maturation [ 32 ]. Our previous analyses also highlight disruptions in amino acid metabolism (urea cycle, glutathione synthesis) in PCOS FF, affecting oocyte quality [ 33 ]. We also compared the amino acid levels between insulin resistant women with PCOS and insulin sensitive women, taurine, arginine, tryptophan, citrulline and sarcosine were also significantly changed, which was consistent with PCOS and control group. These studies reveals that altered amino acid profiles in PCOS FF reflect metabolic disturbances, which could impact folliculogenesis and oocyte competence. In our study, arginine biosynthesis pathway is the most related pathway underlying PCOS. Arginine was positively associated with basal AMH level in control group and negatively associated with basal PRL level in all the patients in study. In the control group, serine and histidine showed a positive correlation with the number of mature MII oocytes, while in the PCOS group, serine and arginine displayed a similar association. Arginine, an essential amino acid, is responsible for the production of various molecules such as nitric oxide (NO) and polyamines, both of which promote vasodilation and nutrient delivery to the follicle, supporting oocyte growth [ 34 ]. Arginine-derived polyamines enhance autophagy and mitochondrial turnover, ensuring energy production and reducing oxidative stress during oocyte maturation [ 35 ]. Dysregulation of NO synthesis via arginine deficiency may impair cumulus cell expansion and meiotic resumption [ 36 ]. Arginine participates in ammonia detoxification via the urea cycle and mTOR signaling in oocyte growth and disrupts insulin signaling, exacerbating follicular arrest and androgen overproduction, the hallmark observed in PCOS ovarie [ 37 ]. Deficiency of arginine is also a common issue that can lead to endothelial dysfunction and/or T-cell dysfunction, depending on the specific clinical situation and disease state [ 38 ]. Supplementation of arginine and glutamate, both part of the arginine family, has been shown to improve fetal growth in complicated pregnancies. Histidine is considered as an anti-inflammatory amino acid and an antioxidant. As a nucleophilic amino acid, it is easily modified by Lipid-peroxidation-derived electrophiles such as 2-enaldehydes, ketoaldehydes and 4-hydroxy-2-enaldehydes [ 39 ]. Therefore, the decreased levels of amino acids discovered in PCOS patients may be a result of their heightened usage as potent antioxidants amidst times of oxidative strain. Histidine serves as a precursor for glutathione (GSH), a key antioxidant that mitigates oxidative stress in oocytes. Oxidative damage disrupts mitochondrial function and DNA integrity, leading to impaired meiotic resumption and chromosomal instability, which defects were observed in PCOS oocytes [ 40 , 41 ]. Histidine also participates in histone acetylation via its metabolite histamine, which regulates chromatin remodeling during oocyte growth. Dysregulated histamine levels may alter histone H3/H4 acetylation, affecting genes critical for follicular development and steroidogenesis in PCOS [ 42 ]. Histidine deficiency disrupts early embryogenesis through proteoglycan synthesis and immune tolerance [ 40 ]. An important participant in a plethora of essential cellular processes - from protein synthesis to neurotransmission to folate and methionine recycling to the creation of sphingolipids, phospholipids, and sulfur-based amino acids - is serine [ 43 ]. Serine serves as a precursor for S-adenosylmethionine(SAM), the universal methyl donor essential for DNA/RNA methylation and histone modification. This process is vital for epigenetic reprogramming during oocyte maturation and early embryonic development [ 44 ]. Serine is also the substrate for phosphatidylserine(PS) and sphingolipid biosynthesis, which are crucial for maintaining mitochondrial membrane integrity and signaling in oocytes [ 45 ]. Follicular Fluid is considered as a powerful tool to understand polycystic ovary syndrome. Numerous scientific analyses have delved into the depths of follicular fluid to diagnose and predict the success of Assisted Reproductive Technology (ART) in PCOS patients [ 46 ]. Wan-Li Xu and colleagues unearthed major metabolic discrepancies in the serum and follicular fluid of PCOS patients through untargeted metabolomics analysis. Three key metabolites, including 1-Methylhistidine, threonine, and Citrate, stood out as being significantly altered in PCOS patients [ 47 ]. Meanwhile, Entai Hou and their team utilized the GC-MS platform to scrutinize follicular fluid samples from both PCOS patients and healthy women, revealing an array of metabolic changes linked to amino acid metabolism [ 48 ]. Yanna Ban [ 49 ] and Ying Ding’s [ 50 ] lipidomic studies further shed light on the differences between PCOS patients and the control group, pinpointing various lipids as potential markers of distinction. Glutamine is the most abundant and versatile amino acid in the body. Immune cells guzzle up glutamine at a rate comparable or even exceeding that of glucose in both health and illness. Studies conducted in test tubes and living organisms have shown that glutamine is indispensable for fueling the proliferation of lymphocytes, the production of cytokines, the activities of macrophages in engulfing and secreting substances, and the ability of neutrophils to eradicate bacteria [ 51 ]. Researchers discovered that the embryos deemed of poor quality were actually consuming more glutamine than their high-quality embryos [ 52 ]. Furthermore, there is growing evidence to suggest that taurine may hold a key role in protecting the reproductive function of male animals. By boosting the activity of the hypothalamus-pituitary-testis axis and maintaining a stable testicular environment, taurine has the potential to revolutionize our understanding of reproductive health [ 53 ]. The depletion of taurine has been shown to lead to a significant decrease in normal embryo numbers and blastocyst formation rate [ 54 ]. These findings have added another layer of complexity to the already intricate web of reproductive biology. High levels of phenylalanine during fetal development have been linked to drastic consequences such as microcephaly, neuronal loss, and corpus callosum hypoplasia [ 55 ]. Tryptophan, a crucial precursor to several bioactive compounds, plays a vital role in maintaining various physiological functions [ 56 ]. From protein biosynthesis to immune system function, its importance cannot be overstated [ 57 ]. Recent studies have even suggested that tryptophan supplementation can improve reproductive performance in sows and enhance the growth of piglets [ 58 ]. Experiments exploring the potential benefits of L-citrulline supplementation have yielded promising results. By increasing nitric oxide availability in the placenta and uterine-placental circulation, this compound may offer new strategies to improve birth outcomes and manage gestational hypertension [ 58 ]. The intricate processes of lysine acetylation and deacetylation serve as critical posttranslational modifications that bridge the gap between external signals and internal responses [ 59 ]. This intricate cellular mechanism continues to captivate researchers seeking to unravel the mysteries of signal transduction. An individual’s capacity for reproduction is mainly restricted by the quality and development potential of their embryos. In PCOS sufferers, stimulated ovarian control processes result in a notably higher number of retrieved cumulus-oocyte complexes compared to non-PCOS individuals. Previous research has reported either a similar [ 24 , 60 ] or significantly higher number of mature oocytes in such cases [ 25 , 61 ]. Our research suggests that the changes in amino acid levels observed in PCOS females differ significantly from those of the control group, indicating that enhanced LH levels, heightened androgen levels, and altered insulin sensitivity may all contribute to compromised oocyte function and early embryo development [ 62 ]. Taurine involved in muscle energy metabolism [ 63 ] and anti-oxidative stress [ 64 ], were decreased in the PCOS group with poor embryo quality. Recent studies have uncovered a fascinating connection between taurine and male reproductive health. It appears that this intriguing compound may play a crucial role in safeguarding the reproductive function of male animals [ 53 ]. Hua Wu and colleagues observed a striking reduction in embryo numbers and blastocyst formation when taurine levels were depleted [ 54 ]. Reduced taurine levels correlate with elevated ROS in oocytes, impairing mitochondrial function and spindle integrity [ 65 ]. Taurine deficiency reduces ATP production, stalling oocyte growth and steroidogenesis [ 66 ]. Additionally, insufficient levels of aspartic acid in oocytes and follicular fluid could lead to poor COC morphology and altered oocyte quality [ 46 ]. The only amino acid that showed changes in both the control and PCOS group was threonine. Jian Wang’s research highlighted the vital role of threonine in embryonic stem cells, suggesting its critical importance for their functioning [ 67 ]. Moreover, glutamic acid levels were notably decreased in the subgroup with lower embryo quality in the control group. This amino acid, abundantly present in female reproductive fluids, was shown by Alexandra Špirková to influence preimplantation embryo development through cell membrane receptors [ 68 ]. Yongjin Lee and colleagues proposed that treating pig oocytes with alanine during development could enhance embryonic competence, corroborating our findings that a deficiency in alanine leads to decreased embryo quality [ 69 ]. Additionally, the essential amino acid tryptophan was found to have a positive impact on mammalian pregnancy [ 70 ], further emphasizing the intricate role of amino acids in reproductive processes. Ahui Liu et al. found the Tryptophan in follicular fluid was positively correlated with the available embryo rate in normal ovarian reserve (NOR) females [ 71 ]. The intriguing findings of Lepeng Gao and colleagues revealed that glycine, a key ingredient in glutathione, is crucial for safeguarding porcine oocytes in a lab setting. Their study showcased how glycine supplementation sparked a remarkable growth in cumulus cells and enhanced oocyte development [ 72 ]. Meanwhile, Shuang Cai and team uncovered a perplexing connection, showing that adding methionine to the diet during pregnancy led to a striking rise in the number of fetuses and their average weight [ 73 ]. Embryo quality is a critical factor in female fertility, but current methods of assessing oocyte competence have limited predictive abilities when it comes to successful pregnancy. Our research aims to reveal the predictive value of follicular fluid as a non-invasive technique for assessing human embryos and their associated cellular and biological fluids that are relevant to their developmental capacity. To effectively analyze metabolite levels, there is a need for the integration of automated, high-throughput targeted metabolomics in real-time with microfluidic platforms. Our study is currently investigating the amino acid composition of follicular fluid. Prospective randomized trials without bias must be conducted in the future to determine the prognostic value of these findings. Due to the small number of participants, our results should be viewed as preliminary until they can be replicated in a larger group. Unlike prior studies focusing on select amino acids (e.g., glutamine or BCAAs), we analyzed 30 amino acids in follicular fluid (FF), including understudied metabolites Like taurine, serine, and 1-methylhistidine. This broader scope revealed unique correlations (e.g., taurine deficiency in PCOS FF correlating with embryo quality) not previously reported. Our identification of 11 amino acids (e.g., glutamine, arginine) associated with embryo quality in PCOS is novel. For example, taurine and aspartic acid were elevated in high-quality embryos within PCOS, contrasting with controls where alanine and glutamic acid dominated poor-quality subgroups. This dual-pattern finding highlights metabolic heterogeneity in PCOS that prior cohort studies overlooked. Reduced glutamine and arginine (involved in insulin signaling) in PCOS FF provide mechanistic context for prior associations between amino acid metabolism and hyperinsulinemia. Our logistic regression further quantifies their predictive value for embryo quality, addressing a gap in translational research. Most previous studies used the Rotterdam criteria to diagnose PCOS and did not limit the study group to those with a more severe oligomenorrhea and anovulatory phenotype. These factors may explain the contradictory results of previous studies that reported different increases and decreases in amino acid metabolites in PCOS patients compared to controls. While our cohort identifies amino acid signatures associated with PCOS and embryo quality, causal inference requires functional validation. Future work should employ Mendelian randomization, dietary interventions, or organoid models to elucidate metabolic pathways. We also recognize that the restriction to dominant follicles may not fully capture the heterogeneity of follicular development in PCOS. Non-dominant follicles (e.g., arrested or atretic follicles) could exhibit distinct metabolic profiles, which were not assessed in this study. Additionally, while our study evaluated the good-quality embryo rate per patient, the use of FF from individual dominant follicles limits direct correlation with the developmental potential of oocytes derived from the same follicle. Future studies profiling FF from matched dominant and non-dominant follicles within the same ovary would elucidate intra-ovarian metabolic heterogeneity in PCOS. However, in our research, we found distinct differences in certain amino acids, and there are several possible explanations for this. Firstly, the variation in PCOS characteristics among different ethnic groups may have influenced the results in various countries and regions. Additionally, the accuracy of detecting amino acids with low abundance may have presented challenges. Lastly, fluctuations in hormone levels during different menstrual cycles could impact metabolic changes. Therefore, our study focused on accurately and thoroughly analyzing targeted amino acids in Chinese Han women, all within the same menstrual cycle. Our goal was to discover potential amino acid markers that may be linked to PCOS and identify any differences in embryo quality.

Infertility affects 10–15% of reproductive-aged couples globally, with assisted reproductive technology(ART) offering the most effective treatment [ 1 ]. Successful ART outcomes critically depend on oocyte quality, a key determinant of embryonic developmental potential [ 2 , 3 ]. Recent advancements have greatly improved the ability to non-invasively assess the quality of oocytes [ 3 , 4 ]. FF, obtained during oocyte extraction, is believed to hold important clues about the quality of the oocyte [ 5 ] and provides crucial nutrients and factors for the oocyte to grow and develop properly [ 6 , 7 ]. For women with polycystic ovary syndrome (PCOS), a leading cause of anovulatory infertility, ART outcomes remain suboptimal. Hormonal imbalances (the excessive secretion of LH [ 8 ], hyperandrogenism) and insulin resistance disrupt the follicular milieu, compromising oocyte quality and reducing pregnancy rates [ 9 ]. Critically, these endocrine disturbances alter metabolic homeostasis within the follicle, a microenvironment essential for oocyte maturation [ 10 ]. Specifically, insulin resistance can impair amino acid uptake/utilization by follicular cells [ 11 ], while hyperandrogenism may disrupt mitochondrial function and redox balance, impacting amino acid-dependent energy metabolism and antioxidant defense [ 12 ]. This dysregulation of amino acid homeostasis likely contributes directly to impaired oocyte competence by affecting energy supply, protein synthesis, and epigenetic regulation [ 13 ]. Amino acids are pivotal in this context: Beyond serving as protein precursors, they regulate metabolic pathways, redox balance, and energy provision essential for oocyte development [ 14 , 15 ]. Yet, while altered serum or FF amino acids in PCOS have been noted [ 16 , 17 ], systematic comparisons of FF amino acid profiles between PCOS and non-PCOS women, and crucially, their direct links to embryo quality, are lacking. Furthermore, how PCOS-specific endocrine-metabolic perturbations shape this FF amino acid signature and its downstream impact on embryogenesis remains poorly defined. This knowledge gap hinders the development of biomarkers to improve ART stratification for PCOS patients. Here, we apply mass spectrometry-based metabolomics to comprehensively define FF amino acid profiles in non-PCOS women (controls) and PCOS patients. Our primary objectives are to (i) characterize and contrast the FF amino acid metabolome between PCOS and control groups; (ii) identify FF amino acid biomarkers significantly associated with embryo quality; and (iii) explore potential correlations between key PCOS endocrine traits and dysregulated amino acids/embryo quality. We hypothesize that specific dysregulation of amino acid metabolism within the PCOS follicle underlies compromised embryo developmental potential. Our findings aim to provide novel mechanistic insights into PCOS-related infertility and identify potential FF amino acid biomarkers. These biomarkers could serve as non-invasive predictors of oocyte/embryo quality for better ART selection, and as targets for personalized interventions aimed at improving the follicular microenvironment and ultimately, pregnancy success rates in PCOS.

Supplementary Material 1. Supplementary Material 1.