Glutamine and norepinephrine in follicular fluid synergistically enhance the antioxidant capacity of human granulosa cells and the outcome of IVF-ET.

This is an observational study of infertile women before and during IVF treatment at the International Peace Maternity and Child Health Hospital (IPMCH), Shanghai, China. Follicular fluid and cumulus GCs on the day of oocyte retrieval were obtained from 27 patients undergoing IVF at the reproductive center. The subjects were recruited with male factor infertility or tubal factor infertility. Subjects with endometriosis and PCOS were excluded. The age of the patients ranged from 25 to 40 years, and the size of the follicles ranged from 19 to 24 mm. The size of the follicle was estimated by ultrasound at the time of oocyte retrieval. Follicular aspirates contain oocytes surrounded by cumulus GCs. After the oocytes were removed by the embryologist, the remaining material was centrifuged at 3000 g for 15 min to isolate the GCs. The fluid from the first aspirated follicle was used to measure hormone and neurotransmitter metabolic levels. Embryos were assessed at Day 3 based on the Istanbul consensus on embryo assessment 20 , and higher quality embryos were defined as Day 3 embryos that had even cleavage, 7–9 blastomeres and < 10% fragments. Human follicular fluid was prepared for neurotransmitters metabolomics detection. Twenty-three metabolites (Ach: Acetylcholine chloride; Gln: Glutamine; Glu: Glutamate; His: L-Histidine; NE: norepinephrine; Tyr: Tyrosine; Trp: Tryptophan; Kyn: Kynurenine; GABA: 4-Aminobutyric acid; HisA: Histamine; PA: Picolinic acid; TyrA: Tyramine; DA: Hydroxytyramine hydrochloride; TrpA: Tryptamine; 5-HT: Serotonin hydrochloride; E: Adrenaline hydrochloride; KynA: Kynurenic acid; 5-HIAA: 5-Hydroxyindole-3-acetic acid; DOPA: Levodopa; XA: Xanthurenic acid; VWA: Vanillylmandelic Acid; 5-HTTP: 5-Hydroxytryptophan; MT: Melatonin) and the internal standards (Tryptophan-D3) were analyzed using LC–MS/MS as described previously 21 , 22 by BioNovo Gene Co., Ltd (Suzhou, China). In brief, LC–MS/MS was conducted by A Waters ACQUITY UPLC coupled with an AB 4000 triple quadrupole mass spectrometer (AB SCIEX, USA) using a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters, USA). The Mobile phase A consisted of methanol and water with 0.1% formic acid (1:9, v/v), whereas mobile phase B consisted of methanol and water with 0.1% formic acid (1:1, v/v). The LC gradient elution, was at a flow rate of 0.3 mL/min at 0–8.0 min and 0.4 ml/min at 8.0–17.5 min, was 10%-30% B at 0–6.5 min; 30%∼100% B at 6.5–7 min; 100% B at 7–14 min; 100%-10% B at 14–17.5 min. The injection volume was 5 μL and column temperature was set to 40 °C. Twenty-three metabolites were simultaneously reported in LC–MS/MS multiple reaction monitoring (MRM) mode. Data analysis was performed with the Analyst 1.6 software (AB SCIEX, USA). GCs were collected in TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA was extracted according to previously described methods 16 . A total of 1 μg RNA was converted to cDNA with a Takara kit (Applied Biosystems Foster City, CA, USA; Takara, Shiga, Japan). The genes of interest were amplified with a 7900HT fast real-time PCR system (Applied Biosystems) and SYBR Green Real-time PCR Master Mix (Applied Biosystems/Takara). The PCR primers were designed according to the cDNA sequences in the NCBI database. The primer sequences used are shown in Supplementary Table 1 . The cycling conditions used for the PCR analysis were as follows: 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s (40 cycles). 18S was used as an internal control. The 2 -ΔΔCt method was employed to determine the relative mRNA expression level. The concentrations of twenty kinds of hormones in follicular fluid were detected by high-performance liquid chromatography–mass spectrometry (HPLC–MS/MS) as described previously 23 by BioNovo Gene Co., Ltd. (Suzhou, China). Briefly, an Agilent 1200 series high-performance liquid chromatography (HPLC) instrument (Agilent, USA) was utilized. A PAL autosampler (CTC, Swiss) and a Gemini-NX-C18 column (2.0 mm × 50 mm, 3 μm, Waters, USA) were also used. The ion source was a quadrupole electrostatic field orbit trap high-resolution mass spectrometer API-4000 (Applied Biosystems, USA). The scanning mode was multiple reaction monitoring (MRM). In addition, the concentration of testosterone and cortisol in follicular fluid was detected by the chemiluminescence method 24 . The human ovarian granulosa cell line was kindly gifted by Dr. Zuwei Yang (IPMCH, School of Medicine, Shanghai Jiao Tong University, Shanghai, China). KGN cells firstly were cultured in the glutamine deprived medium (Advanced DMEM/F12 media, Gibco, 12634010) supplemented with 10% fetal calf serum and antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin) in a 5% CO 2 atmosphere at 37 °C for 24 h in order to consume glutamine stored in cells. And then, glutamine and NE were added into the culture medium of KGN cells for another 48 h. NE (HY-13715, MCE, NJ, USA) was solved in dimethyl sulfoxide (DMSO, Yeasen, shanghai, China) at storage concentrations of 100 mM, and treated cells at a concentration of 10 μM diluted with the advanced DMEM/F12 media. Glutamine (HY-N0390, MCE, NJ, USA) treated cells at a concentration of 2.5 mM or 5 mM diluted with the advanced DMEM/F12 media. Cell culture supernatant samples were collected and centrifuged for 20 min at 1000× g at − 4 °C. The supernatant was stored at − 80 °C for further analysis. The concentrations of 17 β-estradiol and progesterone in cell culture supernatant were measured by ELISA kits (Xinle Biological Technology, Shanghai, China), according to the manufacturer’s instruction. Proteins were extracted from cultured cells with RIPA lysis buffer (Yeasen, Shanghai, China) and protein concentration was measured via a BCA kit (Thermo Scientific, Rockford, IL, USA). The protocol for western blotting was reported previously 16 . The following primary antibodies were used: rabbit antibodies against β-tubulin (1:1000, CST, MA, USA), PCNA (1:1000, CST, MA, USA) and IDH1 (1:1000, Abcam, Cambridge, UK). After incubation with primary antibodies at 4 °C overnight, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2500, CST, MA, USA) for 1 h at room temperature. The bands were detected by electrochemiluminescence (ECL, Millipore, Billerica, MA, USA). The relative intensity of the target proteins was normalized to β-tubulin using ImageJ software (NIH, Bethesda, MD, USA). The proliferation of KGN cell was evaluated by EdU staining (RiboBio, Guangdong, China). Briefly, EdU was added to the cell culture medium at a concentration of 10 µM. Cells were cultured for 24 h and stained with Alexa 594 and DAPI. Images were captured under a microscope (Leica, WetzlaR, Germany) and positive fluorescence signals were counted. The number of EdU-positive cells per 100 cells was evaluated. All statistical analyses were performed using Prism software, and a p value of < 0.05 was considered statistically significant. Means and standard error were used as descriptive statistics. T-tests were used to compare different variables between the low-level and high-level glutamine groups. Pearson's correlation coefficients and corresponding 95% confidence intervals (CIs) were used to assess the correlation between neurotransmitter metabolites in follicular fluid, and the correlation between IDH1 expression in GC and Gln/NE in follicular fluid. Multiple linear regressions were used to test the correlation between Gln/NE in follicular fluid and the ratio of high-quality embryos, as well as IDH1 expression in GC and the ratio of high-quality embryos, adjusting for spouse age, sperm density, and sperm motility. In the current study, when a power analysis was performed with 80% power and an α value of 0.05, the number of samples in each group needed to be 13 to confirm statistical significance. Informed consents for participating in this study were obtained from all the patients and this study was approved by the Institutional Ethics Committee of the International Peace Maternity and Child Health Hospital (Ethical statement number: (GKLW) 2018-43). All experiments were performed in accordance with the relevant guidelines and regulations. All authors have agreed to publish this article.

Dynamic neurotransmitters metabolic changes within follicular fluid are important for cumulus expansion, which represents the consequence of oocyte cumulus complex (OCC) metabolic cooperation 25 , 26 . In this study, we first measured the content of neurotransmitters metabolites in follicular fluid by LC–MS/MS. The results regarding the concentrations of Ach (0.02 ± 0.01 μg/mL), Gln (46.75 ± 7.74 μg/mL), Glu (13.00 ± 3.27 μg/mL), His (1.30 ± 0.49 μg/mL), NE (0.20 ± 0.07 μg/mL), Tyr (6.54 ± 2.91 μg/mL), Trp (6.54 ± 1.33 μg/mL), Kyn (0.31 ± 0.12 μg/mL) in human follicular fluid are shown in Table 1 . The other fifteen neurotransmitters were not detected in most of the follicular fluid samples. Pearson’s correlation analysis showed that Gln was positively correlated with the NE content in follicular fluid (R = 0.5869, P  0.05, Fig.  1 B). Table 1 Concentration of multiple neurotransmitters in human follicular fluid. Sample ID Ach Gln Glu His NE Tyr Trp Kyn (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 01 0.019 45.715 21.989 1.083 0.233 6.431 7.227 0.238 02 0.025 49.37 10.045 1.472 0.281 6.231 7.373 0.548 03 0.080 60.028 16.639 3.620 0.401 19.822 5.658 0.757 04 0.018 49.313 15.104 1.365 0.251 4.751 6.977 0.307 05 0.019 52.869 16.819 1.510 0.240 5.724 7.711 0.350 06 0.016 54.879 13.450 1.273 0.245 6.297 5.830 0.202 07 0.019 35.712 17.795 1.074 0.253 5.903 6.359 0.361 08 0.027 44.439 11.364 1.389 0.244 6.018 4.618 0.307 09 0.018 51.359 12.867 1.329 0.258 6.633 6.925 0.319 10 0.023 60.335 15.089 0.947 0.214 7.698 6.458 0.243 11 0.020 44.826 13.077 1.046 0.202 4.362 5.838 0.284 12 0.026 42.962 14.758 1.339 0.130 5.837 5.290 0.272 13 0.021 58.259 13.474 1.411 0.235 5.413 5.804 0.225 14 0.017 56.594 16.155 1.208 0.285 7.238 10.141 0.241 15 0.02 52.631 6.706 1.455 0.142 10.169 9.157 0.255 16 0.018 44.161 10.595 1.417 0.144 5.729 6.591 0.455 17 0.017 37.091 14.466 1.146 0.231 4.984 7.835 0.346 18 0.021 33.855 8.973 1.014 0.143 4.525 5.623 0.261 19 0.016 50.998 10.091 1.029 0.180 6.335 8.394 0.243 20 0.014 44.261 11.385 1.012 0.186 4.101 6.881 0.284 21 0.018 50.524 11.722 1.249 0.205 6.630 5.096 0.199 22 0.018 38.522 11.873 1.055 0.096 5.849 4.797 0.265 23 0.017 37.578 10.232 1.099 0.113 4.864 5.351 0.248 24 0.018 42.237 14.710 1.106 0.158 6.031 5.536 0.323 25 0.027 44.286 11.252 1.313 0.109 5.851 6.290 0.262 26 0.022 45.651 11.975 1.157 0.218 7.406 7.377 0.336 27 0.026 33.927 8.471 1.103 0.127 5.956 5.352 0.348 Ach acetylcholine chloride, Gln glutamine, Glu glutamate, His l -Histidine, NE norepinephrine, Tyr: tyrosine, Trp tryptophan, Kyn Kynurenine. Figure 1 Correlation analysis of neurotransmitter concentrations in human follicular fluid on the day of oocyte retrieval. ( A ) A positive correlation between the concentration of glutamine (Gln) and norepinephrine (NE) in follicular fluid (n = 27, R = 0.5869, P  0.05). Concentration of multiple neurotransmitters in human follicular fluid. Ach acetylcholine chloride, Gln glutamine, Glu glutamate, His l -Histidine, NE norepinephrine, Tyr: tyrosine, Trp tryptophan, Kyn Kynurenine. Correlation analysis of neurotransmitter concentrations in human follicular fluid on the day of oocyte retrieval. ( A ) A positive correlation between the concentration of glutamine (Gln) and norepinephrine (NE) in follicular fluid (n = 27, R = 0.5869, P  0.05). Gln serves as a substrate for the synthesis of proteins in follicular fluid, which directly promotes oocyte nuclear maturation and enhances embryo development 11 . However, the effect of Gln and NE in follicular fluid on the function of cumulus GCs is still unclear. To further investigate the function of Gln and NE in follicular fluid, 27 samples were divided into two groups according to their concentration of Gln, the high Gln level group (> 45 μg/mL) and low Gln level group (< 45 μg/mL). The function of cumulus GCs involved in proliferative capacity, receptor expression and steroid hormone synthesis was determined by qRT-PCR. Compared with the low Gln level group, those with high Gln level had a significant twofold increase in PCNA and cyclin D2 ( CCND2 ) mRNA levels in the GCs (P < 0.001 and P < 0.01, Fig.  2 A,B). However, there were no statistically significant differences in luteinizing hormone receptor ( LHR ) or follicle stimulating hormone receptor ( FSHR ) mRNA levels between the two groups (P > 0.05, Fig.  2 C,D). Notably, the mRNA levels of the steroid hormone synthesis-related genes, including steroid acute regulatory protein (STAR), cytochrome P450 (CYP11A, CYP19A) and hydroxysteroid dehydrogenase-3 beta ( HSD-3β ) significantly decreased in the low Gln level group (P  0.05, Fig.  2 I). These data demonstrated that the proliferative ability and steroidogenesis of cumulus GCs increased with the high contents of Gln/NE in follicular fluid. Figure 2 The mRNA expression of proliferation, gonadotropin receptor and sex steroid hormone synthesis-related genes in cumulus granulosa cells between high and low glutamine (Gln) level groups. ( A , B ) The column charts show the PCNA and CCND2 mRNA expression in granulosa cells (GCs) in the two groups. ( C , D ) The column charts display the LHR and FSHR mRNA expression in the two groups. ( E – I ) The mRNA expression of sex steroid hormone synthesis-related genes in the two groups, including STAR, CYP11A, CYP19A, HSD-3β and HSD-17β. Data represent the mean values ± standard error of the mean (mean ± SEM). High Gln level group, n = 14; Low Gln level group, n = 13. All indicated P values were determined by Student's t-test. * P value < 0.05; ** P value < 0.01; *** P value < 0.001. The mRNA expression of proliferation, gonadotropin receptor and sex steroid hormone synthesis-related genes in cumulus granulosa cells between high and low glutamine (Gln) level groups. ( A , B ) The column charts show the PCNA and CCND2 mRNA expression in granulosa cells (GCs) in the two groups. ( C , D ) The column charts display the LHR and FSHR mRNA expression in the two groups. ( E – I ) The mRNA expression of sex steroid hormone synthesis-related genes in the two groups, including STAR, CYP11A, CYP19A, HSD-3β and HSD-17β. Data represent the mean values ± standard error of the mean (mean ± SEM). High Gln level group, n = 14; Low Gln level group, n = 13. All indicated P values were determined by Student's t-test. * P value < 0.05; ** P value < 0.01; *** P value < 0.001. To obtain an overview of hormone metabolic characteristics in follicular fluid between high and low Gln level groups, hormone profiling was conducted using HPLC–MS/MS. Nine kinds of hormones were detected in the two groups, including estrone, estriol, 17β-estradiol, progesterone, methyl testosterone, (D)-norgestrel, 19-nortestosterone, epitestosterone and methoxy progesterone. In addition, the concentration of testosterone in follicular fluid was measured by a chemiluminescence immunoassay. The results demonstrated that the concentrations of estriol and 17β-Estradiol were significantly higher in the high Gln level group than in the low Gln level group (P < 0.05 and P  0.05, Fig.  3 C–I). These results revealed that Gln and NE in follicular fluid may play important roles in estrogen synthesis and the production. Figure 3 Metabolic levels of steroid hormones measured by HPLC–MS/MS in human follicular fluid. The graphs show kinds of hormone concentrations in human follicular fluid between high and low glutamine (Gln) level groups, including estriol ( A ), 17β-estradiol ( B ), progesterone ( D ), methyl testosterone ( E ), (D)-norgestrel ( F ), 19-nortestosterone ( G ), epitestosterone ( H ) and methoxy progesterone ( I ). ( C ) The column chart shows the concentration of testosterone in the follicular fluid in the two groups, which was measured by a chemiluminescence immunoassay. Data represent the mean values ± standard error of the mean (mean ± SEM). High Gln level group, n = 14; Low Gln level group, n = 13. All indicated P values were determined by Student's t-test. * P value < 0.05; *** P value < 0.001. Metabolic levels of steroid hormones measured by HPLC–MS/MS in human follicular fluid. The graphs show kinds of hormone concentrations in human follicular fluid between high and low glutamine (Gln) level groups, including estriol ( A ), 17β-estradiol ( B ), progesterone ( D ), methyl testosterone ( E ), (D)-norgestrel ( F ), 19-nortestosterone ( G ), epitestosterone ( H ) and methoxy progesterone ( I ). ( C ) The column chart shows the concentration of testosterone in the follicular fluid in the two groups, which was measured by a chemiluminescence immunoassay. Data represent the mean values ± standard error of the mean (mean ± SEM). High Gln level group, n = 14; Low Gln level group, n = 13. All indicated P values were determined by Student's t-test. * P value < 0.05; *** P value < 0.001. Oxidative metabolism is the major oxygen consumer in the follicle, which could be affected by amino acid metabolism 27 . Antioxidants are indicated as factors that can maintain the balance between ROS reproduction and clearance. Then, we detected the expression levels of the antioxidant genes in GCs via qRT-PCR between the two groups. In the group of high Gln level, antioxidant gene IDH1 and glutathione peroxidase 1 ( GPX1 ) increased significantly in GCs compared with that in group of low Gln level (P < 0.001 and P < 0.0001, Fig.  4 A,B). Moreover, the expression of nuclear factor erythroid 2-related factor 2 ( NRF2 ) and its endogenous inhibitor, kelch-like epichlorohydrin-associated protein-1 ( KEAP1 ), significantly increased in the high Gln level group (P < 0.01, Fig.  4 C,D). Notably, the expression level of IDH1 mRNA in GCs showed a positive correlation with the Gln concentration in follicular fluid (R = 0.3822, P < 0.05, Fig.  4 E). Furthermore, there was a positive correlation between the NE concentration in follicular fluid and IDH1 expression in GCs (R = 0.4009, P < 0.05, Fig.  4 F). To further clarify whether this difference in the expression of antioxidative genes was caused by individual differences, we summarized the clinical data of the IVF patients as Table 2 and found that there were no statistically significant differences in age (P = 0.1601), body mass index (BMI, P = 0.5927), serum Anti-Mullerian Hormone (AMH, P = 0.6033) or thyrotropin (TSH, P = 0.8698) between these two groups. These results demonstrated that the contents of Gln and NE in follicular fluid might increase the antioxidant capacity of GCs. Figure 4 The expression levels of the antioxidative genes in the cumulus granulosa cells between high and low glutamine (Gln) level groups. ( A – D ) The column charts demonstrate the expression of antioxidant genes in the two groups, including IDH1, GPX1, NRF2, and KEAP1. ( E ) Correlation analysis of Gln concentration in follicular fluid and IDH1 mRNA expression in cumulus granulosa cells (GCs) (n = 27, R = 0.3822, P < 0.05). ( F ) Correlation analysis of norepinephrine (NE) concentration in follicular fluid and IDH1 mRNA expression in cumulus GCs (n = 27, R = 0.4009, P < 0.05). Data represent the mean values ± standard error of the mean (mean ± SEM). High Gln level, n = 14; Low Gln level, n = 13. All indicated P values were determined by Student's t test. ** P value < 0.01; *** P value < 0.001; **** P value < 0.0001. Table 2 Characteristics of patients assessed for follicular fluid analysis. Characteristics High level of glutamine Low level of glutamine P-value (n = 14) (n = 13) Age (years) 31.00[28.75–36.00] 31.00[26.50–33.00] 0.1601 BMI (Kg/m 2 ) 21.16[19.52–23.00] 19.95[18.31–21.52] 0.5927 Duration of infertility 2.50[1.00–4.63] 1.00[1.00–3.00] 0.0472 Previous pregnancy 2.00[1.00–3.00] 0[0–1.00] 0.0164 Previous delivery 0[0–0.25] 0[0–0] 0.0820 AMH (ng/ml ) 2.35[1.29–4.29] 2.90[1.93–3.81] 0.6033 TSH (mIU/L ) 1.91[1.45–3.03] 2.18[1.83–2.70] 0.8698 Antral follicle count 9.50[5.75–12.25] 9.00[8.50–12.50] 0.9911 Indication for IVF 0.3289 Idiopathic 1(7.14%) 1(7.69%) Male factor 1(7.14%) 0(0) Endometriosis 4(28.58%) 2(15.38%) Tube factor 6(42.86%) 6(46.15%) Multiple 2(14.28%) 4(30.77%) Data are median (interquartile range (IQR)) or numeric value (%). AMH anti-Mullerian hormone. The expression levels of the antioxidative genes in the cumulus granulosa cells between high and low glutamine (Gln) level groups. ( A – D ) The column charts demonstrate the expression of antioxidant genes in the two groups, including IDH1, GPX1, NRF2, and KEAP1. ( E ) Correlation analysis of Gln concentration in follicular fluid and IDH1 mRNA expression in cumulus granulosa cells (GCs) (n = 27, R = 0.3822, P < 0.05). ( F ) Correlation analysis of norepinephrine (NE) concentration in follicular fluid and IDH1 mRNA expression in cumulus GCs (n = 27, R = 0.4009, P < 0.05). Data represent the mean values ± standard error of the mean (mean ± SEM). High Gln level, n = 14; Low Gln level, n = 13. All indicated P values were determined by Student's t test. ** P value < 0.01; *** P value < 0.001; **** P value < 0.0001. Characteristics of patients assessed for follicular fluid analysis. Data are median (interquartile range (IQR)) or numeric value (%). AMH anti-Mullerian hormone. To determine whether follicular development and oocyte quality were different between the high Gln level and low Gln level groups, we compared several clinical indices in the two groups, including the number of mature follicles (diameters ≥ 14 mm) on the human chorionic gonadotrophin (HCG) injection day, the number of retrieved oocytes, the fertilization rate, and the number and percentage of higher-quality embryos. There were no significant differences between the two groups in the above parameters (P > 0.05, Fig.  5 A). Multiple linear regressions were used to identify the relationship between the Gln/NE concentration in follicular fluid and the ratio of higher-quality/cleaved embryos, and IDH1 expression in GCs and the ratio of higher-quality/cleaved embryos after adjusting for spouse age, sperm density, and sperm motility. Results showed that there were no significant correlations between the Gln/NE concentration in follicular fluid and the ratio of higher-quality/cleaved embryos (R = 0.2430 and R = 0.1243, P > 0.05, Fig.  5 B,C). However, a significantly positive correlation was observed between IDH1 gene expression in cumulus GCs and the ratio of higher-quality/cleaved embryos (R = 0.4480, P < 0.05, Fig.  5 D). These results demonstrated that the IDH1 expression in cumulus GCs may be related with the quality of oocyte and embryo development in IVF patients. Figure 5 The correlation between glutamine (Gln) or norepinephrine (NE) concentration in follicular fluid and the outcome of in vitro fertilization patients. ( A ) Analysis of the treatment variables in the patients between the two groups. ( B ) Correlation analysis of the Gln concentration in the human follicular fluid and the ratio higher-quality/cleaved embryos (n = 27, R = 0.2430, P > 0.05). ( C ) Correlation analysis of the NE concentration in the human follicular fluid and the ratio of higher-quality/cleaved embryos (n = 27, R = 0.1243, P > 0.05). ( D ) A positive correlation between IDH1 gene expression in cumulus granulosa cells (GCs) and the ratio of higher-quality/cleaved embryos (n = 27, R = 0.4480, P < 0.05). The correlation between glutamine (Gln) or norepinephrine (NE) concentration in follicular fluid and the outcome of in vitro fertilization patients. ( A ) Analysis of the treatment variables in the patients between the two groups. ( B ) Correlation analysis of the Gln concentration in the human follicular fluid and the ratio higher-quality/cleaved embryos (n = 27, R = 0.2430, P > 0.05). ( C ) Correlation analysis of the NE concentration in the human follicular fluid and the ratio of higher-quality/cleaved embryos (n = 27, R = 0.1243, P > 0.05). ( D ) A positive correlation between IDH1 gene expression in cumulus granulosa cells (GCs) and the ratio of higher-quality/cleaved embryos (n = 27, R = 0.4480, P < 0.05). Previous studies have reported that Gln is required for oocyte maturation and embryo development 28 and adrenergic activity through NE participates in the control of follicular development and steroidal secretion 29 . To study the roles of Gln and NE in GCs, the proliferation of cultured KGN cells in vitro was evaluated via EdU staining and western blotting. Fluorescent staining showed that the percentage of EdU-positive cells was dramatically reduced in the Gln deprivation group. The addition of NE partially increased the ratio of EdU-positive KGN cells. Importantly, Gln supplementation increased significantly the EdU-positive number (P < 0.0001, Fig.  6 A,B). Moreover, the concentrations of E2 and Prog in the cell culture supernatant were measured by ELISA. The results showed that the concentration of E2 was reduced in the Gln deprivation group, and adding Gln to the medium significantly increased E2 production in KGN cells, which could be further improved by NE supplementation (P < 0.01, Fig.  6 C). Although Gln deprivation reduced the level of Prog in the cell supernatant, Gln and NE supplementation had no obvious effect on Prog production in KGN cells (P < 0.05, Fig.  6 D). The expression of PCNA protein in KGN cells exhibited similar trends to the morphological results in the different treatment groups. Notably, Gln deprivation obviously reduced IDH1 protein expression in GCs, which could be recovered by NE and Gln supplementation (P < 0.0001, P < 0.01 and P < 0.05, Fig.  6 E–G). These results revealed that the neurotransmitters Gln and NE could synergistically enhance the cell proliferation of GCs and increase the E2 production. Figure 6 Effect of glutamine (Gln) and norepinephrine (NE) on the function of granulosa cells in vitro ( A ) Immunofluorescent staining of EdU and DAPI in KGN cells for proliferation analysis. ( B ) The column chart shows EdU-positive cells per 100 cells in different treatment groups. ( C , D ) Column displays the concentrations of E2 and Prog in the conditioned medium from different treatments of KGN cells. ( E – G ) Evaluation of the expression of IDH1 and PCNA protein in different treatment groups by western blotting. All indicated P values were determined by one-way analysis of variance. * P value < 0.05; ** P value < 0.01; **** P value < 0.0001. Effect of glutamine (Gln) and norepinephrine (NE) on the function of granulosa cells in vitro ( A ) Immunofluorescent staining of EdU and DAPI in KGN cells for proliferation analysis. ( B ) The column chart shows EdU-positive cells per 100 cells in different treatment groups. ( C , D ) Column displays the concentrations of E2 and Prog in the conditioned medium from different treatments of KGN cells. ( E – G ) Evaluation of the expression of IDH1 and PCNA protein in different treatment groups by western blotting. All indicated P values were determined by one-way analysis of variance. * P value < 0.05; ** P value < 0.01; **** P value < 0.0001.

Follicular fluid is an important external environment for the oocyte growth and follicular development. Ideally, follicular fluid, a superfluous, abundant and easily available biological sample in IVF cycles, would be the optimum source of noninvasive prediction of oocyte quality 30 . Currently, investigations of follicular fluid biomarkers for oocyte quality mainly focus on measuring steroid hormones secreted by GCs, such as estradiol and progesterone 31 – 33 . Therefore, exploring the relationship between metabolites in follicular fluid and granulosa cell function may be conducive to search reliable biomarkers for assessing oocyte quality and predicting the outcome of IVF patients. Metabolic approach is a powerful tool to analyze the complexities of follicular fluid. Previous study reported the application of gas chromatography-mass spectrometry (GC–MS) to detect polybrominated diphenyl ethers (PBDEs) in follicular fluid and found that the presence of PBDEs could be associated with failed embryo implantation 34 . In the current study, a more simple, fast and efficient LC–MS/MS technology was successfully applied for determination of simultaneous neurotransmitter metabolites in human follicular fluid. Consistent with other studies, we found that many different neurotransmitter metabolites exist in human follicular fluid as shown in Table 1 . Among them, high levels of Gln and NE have attracted our attention; there was a positive relationship between the contents of Gln and NE in follicular fluid. Currently, extensive scientific attention has been given to an important metabolite Gln, which is one of the most abundant amino acid in the human body, such as skeletal muscles, plasma 35 and follicular fluid 36 . It has anti-inflammatory and antioxidant functions as well as effects that modulate the heat-shock protein response during stress; moreover, as a precursor of glutamate, Gln participates in multiple neurotransmitter metabolic pathways 35 , 37 , 38 . In addition, peripheral neurotransmitter NE participates in the control of steroidal secretion from the ovary and follicular development 29 . In this study, we found that the proliferative ability and steroidogenesis of cumulus GCs increased with the high contents of Gln/NE in follicular fluid, which demonstrating that Gln and NE might promote granulosa cell proliferation and up-regulate the expression of steroid hormone syntheses genes. An altered follicular microenvironment may directly damage follicle recruitment and growth. Previous studies show that metabolites in follicular fluid are direct or indirect regulators of oxidative stress and the function of GCs 39 . Gln as the main energy supply substance for mitochondria to form ATP, is an important source of energy for oocyte maturation and the development of embryo 36 . A study reported that human ovarian tissue was cryopreserved using freezing medium supplemented with cryoprotectants and antioxidants ( l -glutamine and taurine) and exhibited good preservation of ovarian vascular integrity and functionality of GCs post thawing 40 . Furthermore, the Gln supplementation could effectively alleviate inflammatory reaction and oxidative stress in PCOS rats 41 . Thus, the relationships among the levels of Gln in follicular fluid, cumulus GC function, and oocyte development are worthy of further study. Excessive ROS damages DNA, reduces cell proliferation and induces mitochondrial-mediated apoptosis in GCs, which further affect oocyte development 42 , 43 . Studies have demonstrated that there is a correlation between the apoptosis of GCs and poor oocyte quality and IVF outcomes in patients with PCOS 9 , 44 . Many environmental stressors may cause inferior oocyte competence and alter oocyte development and growth by disturbing GC function 43 , 45 , 46 . In the current study, we found that Gln and NE not only upregulated IDH1 gene expression but also increased GPX1 expression in cumulus GCs. Notably, the concentrations of Gln and NE in follicular fluid were positively related to IDH1 gene expression in cumulus GCs, which could be a predictor of high-quality embryos. The relation of Gln and NE in follicular fluid and antioxidant gene expression in cumulus GCs will provide more valuable information to understand the molecular mechanism of follicular development. There are several limitations in current study. First, the number of clinical samples was insufficient. Although the number of samples in the two groups met the statistical requirements, the limited number of samples influenced the subsequent analysis of reproductive outcomes of IVF patients. The second limitation was that only antioxidant genes expressions were assayed in cumulus GCs, however, other oxidative stress makers including ROS, MDA, GSH did not detected due to lack of enough samples. Finally, only KGN cells were used to confirm the roles of Gln and NE in maintaining granulosa cell function in vitro experiments. Whether appropriate supplementation of Gln and/or NE in animal models with ovarian dysfunction could improve fertility outcomes needs to be explored in future studies.

In summary, our research demonstrates that the positive association between Gln/NE content in follicular fluid and the function of human cumulus GCs. Moreover, intrafollicular Gln and NE could upregulate the expression of the antioxidative gene IDH1 in cumulus GCs, which may be a potential predictor for evaluating the quality of embryonic development in the future.

Infertility is a reproductive system disease characterized by failure to establish a clinical pregnancy after normal unprotected sexual intercourse for 12 months or more, or impairment of the reproductive capacity of an individual or with his or her partner 1 , 2 . Infertility has been recognized as a worldwide public health problem by the World Health Organization (WHO), and approximately half of these cases lack an definitive explanation for pregnancy failure 3 . It is estimated that in western countries, one in six couples has infertility, which is recognized as the cause of psychological distress in both men and women 4 . In vitro fertilization (IVF) offers a possibility for achieving pregnancy, but the success rate is still suboptimal 5 , 6 . As an important biofluid, follicular fluid provides a special microenvironment for follicular development and impacts oocyte quality, implantation and early embryo development. Follicular fluid is rich in proteins, including sex hormones, nutrient substances, cytokines, growth factors and neurotransmitters, which is critical for oocyte development 7 . Cumulus GCs represent the predominant somatic cell type of follicle development and are involved in steroidogenesis and folliculogenesis. Furthermore, they connect with oocytes via direct gap junctions to support the developing oocyte 8 – 10 . The process of follicular development involves local biochemical exchanges and substantial modifications in cellular metabolism. Amino acids serve as substrates for the synthesis of proteins, not only enhance embryonic development but also affect blastocyst formation in mice. A study has demonstrated that Glycine (Gly), Glutamine (Gln), alanine (Ala), Glutamate (Glu) and Proline (Pro) are the most abundant amino acids in follicular fluid 11 . The disturbance of follicular amino acid metabolism is related to women with polycystic ovary syndrome (PCOS), which may be the reason for the poor pregnancy outcome in obese patients and increased risk of abortion in PCOS patients 12 . In addition, they are not only components of proteins, but also regulate neurotransmitter homeostasis, such as a metabolite shuttle known as the glutamate/GABA-glutamine cycle describes the release of neurotransmitter glutamate or GABA from neurons and subsequent uptake into astrocytes, play import roles in nervous system 13 , 14 . However, whether the neurotransmitters metabolic levels provided by follicular fluid influences the function and hormone synthesis of cumulus GCs need to be further investigated. Oxidative stress plays an important role in reproductive processes, including follicular development, oocyte maturation, ovulation and fertilization, as well as embryo implantation and embryo development 15 . Excessive generation of ROS, if not efficiently counterbalanced by antioxidative enzymes, may cause damage to oocytes and GCs, leading to poor oocyte quality 9 . In our previous study, psychological stress-induced oxidative stress decreased the expression of the antioxidant gene IDH1 to disturb and compromise the function of GCs, accelerating ovarian senescence 16 . Antioxidant supplementation in IVF culture medium could increase fertilization rates and subsequent healthy embryo development 17 . Previous studies reported that NE and its metabolites could induce the generation of reactive oxygen species (ROS) in human GCs 18 . Once the level of NE concentration exceeds the physiological concentration, it may lead to ROS-related pathophysiology events including oxidative stress and cell death 19 . However, little is known about the impact of neurotransmitters in follicular fluid on oxidative capacity of human GCs. During the process of follicle growth to ovulation, which is mediated by the preovulation production of gonadotropins, metabolic profiles change dynamically in follicular fluid. Thus, a better understanding of the neurotransmitters metabolic levels in follicular fluid is important for exploring the metabolic cooperativity between oocytes and cumulus GCs. In this study, we quantified the neurotransmitters metabolites and steroid hormones in follicular fluid from IVF patients by LC–MS/MS and HPLC–MS/MS technology, and evaluated the impact of Gln and NE in follicular fluid on the function and antioxidative capacity of cumulus GCs, which could be related with the clinical outcome of IVF patients. Additional in vitro cell experiments were performed to further explore the effects of Gln and NE on the proliferation and steroidogenesis of GCs.

Supplementary Figure. Supplementary Table. Supplementary Figure. Supplementary Table.