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

An increasing number of studies demonstrate that changes in neurotransmitters metabolic levels in follicular fluid are directly related to oocyte maturation, fertilization, the quality of embryo and pregnancy rates. However, the relationship between the intra-follicular neurotransmitters and the function of granulosa cells (GCs), and the outcome of in vitro fertilization-embryo transfer (IVF-ET) is not clear. Human follicular fluid and cumulus GCs were harvested from large follicles obtained from patients undergoing IVF. Neurotransmitters and steroid hormones in follicular fluid were measured through liquid chromatography-tandem mass spectrometry (LC–MS/MS) and high-performance liquid chromatography-mass spectrometry (HPLC–MS/MS). Based on the content of glutamine (Gln) in follicular fluid, the samples were divided into two groups: high Gln level group and low Gln level group. The expression of proliferation-, steroidogenesis- and antioxidant-related genes in GCs was detected by qRT-PCR. In vitro, KGN cells were used to further verify the effects of Gln and NE on GCs function. Primary and secondary outcomes were the number of mature and retrieved oocytes, and the ratio of high-quality embryos, respectively. Gln (46.75 ± 7.74 μg/mL) and norepinephrine (NE, 0.20 ± 0.07 μg/mL) were abundant neurotransmitters in follicular fluid, and exhibited a significantly positive correlation (R = 0.5869, P < 0.005). In high Gln level group, the expression of proliferation, steroidogenesis and antioxidant-related genes in GCs were higher than those in low Gln level group, and the contents of estriol and E2 in follicular fluid were more abundant. Moreover, the concentrations of Gln and NE in follicular fluid showed significantly positive correlation with IDH1 expression in GCs (R = 0.3822, R = 0.4009, P < 0.05). Importantly, a significantly positive correlation was observed between IDH1 expression in GCs and the ratio of higher-quality/cleaved embryos (R = 0.4480, P < 0.05). In vitro studies further demonstrated that Gln and NE played synergistically function in improving GCs proliferation and E2 production by upregulating IDH1 expression. These data demonstrate that Gln and NE in follicular fluid might play significant positive roles in GCs function, and may be potential predictors for selecting optimal quality oocytes and evaluating the quality of embryonic development.

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][9][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.

Methods
Study design. 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.
Collection of GCs and follicular fluid. 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.
Embryo quality assessment. 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. 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). www.nature.com/scientificreports/ RNA extraction and qRT-PCR. 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 Hormone metabolic levels in follicular fluid. 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 highperformance 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 . EdU staining. The proliferation of KGN cell was evaluated by EdU staining (RiboBio, Guangdong, China).

Quantification of neurotransmitters metabolites in follicular fluid by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
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.
Statistical analysis. 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.
Ethics approval and informed consent. Informed

Results
Glutamine is positively correlated with norepinephrine content in follicular fluid of IVF patients. 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 < 0.005, Fig. 1A). However, there was no relationship between the contents of Gln and Glu in follicular fluid (R = 0.2374, P > 0.05, Fig. 1B). 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).

Effect of intra-follicular glutamine/norepinephrine on the function of granulosa cells. 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. 2A,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. 2C,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. 2E-H). However, there was no significant difference in hydroxysteroid dehydrogenase-17 beta (HSD-17β mRNA levels between the two groups (P > 0.05,

Glutamine/norepinephrine promote estrogen synthesis in follicular fluid of IVF patients.
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 < 0.001, Fig. 3A,B). There were no statistically significant differences in other hormone contents between the two groups (P > 0.05, Fig. 3C-I). These results revealed that Gln and NE in follicular fluid may play important roles in estrogen synthesis and the production.

Glutamine/norepinephrine in follicular fluid increase the expression of antioxidant genes in granulosa cells.
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. 4A,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. 4C,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. 4E). 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. 4F). 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.

IDH1 expression in granulosa cells is positively related with the quality of embryo development of IVF patients.
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 higherquality embryos. There were no significant differences between the two groups in the above parameters (P > 0.05, Fig. 5A). 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. 5B,C). However, a significantly positive correlation was observed between IDH1 gene expression in cumulus GCs and the ratio of higherquality/cleaved embryos (R = 0.4480, P < 0.05, Fig. 5D). These results demonstrated that the IDH1 expression in cumulus GCs may be related with the quality of oocyte and embryo development in IVF patients.  www.nature.com/scientificreports/

Glutamine/norepinephrine improve the function of granulosa cells in vitro. 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. 6A,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. 6C). 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. 6D). The expression of PCNA protein in KGN cells exhibited similar trends to the morphological results in the different treatment groups. Notably, www.nature.com/scientificreports/ 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. 6E-G). These results revealed that the neurotransmitters Gln and NE could synergistically enhance the cell proliferation of GCs and increase the E2 production.

Discussion
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][32][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  www.nature.com/scientificreports/   www.nature.com/scientificreports/ 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 www.nature.com/scientificreports/ 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.

Conclusions
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.