{"paper_id":"f98ef6a8-bc03-4599-bbc2-1a7ddb34eddf","body_text":"1Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreports\nDetection of t lymphocyte subsets \nand related functional molecules \nin follicular fluid of patients with \npolycystic ovary syndrome\nZitao Li1, Anping peng2, Yuanfa Feng3, Xiaona Zhang4, Fenghua Liu1, Chuangqi Chen1, Xin Ye3, \nJiale Qu3, Chenxi Jin3, Mei Wang3, Huaina Qiu3, Yanwei Qi3, Jun Huang  3,5 & Quan Yang1,3,5\nImmune responses play an important role in the pathogenesis of polycystic ovary syndrome (pCos). \nHowever, the characteristics of T lymphocyte subsets in PCOS remain insufficiently understood. In \nthis study, lymphocytes of follicular fluid (FF) were obtained from oocyte retrieval before in-vitro \nfertilization (IVF) in infertile women with or without PCOS. The levels of cluster of differentiation 25 \n(CD25), CD69, programmed death 1 (PD-1), interferon-γ (IFN-γ), interleukin 17A (IL-17A) and IL-10 in T \nlymphocytes were determined by flow cytometry. Our results showed that the percentage of FF CD8\n+ \nT cells was significantly decreased in infertile patients with PCOS (P < 0.05). Furthermore, the levels of \nCD69 and IFN-γ were significantly decreased and the level of PD-1 was increased in both CD4+ and CD8+ \nT cells from infertile patients with PCOS (P < 0.05). Moreover, the expression of PD-1 on CD4+ or CD8+ \nT cells was positively correlated with the estradiol (E2) levels in the serum and reversely correlated with \nthe expression of IFN-γ in CD4\n+ or CD8+ T cells in infertile patients with PCOS. These results suggested \nthat t cell dysfunction may be involved in the pathogenesis of pCos.\nPolycystic ovary syndrome (PCOS), as a common female endocrinopathy at reproductive age, is a heterogene-\nous condition characterized by clinical symptoms, including reproductive, cardiometabolic, and psychological \ndisorders\n1,2. Patients with PCOS are at a significantly higher risk for the development of endometrial, breast and \novarian cancers and symptomatic atherosclerotic cardiovascular diseases (CVD)3,4. Other manifestations include \nhyperinsulinism, insulin resistance, obesity, diabetes, hirsutism, endothelial dysfunction, and a state of low-grade \ninflammation\n5–9. Besides, recent study have reported that insulin resistance, compensatory hyperinsulinemia \nand increased androgen production have potential effected on the pathogenesis of PCOS10,11. Although previous \nstudies show that both environmental and genetic factors play roles in the etiology of PCOS12,13, the pathogenesis \nof PCOS is not fully understood.\nThe immune system is a defense system, which comprises many biological structures that protect the host \nagainst disease. Once the body’s immune system is dysfunctional, it can lead to various diseases. A recent study \nhas reported that immunological mechanisms are involved in the regulation of polycystic ovary syndrome\n14. \nPatients with PCOS have been found to be under a chronic low-grade inflammation status, including high levels \nof leukocytes, endothelial dysfunction, and disorder of the proinflammatory cytokines\n15–17. Large amounts of \nimmunocompetent cells, including T cells, B cells, macrophages and dendritic cells, have been found in human \npreovulatory follicles\n18–20. As the main component of lymphocytes, T cells have various biological functions, \nwhich are mainly involved in the cellular immune response of the body. They can kill target cells directly or \nthrough the release of lymphatic factor to enhance and expand the immune effect\n21,22. According to different \n1Reproductive medical center, Guangdong Women and Children Hospital, Guangzhou Medical University, 511400, \nGuangzhou, china. 2Clinical laboratory, Traditional Chinese Medicine Hospital of Guangdong province, 510120, \nGuangzhou, c hina. 3Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, \nGuangzhou Medical University, 511436, Guangzhou, China. 4The Sixth Affiliated Hospital of Sun Yat-Sen University, \n510655, Guangzhou, China. 5State Key Laboratory of Respiratory Disease, Guangzhou i nstitute of Respiratory \nHealth, The First Affiliated Hospital of Guangzhou Medical University, 510120, Guangzhou, China. Zitao Li, Anping \nPeng and Yuanfa Feng contributed equally. Correspondence and requests for materials should be addressed to J.H. \n(email: hj165@sina.com) or Q.Y . (email: yquangy2015@gzhmu.edu.cn)\nReceived: 19 September 2018\nAccepted: 4 April 2019\nPublished: xx xx xxxx\nopeN\n\n\n2Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nfunctions, T cells can be classified into three subtypes: helper T cells (CD3 +CD4+ Th) and cytotoxic T cells \n(CD3+CD8+ Tc). CD4+ T helper cells can be subdivided into different subsets, including Th17 cells, which pro-\nduce IL-17A, and IL-17F , Th1 cells, which produce IFN-γ, IL-2, and TNF-α , Th2 cells, which secrete IL-4, IL-5, \nand IL-13, and Regulatory T cells, which express Foxp3, IL-10, and TGF-β23. Although T cells have been reported \nto exist in pre-ovulation follicles in humans and the interaction between subtypes is very active24,25, the role of T \nlymphocyte subsets in the pathogenesis of PCOS remains unclear.\nAlthough substantial evidence has indicated that inflammation, as well as immune regulation might play \nimportant roles in the cause of PCOS, the underlying regulatory mechanisms have remained unclear14,15. The aim \nof this study is to investigate the subpopulations and related functional molecules of T lymphocytes in the FF of \ninfertile women with or without PCOS. Our results will provide a better understanding of the immunoregulatory \nmechanism in the pathogenesis of PCOS.\nMethods\nethics statement. The research followed the tenets of the Declaration of Helsinki. Informed consents were \nobtained from all patients. And all the enrolled patients participated in the research voluntarily and freely. Our \nresearch were approved by the Institutional Review Board (IRB approval number: 201701042) of the Guangdong \nWomen and Children Hospital. Our study conformed to the international guidelines available through the \nEnhancing the QUAlity and Transparency Of health Research (EQUATOR) network.\npatient characteristics. Sixty-six primary infertile women undergoing in vitro fertilization (IVF) or intra-\ncytoplasmic sperm injection (ICSI) were enrolled in the study (age range: 23–37 years). Among all patients, \n36 cases enrolled in the study were normally ovulating women (NOW , group A) and 30 cases were affected by \nPCOS (group B). Patients affected by other significant gynecological and non-gynecological comorbidities were \nexcluded. Before admission to the study, each woman underwent clinical and transvaginal ultrasonography. Basic \nsexual hormones, including estradiol (E2), androstenedione (A), progesterone (P), testosterone (T), cortisol, lute-\ninizing hormone (LH) and follicle stimulating hormone (FSH), were evaluated. The characteristics of all patients \nare summarized in Table 1.\nThe control group and PCOS group were in line with the normal distribution tested with SPSS. The inclusion \ncriteria in group A were: the absence of endocrinological disorders of the pituitary or ovary, such as hyperprolac-\ntinemia, hypogonadotropic hypogonadism, premature ovarian failure and premature menopause, or of abnormal \nadrenal or thyroid function. Previously reported criteria for PCOS were employed\n26–28, which include at least two \nof the following three criteria: 1. Oligo- and/or anovulation; 2. Clinical and/or biochemical signs of hyperandro-\ngenism; or 3. Polycystic ovaries (presence of 12 or more follicles in each ovary measuring 2 ± 9 mm in diameter \nand/or increased ovarian volume), as well as the exclusion of nonclassic congenital adrenal hyperplasia, Cushing’s \nSyndrome, hyperprolactinemia and thyroid diseases.\nsample size. In our study, a sample size of 30 cases in PCOS group and 36 cases in NOW group with infer-\ntility was obtained from the two groups whose T cell subsets frequencies were compared. We performed a sam-\nple size calculation according to two independent design data calculation formulas post-hoc. We calculated the \nsample size according to the difference between the two groups of T cells and the power of this study was 0.8. The \nresult we got was that each group needed 35 cases. We tried our best to collect the total number of cases close \nto our expected sample size. Due to the large number of testing items, we have collected relatively few complete \ntesting cases. However, the data were true and reliable. Owing to the small sample size of our study, we could not \nexclude that a type 1 error might occur in our statistical analysis.\nControlled ovarian hyperstimulation (C o H). In PCOS patients with infertility, rFSH [Gonal-F alfa \n(Merck Serono, Geneva, Switzerland) or Puregon beta (MSD, New Jersey, USA)] treatment was initiated on men-\nstrual cycle day 2 or day 3. The starting doses were 112.5–300 IU per day selected based on the age, circulating \nbasal FSH level and BMI of patients. The rFSH doses were adjusted according to growing follicles and E2 con-\ncentration during the stimulation monitoring. The GnRH antagonist treatment (Ganirelix 0.25 mg, Orgalutran\n®, \nOrganon, Italy) was initiated on stimulation day 5–7 as the growing follicles 10–12 mm in diameter.When at least \nthree dominant follicles (diameter ≥ 17 mm) were observed by ultrasound, 250 μg rhCG (Choriogonadotropin \ngroup B \n(PCOS, n = 30)\ngroup A \n(NOW , n = 36) t value p value\nAge (year) 30.30 ± 6.23 29.50 ± 4.22 0.345 0.731\nFSH (IU/L) 6.15 ± 1.95 6.92 ± 1.93 −1.297 0.199\nLH (mIU/L) 10.78 ± 5.91 5.54 ± 3.65 3.049 0.008\nLH/FSH 1.75 ± 0.69 0.85 ± 0.58 4.826 <0.001\nE2 (pg/L) 58.70 ± 52.75 53.57 ± 56.06 0.299 0.766\nT (ng/mL) 0.61 ± 0.48 1.08 ± 4.95 −0.341 0.734\nTable 1. Comparison of related indicators for two groups. *p-values reported are the results of independent-\nsample t-tests or γ2 tests for dichotomous variables; x ± s; *p < 0.05. PCOS: polycystic ovary syndrome; NOW: \nnormally ovulating women; FSH: follicle stimulating hormone; LH: luteinizing hormone; E2: estradiol; T: \ntestosterone.\n\n3Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nalfa, Merck Serono, Geneva, Switzerland) was administered to trigger the final oocyte maturation. Oocyte \nretrieval was performed 34–36 hour after rhCG administration. One or two embryos were transferred 3–5 days \nafter oocyte retrieval. Luteal phase progesterone support (Progesterone Vaginal Gel, Merck Serono, Geneva, \nSwitzerland) was provided.\nFollicular fluid aspiration. Individual follicles were measured before aspiration in two dimensions. When \nthe leading follicle was calculated greater than or equal to 17 mm in diameter, 250 μg of human chorionic gonado-\ntropin (hCG) (Merck Serono, Geneva, Switzerland) were administered to induce ovulation. Transvaginal follicu-\nlar aspiration was scheduled after 36 h and performed under general anesthesia. The follicles were aspirated with \na 16-gauge single lumen needle, and each follicle was emptied completely. The follicular fluid (FF) of the leading \nfollicle was collected into a dry tube (without medium) (BD Falcon #352057; BD Biosciences, Boston, MA). In the \nlaboratory, the cumulus oophorus complex was isolated, and the follicular fluid was subsequently centrifuged at \n600 × g. The pellet was resuspended in 1 ml of RPMI 1640 medium (Invitrogen Life Technologies, Grand Island, \nNY , USA) and placed into a tube for further experiments. The experiments were performed within 6 hours.\nCell surface staining. This experiment was performed following previously described procedures 29. \nSingle-cell suspensions from the follicular fluid of infertile women with and without PCOS were adjusted to \n0.3 × 10\n6/ml, washed twice in phosphate-buffered saline (PBS) (Invitrogen Life Technologies, Grand Island, NY , \nUSA) and blocked in PBS buffer that contained 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, \nMO, USA) for 30 min. The cells were then stained for 30 min at 4 °C in the dark with conjugated antibodies spe-\ncific for the following cell surface antigens: anti-CD3 PerCP , anti-CD4 FITC, anti-CD8 PE, anti-CD25 PE-CY7, \nanti-CD69 APC and anti-PD-1 Brilliant Violet 421 (eBioscience, San Diego, CA, USA). The phenotypic charac-\nteristics of the antibody-labeled lymphocytes were analyzed using flow cytometry (Beckman Coulter, Fullerton, \nCA, USA), and the results were analyzed using FlowJo version 6.0 software (TreeStar Inc., Ashland, OR, USA). \nIsotype-matched controls were included in each staining protocol.\nIntracellular cytokine staining. Previously described procedures were employed 30. Cells (1 × 106/ml) \nfrom the follicular fluid of infertile women with and without PCOS were stimulated with propylene glycol mon-\nomethyl acetate (PMA) (at a final concentration of 20 ng/ml, Sigma-Aldrich, St. Louis, MO, USA) plus iono-\nmycin (at a final concentration of 1 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) for 5 hours at 37 °C under a 5% \nCO\n2 atmosphere. Brefeldin A (a final concentration of 10 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) was added \nduring the last 4 hours of incubation. The cells were washed twice in PBS and then stained for 30 min at 4 °C in \nthe dark with conjugated antibodies specific for cell surface antigens: anti-CD3 PerCP , anti-CD4 FITC, anti-CD4 \nPE-cy5, anti-CD8 PE, and anti-CD8 FITC (eBioscience, San Diego, CA, USA). The cells were washed twice in \nPBS again, fixed with 4% paraformaldehyde and permeabilized overnight at 4 °C in PBS buffer that contained \n0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA), 0.1% BSA and 0.05% NaN\n3 (Sigma-Aldrich, St. Louis, MO, \nUSA). The cells were then stained for 30 min at 4 °C in the dark with conjugated antibodies specific for cytokines: \nanti-IFN-γ APC, anti-IL-10 PE-cy7, anti-IL-4 PE, and anti-IL-17A APC-CY7 (eBioscience, San Diego, CA, USA). \nThe expressions of cytokines secreted by antibody-labeled lymphocytes were analyzed using a FACS, and the \nresults were analyzed using FlowJo version 6.0 software. Isotype-matched controls for cytokines were included \nin each staining protocol. For the staining of Foxp3, the Foxp3/Transcription Factor Staining Buffer Set and the \nconjugated antibodies specific for Foxp3, anti-Foxp3 APC (eBioscience, San Diego, CA, USA), were used. After \nstaining, the cells were washed and resuspended in PBS for flow cytometric analysis using a FACS. The data were \nthen analyzed using FlowJo version 6.0 software.\nCytometric bead array (CBA). Previously described procedures were employed17. Cells were isolated from \nthe follicular fluid of infertile patients with PCOS or NOW and resuspended in 200 μl of RPMI 1640 medium. \nThe cells were then stimulated with 20 ng/ml PMA and 1 μg/ml ionomycin and incubated for 48 h. The levels of \nIFN-γ, IL-10, IL-4 and IL-17A in the cell culture supernatants were analyzed using a cytometric bead array kit \n(CBA) (Human Th1/Th2/Th17 Cytokine Kit, Becton Dickinson, San Jose, CA). Briefly, the supernatants were \nharvested and stored at −80 °C until cytokine determination. Then, 50 μl of each sample was mixed with 50 μl of \nmixed capture beads and 50 μl of the human Th1/Th2/Th17 PE detection reagent that consisted of PE-conjugated \nanti-human cytokines. The samples were incubated at room temperature for 3 h in the dark. After incubation \nwith the PE detection reagent, the samples were washed once and resuspended in 300  μl of washing buffer \nbefore acquisition on a FACSCalibur cytometer (BD Biosciences). Data were analyzed using CBA software (BD \nBiosciences). Standard curves were generated for each cytokine using the cytokine standard provided by the kit. \nThe concentration of each cytokine in the cell supernatant was determined by interpolation to the corresponding \nstandard curve. The assay sensitivity is denoted by 3.7 pg/ml for IFN-γ, 4.5 pg/ml for IL-10, 4.9 pg/ml for IL-4 and \n18.9 pg/ml for IL-17A. We refrained from adjusting the cell number prior to the in vitro stimulation because we \naimed to obtain the net production of cytokines for each individual.\nstatistics. Statistical analyses of the differences between means were performed using unpaired, two-tailed \ntests. If the data is non-normally distributed, we used a nonparametric test to compare the difference. Statistical \ntests were performed using GraphPad Prism version 5.0 and SPSS Statistics 17.0. P-values of <0.05 were consid-\nered significant.\n\n4Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nResults\nCharacteristics of infertile patients with PCOS. Before oocyte retrieval, a general clinical examination \nwas performed. The plasma hormones and biochemical indicators were determined, including the baseline lev-\nels of T, E2, LH and FSH. Moreover, the ratio of LH to FSH was calculated. The results of the study showed that \nthere were no significant differences in age, infertility years, or levels of T or FSH between the infertile patients \nwith PCOS and the controls (Table 1, P > 0.05). However, the levels of LH and LH/FSH ratio were significantly \nincreased in group B (Table  1, P < 0.01). Our data showed disparities compared with previous reports\n17. This \ndiscrepancy might be the result of the hereditary and demographic differences between Asian and European or \nAmerican individuals.\nPercentages of T lymphocyte subsets in follicular fluid of infertile women with and without \npCos. To observe the changes in the T lymphocyte subsets between group A and group B, lymphocytes were \nisolated from the follicular fluid. The cells were quantified, and the expressions of CD14, CD45, CD3, CD4 and \nCD25 were subsequently detected by flow cytometry. Anti-CD14 and anti-CD45 antibodies were used to confirm \nthe population of lymphocytes (CD45\n+CD14−  cells). The flow cytometric analysis showed that the percentages \nof CD3+ and CD8+ (CD3+CD8+) T lymphocytes were significantly reduced in the follicular fluid of the infertile \nwomen with PCOS compared with the infertile women with normal ovulation (66.2% ± 2.1% vs. 54.8% ± 2.8%, \nP < 0.01; 28.4% ± 1.2% vs. 16.8% ± 1.4%, P < 0.01). However, the differences in the relative percentages of CD4+ \n(CD3+CD4+) between the PCOS and control group were not robust (Fig. 1a,b).\nExpression of CD25 and CD69 on the surface of CD4 + and CD8+ t  cells. To further explore the \nactivation state of the T lymphocyte subsets, the expressions of the activated molecules CD25 and CD69 were \nmeasured by cell surface staining. CD3\n+CD4+ cells and CD3+CD8+ cells were first gated, and the percentages \nof CD25 and CD69 on these cell populations were subsequently analyzed. As shown in Fig.  2a,b, there was no \ndifference in the expression of CD25 on CD4+ or CD8+ T cells between the PCOS and NOW (P > 0.05); however, \nthe expressions of CD69 in the PCOS group with infertility were significantly decreased both on CD4 + T cells \n(P < 0.05) and CD8+ T cells (P < 0.01) compared to the infertile patients with normal ovulation.\nFigure 1. Percentages of T lymphocyte subsets in follicular fluid of infertile women with and without PCOS. \nFollicular fluid samples were collected from normally ovulating infertile women (NOW , n = 36) and infertile \npatients with PCOS (n = 30). CD45+CD14−  lymphocyte cells were first gated, and the levels of T lymphocytes \nand subsets were determined by flow cytometry. Representative results (a) and mean ± s.e.m. (b) are shown. \n**P < 0.05, ***P < 0.01, no significant difference (ns) was P > 0.05 compared with control group.\n\n5Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nExpressions of IFN-γ, IL-17, IL-10, IL-4 and PD-1 by CD4+ and CD8+ t cells. As the percentage and \nactivation state of the T lymphocyte subsets were different, we investigated the cytokine production of CD4+ and \nCD8+ T cells. Lymphocytes from the follicular fluid were isolated and adjusted to 1 × 106/ml. After stimulation by \nPMA and ionomycin, intracellular cytokines were stained. CD3+CD4+ cells and CD3+CD8+ cells were first gated, \nand the results indicated that the percentages of IFN-γ-expressed CD4+ and CD8+ T cells in the infertile patients \nwith PCOS were significantly lower than those in the infertile patients with normal ovulation (23.6% ± 3.4% vs. \n16.9% ± 2.6%, P < 0.05; 31.8% ± 2.5% vs. 22.5% ± 2.2%, P < 0.01). However, no changes were identified in the \npercentage of IL-17 and IL-4 expressed CD4+ or CD8+ T cells between the two groups (P > 0.05) (Fig. 3a,b).\nTo examine the effect of PD-1 and IL-10 engagement on CD4 + and CD8+ T cell activation, cells from the \nfollicular fluid of the patients with PCOS and the patients with normal ovulation were isolated. The expression \nof PD-1 was assayed by cell surface staining, while intracellular staining was used to detect IL-10 after stimu -\nlation with PMA plus ionomycin. As shown in Fig.  4a,b, the expression of PD-1 on CD4 + T cells in the PCOS \ngroup with infertility was significantly higher than that in the control group (13.80% ± 3.18% vs. 26.13% ± 3.31%, \nP < 0.05; 10.31% ± 2.34% vs. 19.30% ± 2.50%, P < 0.01). The percentages of IL-10-expressed CD4+ and CD8+ T \ncells in the PCOS group with infertility were slightly increased compared with the control group; however, there \nwere no significant differences between the two groups (P > 0.05).\nWe further confirmed the presence of intracellular cytokines using a cytometric bead array (CBA). Cells from \nthe follicular fluid of the patients with PCOS or NOW were stimulated with 20 ng/ml PMA and 1 μg/ml iono-\nmycin and incubated for 48 h. The levels of the cytokines IFN-γ, IL-10, IL-4 and IL-17A were analyzed in the cell \nculture supernatants by CBA. The results indicated that the level of IFN-γ was significantly reduced (423.6 ± 61.3 \nvs. 208.9 ± 53.5, pg/ml, P < 0.05) (Fig. 5a), while the levels of IL-4, IL-17A and IL-10 did not differ between the \ngroups (P > 0.05) (Fig. 5b–d).\nCorrelations between the PD-1 expression on CD4+ or CD8+ T cells in FF and serum E2 level, the \nIFN-γ expression in FF CD4+ or CD8+ T cells in infertile patients with PCOS. While the importance \nof T cells in the immune response has been demonstrated, a potential correlation of T cell exhaust with the ovar-\nian response to gonadotropin stimulation is unknown. Our results showed that the PD-1 expression on CD4+ or \nCD8+ T cells, which reflects T cell exhaust, positively correlated with the serum E2 level in the infertile patients \nwith PCOS (r2 = 0.424, P < 0.05 and r2 = 0.431, P < 0.05, respectively, Fig. 6a,b). The results further indicate that \nthe exhaustion of T cells might be related to the development of oocytes and ovulation. Interestingly, inverse \ncorrelations between the expressions of PD-1 and IFN-γ  in the FF CD4\n+ and CD8+ T cells were found in the \ninfertile patients with PCOS (r 2 = 0.418, P < 0.05 and r2 = 0.387, P < 0.05, respectively, Fig. 6c,d). These results \nindicated that the secretion of IFN-γ in T cells in PCOS patients with infertility may be suppressed by increased \nexpression of PD-1.\nFigure 2. Activated CD4+ and CD8+ T cells in follicular fluid of infertile women with and without PCOS. \nSingle cell suspensions were isolated from follicular fluid of NOW (n = 36) and patients with PCOS (n = 30). \nThe expressions of CD25 and CD69 on T lymphocyte subsets were detected using cell surface staining, as \npreviously described in the “Materials and methods” . The expressions of CD25 and CD69 were analyzed using \nflow cytometry. A representative result (a) and mean expressions of CD25 and CD69 were calculated from \nFACS data (b). *P < 0.05, **P < 0.01, compared with control group.\n\n6Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nDiscussion\nOur results showed a significant reduction in the percentages of total CD3+ T cells and CD8+ T cells in the fol-\nlicular fluid of the PCOS group with infertility compared with the control group (P  < 0.05, Fig. 1). Consistent \nwith previous reports, there were no disturbances in the percentages of CD4 + T cells in PCOS patients with \ninfertility31. Although the percentage of CD4+ T cells did not change in PCOS patients with infertility, both CD4+ \nand CD8+ T cells expressed significantly lower levels of CD69 in the PCOS group (P < 0.01, Fig. 2). CD69 is the \nearliest molecule expressed on the cell surface of lymphocytes after activation32. These results further confirmed \nthe existence of CD4+ and CD8+ T cell responses in PCOS patients with infertility, and the dysfunction of T cells \nmight be associated with the pathogenesis of PCOS.\nRecent studies have shown that several cytokines, including IFN-γ, TNF-α , IL-2, IL-4, IL-5, and IL-10, were \nproduced by immunocompetent cells in the blood from PCOS with infertility in vitro, which might be involved \nin chronic inflammation 17. Moreover, previous reports used a Cytometric Bead Array kit (CBA) to detect the \nproduction of these cytokines in cell culture supernatants. Our study is the first study to enrich lymphocytes from \nthe follicular fluid and analyze the expression of IFN-γ , IL-4, IL17A, and IL10 in lymphocyte subsets of PCOS \npatients with infertility using flow cytometry.\nIFN-γ has the ability to ‘interfere’ with the replication of virus in infected cells. Other effects of IFN-γ include \nthe activation of macrophages, enhancing the activity of natural killer cells, synergy with cytokines and facili-\ntating antibody production by cells\n33. IFN-γ-induced chemokines and their receptors play important roles in \nthe pathogenesis of autoimmune endocrine diseases34. However, whether IFN-γ is involved in the pathogenesis \nof PCOS is not clear. Consistent with previous reports17, our results showed that untreated PCOS patients with \ninfertility demonstrated significantly decreased expression of IFN-γ compared to women with normal ovulation \n(P < 0.05, Fig. 3a,b). Thus, the disordered levels of T cells and IFN-γ observed might cause the alteration of the \nFigure 3. IFN-γ, IL-4, L-17A and Foxp3 expressed by CD4+ and CD8+ T cells. Cytokine expression profiles \nof CD4+ and CD8+ T cells from follicular fluid of NOW (n = 20) and patients with PCOS (n = 20) were \ndetermined. Single cell suspensions were stimulated with PMA plus ionomycin. CD3+CD4+ cells and \nCD3+CD8+ cells were first gated, and the expressions of IFN-γ, IL-4, L-17A and Foxp3 by CD4+ and CD8+ \nT cells were examined using intracellular cytokine or nuclear protein staining. A representative result (a) and \nmean ± s.e.m. (b) are shown. *P < 0.05, **P < 0.01, compared with control group.\n\n7Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nmechanisms that regulate the expression of proteolytic enzymes, including collagenase and elastase, which can \ndigest extracellular matrix proteins and thereby lead to follicular rupture and ovulation. As established, IL-4 can \ndecrease the production of Th1 cells. However, our results showed that IL-4 could be rarely produced by CD4\n+ \nand CD8+ T cells in infertile patients with and without PCOS, and no significant difference was identified in the \nexpression of IL-4 between the two groups (P > 0.05, Fig. 3a,b). Consistent with previous reports35, these results \nshow that IL-4 might not be involved in the pathogenesis of PCOS.\nRecent studies have shown that IL-17A is a major pro-inflammatory cytokine, which is associated with the \ninteraction between PCOS and gingival inflammation36. However, in previous reports, ELISA was used to detect \nthe production of IL-17A in gingival crevicular fluid (GCF), saliva, or serum. Our study is the first study to enrich \nlymphocytes from follicular fluid and analyze the IL-17A expression in lymphocyte subsets via flow cytometry \nanalysis in PCOS patients in real time. Moreover, we found that IL-17A could be produced by CD4\n+ and CD8+ T \ncells in patients with and without PCOS; however, there was no significant difference in the expression of IL-17A \nbetween the two groups (P > 0.05, Fig. 3a,b). The results show that IL-17A might not be involved in the patho-\ngenesis of PCOS.\nPD-1 is crucial in mediating immune tolerance, infection, and cancer immunity\n37. As an inducible receptor, it \nhas been reported to be expressed on peripheral T lymphocytes following activation. PD-1 inhibits antiviral T cell \nresponses via the interaction with two ligands, PD-L1 and PD-L2\n37,38. As shown in Fig. 4a,b, our results indicated \nthat the expression of PD-1 in FF CD4+ or CD8+ T cells from the PCOS group with infertility was significantly \nhigher than that from the control group (P < 0.05). Furthermore, patients with PCOS showed an inverse correla-\ntion between the expression of PD-1 and IFN-γ in FF CD4+ or CD8+ T cells (P < 0.05, Fig. 6c,d). These findings \nindicated that the survival and activation of T cells in PCOS patients with infertility might be suppressed by \nincreased expression of PD-1. IL-10 markedly inhibits the functions of monocytes-macrophages, such as antigen \npresentation\n39. As a potent inhibitory molecule, IL-10 restrains the lytic activity of CD4 + and CD8+ T cells40. \nFigure 4. PD-1 and IL-10 expression on CD4+ and CD8+ T cells. Single cell suspensions were isolated from \nfollicular fluid of NOW (n = 20) and infertile patients with PCOS (n = 20). CD3+CD4+ cells and CD3+CD8+ \ncells were first gated, and the expression of PD-1 on CD4+ and CD8+ T cells was detected using cell surface \nstaining, while IL-10 expression was examined using intracellular cytokine staining, as previously described \nin the “Materials and methods” section. A representative result is shown (a). Average expressions of PD-1 and \nIL-10 on CD4\n+ and CD8+ T cells were calculated from the FACS data (b). *P < 0.05, compared with control \ngroup.\n\n8Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nFigure 5. Cytokine expression profiles on CD4+ and CD8+ T cells by cytometric bead array kit (CBA). (a–c) \nCells from follicular fluid of infertile patients with PCOS (n = 10) or NOW (n = 10) were stimulated with 20 ng/\nml PMA and 1 μg/ml ionomycin and incubated for 48 h. The levels of IFN-γ (a), IL-4 (b), IL-17A (c) and IL-10 \n(d), all measured with cytometric bead array kit (CBA). *P < 0.05, compared with the control group.\nFigure 6. Correlations between the PD-1 expression on CD4+ or CD8+ T cells and the serum E2 level or \nIFN-γ expression on CD4+ or CD8+ T cells in infertile patients with PCOS. The PD-1 expression on CD4+ or \nCD8+ T cells was positively correlated with the E2 levels in serum (a,b) and reversely correlated with the IFN-γ \nexpression on CD4+ or CD8+ T cells in infertile patients with PCOS (n = 20) (c,d). Pearson’s correlation test was \nused.\n\n9Scientific  RepoRts  |          (2019) 9:6040  | https://doi.org/10.1038/s41598-019-42631-x\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nMoreover, IL-10 could be detected in both infertile patients with and without PCOS; however, no difference was \nobserved in the percentage of IL-10+CD4+ or IL-10+CD8+ cells between the two groups (P > 0.05, Fig. 4a,b).\nFollicular granulosa cells can produce a supraphysiological level of serum E2 during controlled ovarian hyper-\nstimulation, which is associated with the development of multiple ovarian follicles, and the level of serum E2 \ncorrelated with the maturity and quality of ovarian follicles\n41. Interestingly, we found that the serum E2 level \npositively correlated with the expression of PD-1 in FF CD4+ or CD8+ T cells. Furthermore, an inverse correla-\ntion between the expression of PD-1 and IFN-γ in FF CD4+ or CD8+ T cells was found in infertile patients with \nPCOS (P < 0.05, Fig. 6). The results showed that abnormal activation of T cells and cytokine production might \nlead to the abnormal oocyte development observed in PCOS patients. Recent studies have shown that metabolic \ndysbalance plays a key role in PCOS pathogenesis, and inositol supplementation could reduce the amount of \ngonadotropins and the length of ovarian stimulation in women undergoing IVF\n42–44. Consequently, we propose \nthat the correction of T cell dysfunction may re-address hormonal and clinical parameters to restore homeostasis.\nPCOS is the most prevalent endocrinopathy of reproductive-aged women. However, infertility occurs in about \n10–20% of patients with PCOS45,46. In our study, there is a potential selection bias since the study only included \ninfertile patients with or without PCOS, which might be a surrogate for the severity/chronicity of the disease. To \nget more reliable results, the study should include a random sample of all PCOS patients. However, the depart-\nment we work in is the reproductive medical center, it is difficult to obtain clinical samples from PCOS patients \nwith fertility. Furthermore, even in PCOS patients, follicular fluid samples will not be taken during examination \nand treatment if the pregnancy is normal. Thus, we only focused on PCOS in infertile patients and limited our \nfindings to infertile patients with PCOS in this study.\nIn summary, this report found that increased expression of PD-1 and significantly decreased expression of \nIFN-γ were detected in CD4\n+ T and CD8+ T cells in infertile patients with PCOS (P < 0.05). We speculate that \nthe higher expression of PD-1 in CD4+ T and CD8+ T cells in the FF in PCOS patients with infertility probably \ncannot induce T cell activation or recruitment, which, in turn, leads to the failure of dominant follicle selection \nand development. It is concluded that the dysfunction of T cells, which may be an immunological feature, might \nparticipate in the immune pathogenesis in the ovary of PCOS patients with infertility. These results suggest that \nchronic inflammation may be one of the underlying mechanisms for the pathogenesis of PCOS.\nReferences\n 1. Barthelmess, E. K. & Naz, R. K. Polycystic ovary syndrome: current status and future perspective. Front Biosci (Elite Ed). 6, 104–119 \n(2014).\n 2. Goodarzi, M. O. et al. Polycystic ovary syndrome: etiology, pathogenesis and diagnosis. Nat Rev Endocrinol. 7(4), 219–231. (2011).\n 3. Harris, H. R. & Terry, K. L. 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Open Journal of Endocrine and Metabolic Diseases 6, 58–65 \n(2016).\nAcknowledgements\nThis work was supported by a grant from the Natural Science Foundation of Guangdong Province \n(2016A030310282, 2018A0303130317), Natural Science Foundation of China (31800739, 81771696), the \nGuangdong provincial education department (2016KZDXM033, 2017KTSCX157), and the Science and \nTechnology Planning Project of Guangdong Province (2016A020215164).\nAuthor Contributions\nDesign of the research, Quan Y ang; performing experiments, Zitao Li, Anping Peng, Xiaona Zhang; data \ninterpretation, Fenghua Liu, Chuangqi Chen, Xin Y e; supplying materials, Yuanfa Feng, Jiale Qu, Mei Wang, \nChenxi Jin, Huaina Qiu, Y anwei Qi; writing manuscript, Quan Y ang, Jun Huang. All authors read and approved \nthe final manuscript.\nAdditional Information\nCompeting Interests: The authors declare no competing interests.\nPublisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-\native Commons license, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the \nmaterial. 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