Ovarian leukocytes: Association with follicular persistence and cyst formation in dairy cows.

One of the initial causes of cystic ovarian disease (COD) is a failure in the normal ovulation mechanism. This study aimed to characterize the populations of immune cells (T-lymphocytes, B-lymphocytes, monocytes-macrophages and granulocytes) present in the ovary of cows with COD and induced follicular persistence, and evaluate their relation with follicular persistence and cyst formation. The follicular persistence model was developed using a progesterone (P4) slow-release intravaginal device, to obtain subluteal concentrations of P4. Results evidenced that T-lymphocytes, B-lymphocytes and monocytes-macrophages in the cortex, medulla, and theca externa and interna of dominant follicles were higher in the control group than in the COD and all persistence groups. Granulocytes in the medulla and theca externa of dominant follicles were lower in the control group than in the COD group, and those in the cortex and medulla were lower in the control group than in the persistence groups. The presence of T-lymphocytes, B-lymphocytes and granulocytes in the follicular fluid was abundant, especially that of granulocytes, without differences between control and COD cows. These results suggest that the immune system potentially plays a role in the local mechanisms of COD pathogenesis in dairy cows. In spontaneous COD and in our follicular persistence model, the distribution of the cells studied was different from that in the control group. However, to our knowledge, this is the first report describing the presence of immune cells in bovine follicular fluid samples and the expression of steroid hormone receptors in infiltrating immune cells in the bovine ovary.

Differential proteomic analysis demonstrates follicle fluid participate immune reaction and protein translation in yak.

BACKGROUND: Ovarian follicle fluid (FF) as a microenvironment surrounding oocyte plays critical roles in physio-biochemical processes of follicle development and oocyte maturation. It is hypothesized that proteins in yak FF participate in the physio-biochemical pathways. The primary aims of this study were to find differentially expressed proteins (DEPs) between mature and immature FF, and to elucidating functions of the mature and immature FF in yak. RESULTS: The mature and immature FF samples were obtained from three healthy yaks that were nonpregnant, aged from four to five years, and free from any anatomical reproductive disorders. The FF samples were subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ). The FF samples went through correlation analysis, principle component analysis, and expression pattern analysis based on quantification of the identified proteins. Four hundred sixty-three DEPs between mature and immature FF were identified. The DEPs between the mature and immature FF samples underwent gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) analysis. The DEPs highly expressed in the mature FF mainly took parts in the complement and coagulation cascades, defense response, acute-phase response, response to other organism pathways to avoid invasion of exogenous microorganisms. The complement activation pathway contains eight DEPs, namely C2, C5, C6, C7, C9, C4BPA, CFH, and MBL2. The three DEPs, CATHL4, CHGA, and PGLYRP1, take parts in defense response pathway to prevent invasion of exogenetic microorganism. The coagulation cascades pathway involves many coagulation factors, such as F7, F13A1, FGA, FGB, FGG, KLKB1, KNG1, MASP1, SERPINA1, and SERPIND1. While the DEPs highly expressed in the immature FF participated in protein translation, peptide biosynthetic process, DNA conformation change, and DNA geometric change pathways to facilitate follicle development. The translation pathway contains many ribosomal proteins, such as RPL3, RPL5, RPS3, RPS6, and other translation factors, such as EIF3J, EIF4G2, ETF1, MOV10, and NARS. The DNA conformation change and DNA geometric change involve nine DEPs, DDX1, G3BP1, HMGB1, HMGB2, HMGB3, MCM3, MCM5, MCM6, and RUVBL2. Furthermore, the expressed levels of the main DEPs, C2 and SERPIND1, were confirmed by western blot. CONCLUSIONS: The differential proteomics revealed the up-regulated DEPs in mature FF take parts in immunoreaction to prevent invasion of microorganisms and the up-regulated DEPs in immature FF participate in protein synthesis, which may improve our knowledge of the follicular microenvironment and its biological roles for reproductive processes in yak. The DEPs, C2 and SERPIND1, can be considered as protein markers for mature yak follicle.

Immunological Microenvironment Alterations in Follicles of Patients With Autoimmune Thyroiditis.

Autoimmune thyroiditis (AIT) is the most prevalent autoimmune endocrine disease, with a higher incidence in women than in men. Immunological abnormalities may lead to the impairment of ovarian folliculogenesis; however, whether the presence of AIT affects immunological microenvironment in follicles remains controversial. We performed a cross-sectional study including 122 patients, aged 20-40 years, who underwent IVF/ICSI treatment owing to isolated male or tube factor infertility. Patients were divided into AIT and control groups according to clinical presentation, thyroid function, and thyroid autoantibody measurements. Follicular fluid was collected and the distribution of cytokines/chemokines in follicular fluid was measured by flow cytometry using multiplex bead assays between the two groups. Based on differences in levels of intrafollicular chemokines and cytokines between the AIT and control groups, the relevant inflammatory cascade was further demonstrated. Among the 12 chemokines analyzed, three (CXCL9, CXCL10, and CXCL11) showed significantly elevated levels in the follicular fluid of patients with AIT. Among the 11 cytokines detected, compared with those in the control group, significantly higher levels of IFNγ were observed in patients with AIT. IFNγ dose-dependently stimulated the expression and secretion of CXCL9/10/11 in cultured primary granulosa cells. The percentage of CXCR3+ T lymphocytes was significantly elevated in the follicular microenvironment of patients with AIT. We concluded that the IFNγ-CXCL9/10/11-CXCR3+ T lymphocyte inflammatory cascade is activated in the follicular microenvironment of patients with AIT. These findings indicate that a considerable immune imbalance occurred in the follicular microenvironment of patients with AIT.

Anticentromere antibody induced by immunization with centromere protein and Freund's complete adjuvant may interfere with mouse early-stage embryo.

BACKGROUND: Anticentromere antibody (ACA) is a member of the antinuclear antibody spectrum (ANAs) which has been speculated to be associated with subfertility. Thus, the present study aimed to investigate the induction of ACA production and its potential interference with early-stage embryos. METHODS: Recombinant centromere protein-A (CENP-A) or centromere protein-B (CENP-B) and complete Freund's adjuvant (CFA) were used to immunize mice. Serum ACA level was then evaluated by using an indirect immunofluorescence test. Immunofluorescence assay was performed to detect IgG in follicles in ovarian tissues and early-stage embryos. RESULTS: Following treatment, serum positive ACA was observed in mice treated with CENP and CFA. Furthermore, IgG were detected in follicular fluid and early-stage embryos from mice treated with CENP and CFA. CONCLUSIONS: This study preliminarily indicated that ACA induced by CENP and CFA may penetrate into the living embryos of early-stage in mice.

Leptin concentrations in endometriosis: A systematic review and meta-analysis.

INTRODUCTION: Endometriosis is an inflammatory condition, affecting mainly women of reproductive age. Leptin is a regulator of food intake and energy expenditure, posing pleiotropic actions, and regulating immunity and fertility. The aim of this study was to systematically review the literature regarding leptin concentrations in biological fluids and tissues of women with endometriosis, and to investigate and propose a possible role of leptin in the pathophysiology of endometriosis. MATERIALS AND METHODS: A systematic search of the literature was conducted in two electronic databases (MEDLINE, COCHRANE) and grey literature for original research articles on humans, published in any language. RESULTS: Twenty-nine studies with 1291 women with endometriosis and 1664 controls were included in the systematic review. Peritoneal fluid and follicular fluid leptin concentrations were higher in endometriosis compared with control group [mean difference (MD) 7.10, 95 % confidence interval (CI) 4.76 to 9.44 ng/mL, 18 studies), (MD 1.35, 95 % CI 0.54-2.17 ng/ml, 2 studies) respectively. No differences were evident in serum (MD 0.92, 95 % CI -0.84 to 2.68 ng/mL, 12 studies) or plasma (MD -0.95, 95 % CI -4.63 to 2.72 ng/mL, 3 studies) between the groups. No meta-analysis was conducted for ovarian tissue leptin (2 studies). CONCLUSIONS: This meta-analysis provided evidence for increased leptin concentrations in both peritoneal fluid and follicular fluid of women with endometriosis compared with control; these differences were not present in the serum or plasma. The above results support a potential pathophysiologic role for leptin in the local microenvironment while declines its use as a blood diagnostic marker. Furthermore, we propose a possible role of leptin in the pathophysiology of endometriosis.

IL-18 and IL-18 binding protein concentration in ovarian follicular fluid of women with unexplained infertility to PCOS during in vitro fertilization.

The correlation between the concentration of interleukin (IL) 18 in follicular fluid and the pathogenesis of Polycystic Ovary Syndrome (PCOS) is unclear. Therefore, we tested the IL-18 and IL-18 binding protein (IL-18BP) levels in follicular fluid (FF) and serum in PCOS women undergoing reproductive measures and to explore their possibly correlation with PCOS. Serum and pooled follicular fluid levels of IL-18, IL-18BP and IL-18/IL-18BP ratios were evaluated in sixty patients with PCOS and sixty women with unexplained infertility undergoing in vitro fertilization. The FF IL-18 levels were increased in PCOS group than the CON group (p < 0.01), and the IL-18 levels were significantly higher in the FF than in serum in PCOS group. Furthermore, the elevated FF IL-18 levels have no correlation with the serum IL-18 levels. Additionally, the expression of IL-18 in the follicular fluid of the overweight PCOS patients was increased compared to the normal weight PCOS patients, while in the overweight patients, FF IL-18 was significantly higher in the PCOS group than in the control group. The FF IL-18 and IL-18BP may have a local involvement in the pathogenesis of PCOS. The PCOS itself and overweight will aggravate the local inflammatory response in the ovary. Further studies are needed to elucidate this issue.

Is there an immune modulating role for follicular fluid in endometriosis? A narrative review.

Follicular fluid (FF) surrounds the granulosa cell-oocyte complex and is one of the mediating factors in the communication between the cells within the follicle. Literature reveals that human FF and its components are key factors to the success of natural fertilization. Among other substances, FF consists of multiple cytokines and immune cells, including interleukin 6 (IL6), IL12, sHLA-G, macrophages, NK cells and lymphocytes. Together, these cells and cytokines might influence the oocyte-granulosa-cell complex. Altered balances of immune content might be involved in changes on folliculogenesis, oocyte maturation, oocyte quality and ovulation. Furthermore, these altered balances are possibly involved in infertility associated with immune-mediated diseases such as endometriosis. The aim of this narrative review is to elaborate on the function and contents of FF and its immunological profile in patients with endometriosis. A comprehensive literature search was performed for the published literature on FF (immune) contents, FF function and FF content alterations in endometriosis patients. In FF of patients with endometriosis, elevated levels of macrophages and several cytokines have been reported. The role of specific immune cells in FF and a clarification of the biological mechanism in healthy women and endometriosis patients remain largely unknown. Future studies in this field will give us more insight in the role of FF immune cells and the effect of altered balances in patients with endometriosis.

Association of IL-17 and IL-23 follicular fluid concentrations and gene expression profile in cumulus cells from infertile women at risk for ovarian hyperstimulation syndrome.

This study determined the association between the levels of interleukin (IL)-17 and IL-23 in follicular fluid (FF), as well as their mRNA levels in cumulus cells from infertile women at risk for ovarian hyperstimulation syndrome (OHSS). In this case-controlled study, the control group (n = 40) was infertile women whose partners had male factor infertility, whereas the case group (n = 40) was infertile women at risk of OHSS. IL-17 and IL-23 concentrations in FF were measured using an enzyme-linked immunosorbent assay method, whereas the mRNA expression levels of IL-17 and IL-23 of cumulus cells were determined using RT-PCR. Significantly higher levels of IL-17 were seen in the case group (p = 0.04), whereas there was no significant difference in IL-23 concentrations between the two groups (p = 0.3). The mRNA levels of IL-17 and IL-23 showed no significant differences. In the case group, there was a positive significant correlation between the IL-23 concentration in FF and the oocyte maturation rates (p = 0.01). In the case group, the number of follicles, MII oocytes, immature oocytes, fertilized oocytes and number of embryos were significantly higher than the control group (p < 0.05). Our findings showed that the mRNA expressions of IL-17 and IL-23 were similar in the two groups, and IL-17 was increased in the case group.

The clinical outcome of Dienogest treatment followed by in vitro fertilization and embryo transfer in infertile women with endometriosis.

BACKGROUND: Endometriosis is considered to be the most intractable cause of female infertility. Administering any type of treatment for endometriosis before in vitro fertilization and embryo transfer (IVF-ET) is an important strategy for improving the IVF-ET outcomes for infertile women with endometriosis. In fact, treatment with a gonadotropin-releasing hormone (GnRH) agonist just before IVF-ET has been reported to improve the clinical outcome in endometriosis patients. However, the benefit of Dienogest (DNG), a synthetic progestin, treatment just before IVF-ET remains unclear. METHODS: Sixty-eight infertile women with Stage III or IV endometriosis (ovarian endometrial cyst < 4 cm) were recruited for this study. The subjects were divided into 2 groups: a DNG group (n = 33) and a control group (n = 35). DNG was administered orally every day for 12 weeks prior to the conventional IVF-ET cycle in the DNG group. Standard controlled ovarian hyperstimulation with the GnRH agonist long protocol was performed in the control group. The numbers of mature follicles and retrieved oocytes, fertilization rates, implantation rates, and clinical pregnancy rate were compared between the two groups. In addition, the concentrations of inflammatory cytokines, oxidative stress markers, and antioxidants in follicular fluids were also measured. RESULTS: The numbers of growing follicles, retrieved oocytes, fertilized oocytes, and blastocysts were significantly lower in the DNG group than in the control group. The fertilization and blastocyst rates were also lower in the DNG group than in the control group. Although there was no significant difference in the implantation rate between the groups, the cumulative pregnancy rate and live birth rate were lower in the DNG group than in the control group. There was no significant difference in the abortion rate. Our results failed to show that DNG reduces the inflammatory cytokine levels and oxidative stress in follicular fluids. CONCLUSIONS: Administering DNG treatment just before IVF-ET did not provide any benefits to improve the clinical outcomes for infertile women with endometriosis.

Immunological microenvironment alterations in follicles of women with proven severe endometriosis undergoing in vitro fertilization.

The purpose of this study was to test the hypothesis that different cytokine profiles may exist in the follicular fluid of endometriosis (EM) patients undergoing in vitro fertilization (IVF), as these differences may provide insights into the pathogenesis of the disease. This was a cross-sectional study conducted at the reproductive center of a medical university hospital. The study included 49 patients receiving IVF. 20 infertile women with proven EM and 29 women without diagnosed EM (control group) were evaluated. Follicular fluid (FF) and serum were collected at the time of follicle aspiration and the concentrations of 38 cytokines were determined by multiplexed immunoassay. The results indicated that the levels of IL-4, IL-13, IL-3 and IL-1α were significantly increased in the FF of women with EM, while levels of IFN-γ, IL-17A, MDC and MIP-1α were decreased compared with in the control subjects. In conclusions, the immune microenvironment of the FF in patients with EM is altered. This may contribute to the pathologic mechanism responsible for the poor outcome of IVF in patients with EM.

Comprehensive immune complexome analysis detects disease-specific immune complex antigens in seminal plasma and follicular fluids derived from infertile men and women.

BACKGROUND: Autoimmune reactions and subsequent inflammation may underlie spermatogenic dysfunction and endometriosis-related infertility. The aim of this study is to identify disease-specific antigens in immune complexes (ICs) in seminal plasma (SP) and in follicular fluid (FF). METHODS: Immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, was performed for specimens collected from infertile couples undergoing assisted reproduction. Forty-two male patients consisting of subjects with oligozoospermia (n = 6), asthenozoospermia (n = 8), and normal semen analysis (n = 28). Fifty-eight female patients consisting of subjects with ovarian endometriosis (n = 10) and control women without disease (n = 48). RESULTS: Four disease-specific antigens were identified in subjects with oligozoospermia, while five disease-specific antigens were detected in subjects with asthenozoospermia, some of which are involved in sprematogenesis. Eight antigens were detected only in subjects with endometriosis. CONCLUSION: Functional characteristics of disease-specific antigens were found to correspond to the pathogenesis of male and female infertility. The formation of ICs may contribute to spermatogenic dysfunction and endometriosis-related infertility via loss of function of the related proteins. Immune complexome analysis is expected to be a valuable tool for the investigation of novel diagnostic methods and treatment strategies for infertility.

Ovulatory follicular fluid induces sperm phagocytosis by neutrophils, but oviductal fluid around oestrus suppresses its inflammatory effect in the buffalo oviduct in vitro.

We have recently shown that the conditioned media from bovine oviductal epithelial cell culture suppress sperm phagocytosis by neutrophils, suggesting that the oviduct around oestrus supplies the anti-inflammatory microenvironment. To investigate the immune response of neutrophils toward the sperm at ovulation in the buffalo oviduct, we examined (a) a detailed distribution of neutrophils in the oviduct in buffaloes, (b) the effect of ovulatory follicular fluid (FF) and oviductal fluid (OF) on sperm phagocytosis by neutrophils, and (c) the interaction of the ovulatory FF with OF on sperm phagocytosis by neutrophils in vitro. Buffalo oviducts were collected from healthy reproductive tracts at a local slaughterhouse. A detailed observation by histological examination and transmission electron microscopy revealed that neutrophils exist in the oviduct epithelium and lumen throughout the oestrous cycle in buffaloes. The number of neutrophils at the oestrus stage was higher in ampulla compared with those in isthmus, whereas they remained relatively constant at the dioestrus stage. Two hours of preincubation of neutrophils with FF enhanced sperm phagocytosis through the formation of neutrophil extracellular traps (NETs) together with H2 O2 production, whereas OF around oestrus (eOF) suppressed sperm phagocytosis, NETs formation, and H2 O2 production and relieved the above FF-induced inflammatory response. Our findings show that neutrophils exist in the healthy cyclic oviduct across bovine species, and the OF supplies a strong anti-inflammatory environment that could minimize the inflammatory effect of the FF that flows into the oviduct lumen after ovulation and supports the occurrence of fertilization.

Detection of T lymphocyte subsets and related functional molecules in follicular fluid of patients with polycystic ovary syndrome.

Immune responses play an important role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the characteristics of T lymphocyte subsets in PCOS remain insufficiently understood. In this study, lymphocytes of follicular fluid (FF) were obtained from oocyte retrieval before in-vitro fertilization (IVF) in infertile women with or without PCOS. The levels of cluster of differentiation 25 (CD25), CD69, programmed death 1 (PD-1), interferon-γ (IFN-γ), interleukin 17A (IL-17A) and IL-10 in T lymphocytes were determined by flow cytometry. Our results showed that the percentage of FF CD8+ T cells was significantly decreased in infertile patients with PCOS (P < 0.05). Furthermore, the levels of CD69 and IFN-γ were significantly decreased and the level of PD-1 was increased in both CD4+ and CD8+ T cells from infertile patients with PCOS (P < 0.05). Moreover, the expression of PD-1 on CD4+ or CD8+ T cells was positively correlated with the estradiol (E2) levels in the serum and reversely correlated with the expression of IFN-γ in CD4+ or CD8+ T cells in infertile patients with PCOS. These results suggested that T cell dysfunction may be involved in the pathogenesis of PCOS.

Follicular fluid thyroid autoantibodies, thyrotropin, free thyroxine levels and assisted reproductive technology outcome.

OBJECTIVE: Although there are substantial data linking thyroid autoimmunity (TAI) and infertility, data regarding assisted reproductive technology (ART) outcomes and TAI markers in follicular fluid (FF) of women undergoing ART are scarce. Objective of the study was to assess the association of the levels of thyroid autoantibodies in FF and ART outcome expressed as the achieved pregnancies. METHODS: This study enrolled 52 women undergoing ART (26 TAI positive subjects and 26 age and body mass index matched TAI negative controls). Blood samples were drawn before the initiation of protocol for controlled ovarian stimulation, and thyrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), thyroid peroxidase antibodies (TPOAbs) and thyroglobulin antibodies (TgAbs) levels were measured. TSH, fT4, TPOAbs, TgAbs and progesterone levels were also measured in FF. RESULTS: There were no significant differences between the groups regarding mean levels of FF TSH and FF fT4. Statistically significant correlation was discovered regarding the levels of serum and FF TPOAbs (0,961, p<0.001 in TAI positive, 0,438, p = 0.025 in TAI negative group) and TgAbs (0,945, p<0.001 in TAI positive, 0,554, p = 0.003 in TAI negative group). Pregnancies rates per initiated cycle and per embryotransfer cycle were significantly different between TAI positive and TAI negative group, (30.8% vs 61.5%), p = 0.026 and (34.8% vs 66.7%), p = 0.029, respectively. Multivariate analysis showed that TAI positive women had less chance to achieve pregnancy (p = 0.004, OR = 0.036, 95% CI 0.004-0.347). CONCLUSIONS: Higher levels of thyroid autoantibodies in FF of TAI positive women are strongly correlated with serum levels and may have effect on the post-implantation embryo development.

Inflammatory markers in human follicular fluid correlate with lipid levels and Body Mass Index.

The detrimental consequences of obesity on female fertility are well known, but the functional changes that occur in the ovary in response to elevated BMI are not clear. Obesity induces multiple components of a systemic inflammatory state that is a key pathway by which it initiates tissue dysfunction in adipose, liver and muscle; however whether obesity induces similar inflammatory changes in the ovary has not been fully investigated. This is important to understand because it is increasingly clear that obesity at conception impacts not only pregnancy rates but also influences pre-implantation embryo development. To further understand the characteristics of inflammation in the ovaries of obese women we analysed a panel of cytokines (IL6, IL10 and TNFα), adipokines (adiponectin, leptin and monocyte chemotactic factor 1 (MCP-1)) and acute phase proteins (C-Reactive Protein (CRP) and sICAM-1) in the ovarian follicular fluid obtained at oocyte aspiration from women (n = 48) who were lean, overweight or obese. We hypothesised that adipokines and pro-inflammatory cytokines would be correlated with and/or dysregulated by increasing Body Mass Index (BMI). Surprisingly however, the majority were not related to BMI but instead were positively correlated with lipid levels in follicular fluid, namely triglycerides and free fatty acids. Further, as is typical of metabolic inflammation, the inflammatory markers that were associated with intra-follicular lipids included both pro-inflammatory (CRP, IL6, TNFα) and anti-inflammatory (adiponectin, IL10) mediators. The direct consequences of an ovarian microenvironment containing high levels of lipids and inflammatory mediators are not known but could impact luteinisation, ovulation and/or oocyte developmental competence.

Evaluation of pro-oxidant-antioxidant balance (PAB) and its association with inflammatory cytokines in polycystic ovary syndrome (PCOS).

Chronic low-grade inflammation has been suggested as a key contributor of the pathogenesis and development of polycystic ovary syndrome (PCOS). To investigate the association between oxidative stress status and inflammatory cytokines in follicular fluid of 21 PCOS women compared to 21 women with normal ovarian function who underwent intra-cytoplasmic sperm injection. Concentration of IL-6, IL-8, IL-10, and TNF-α was measured using sandwich ELISA. Oxidative stress was examined by measuring total oxidant status (TOS), malondialdehyde (MDA), total antioxidant capacity (TAC), and thiol groups. PCOS women had an elevated concentration of MDA and TOS compared to controls. Levels of TAC and thiol groups were lower in PCOS compared to controls. PCOS patients had a higher concentration of IL-6, IL-8, and TNF-α compared to controls. Concentration of IL-10 was lower in PCOS compared to controls. Significant correlations were found between MDA and TOS concentration with TNF-α and between IL-6 and MDA, IL-8 and TAC, IL-10 and TOS levels and also between IL-10 and TAC levels. TAC and thiol groups were negatively correlated with TNF-α. Increased oxidative stress in PCOS is associated with inflammation which is closely linked. Inflammation can induce production of inflammatory cytokines in this syndrome and directly stimulates excess ovarian androgen production.

Gonadotrophin stimulation in IVF alters the immune cell profile in follicular fluid and the cytokine concentrations in follicular fluid and serum.

Study question: Are the immune cell profiles and the cytokine concentrations in follicular fluid (FF) and serum at the preovulatory stage different in conventional exogenous gonadotrophin stimulated IVF (c-IVF) compared with natural cycle IVF (NC-IVF)? Summary answer: The cell counts of CD45+ leucocytes and T cell subpopulations and the cytokine concentrations in FF and serum are different in c-IVF compared to NC-IVF. What is known already: FF-derived cells are heterogeneous. Immune cells are involved in intra-ovarian processes and cytokines are required for normal follicular development. Gonadotrophins stimulate the regulatory intrafollicular system and influence the local distribution of immune cells and the intrafollicular release of cytokines. Administration of exogenous gonadotrophins may have a significant effect on this local regulatory system, which then in turn could influence oocyte quality. Study design, size, duration: The study included 105 patients, 69 undergoing c-IVF and 36 undergoing NC-IVF. c-IVF was performed by exogenous ovarian stimulation with hMG and GnRH antagonists. Participants/materials, setting, methods: FF samples were collected from the first dominant follicle in c-IVF without pooling and from single leading preovulatory follicles in NC-IVF. Three different approaches were used to analyze FF samples: (i) microscopic investigation of CD45+ leucocytes, (ii) fluorescence-activated cell sorting to determine CD19+ B cells and CD3+ T cells including T cell subpopulations (CD4+, CD8+), and (iii) evaluation of tumour necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), interleukins (IL)-2, -6, -8, -10 and vascular endothelial growth factor (VEGF) levels in matched FF and serum samples using the Bio-Plex® platform. Main results and the role of chance: FF obtained from c-IVF contained proportionally more CD45+ leucocytes (P = 0.0384), but fewer CD8+ cytotoxic T cells than FF from NC-IVF. CD3+ T lymphocytes were the most common type of lymphocytes, and the number thereof was comparable in the two study groups. In c-IVF, serum VEGF levels were higher (P = 0.007) than in NC-IVF while FF contained marginally decreased concentrations of IL-8 in c-IVF in comparison to NC-IVF. The cytokine concentration gradient between FF and serum in c-IVF was 10-fold for IL-8 and 8-fold for VEGF and thereby markedly lower than in NC-IVF, where the differences were 32-fold and 30-fold, respectively. Strong positive correlations were determined between FF- IL-10 and FF- VEGF in c-IVF (r = 0.85, P < 0.0001) and in NC-IVF (r = 0.81, P < 0.0001). Large scale data: N/A. Limitations, reasons for caution: The ovulation of NC-IVF follicles was induced by the exogenous administration of hCG, which means that the environment did not fully correspond to the physiological situation. Wider implications of the findings: The differences in the immune profile and the cytokine concentrations in c-IVF and NC-IVF follicles support the hypothesis that conventional ovarian stimulation affects indirectly and heterogeneously the intrafollicular milieu, and thereby possibly affects the oocyte quality and the IVF outcome. However, further studies are needed to confirm our findings and to refine stimulation protocols in the context of optimizing the intrafollicular environment during oocyte maturation. Study funding/competing interest(s): The study was supported by a research grant from IBSA Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. The authors are clinically involved in low dose mono-follicular stimulation and IVF-therapies, using gonadotrophins from all gonadotrophins distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH.

Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells.

OBJECTIVE: Ovarian steroid hormones (mainly E2 and P4) regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models. METHODS: A cell line derived from the porcine oviductal epithelium (CCLV-RIE270) was characterized (lineage markers, proliferation characteristics and transformation status). Primary porcine oviduct epithelial cells (POEC) were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml). Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR) and inflammation was quantified. RESULTS: CCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml) upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8). Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2. CONCLUSIONS: CCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells.

Obesity is not a descriptive factor for oxidative stress and viscosity in follicular fluid of in vitro fertilization patients.

BACKGROUND: Obesity's impact on micro-environmental oxidative stress and follicular fluid (FF) viscosity and whether or not it has any effect on in vitro fertilization (IVF) success is a matter of debate. AIMS: In this study, our aim was to evaluate the levels of oxidative stress markers and the FF viscosity in obese and non-obese patients. METHODS: Eighty norm-responder patients undergoing IVF were prospectively grouped according to their body mass indexes (BMI). Group 1 (n = 40) and group 2 (n = 40) had BMI values of ≤24.9 and ≥25.0, respectively. Total sulfhydryl (RSH) levels (nmol/m) and the formation of thiobarbituric acid-reactive substances (malondialdehyde, or MDA) (µmol/ml) in FFs were quantified. For the first time in our study, FF viscosity with changing BMI values was also determined. RESULTS: The mean levels of MDA (µmol/ml) and RSH (nmol/ml) were not significantly different between groups (1.37 ± 0.51; 1.51 ± 0.51; p > 0.05 for MDA and 0.42 ± 0.30; 0.41 ± 0.20; p > 0.05 for RSH, respectively). Similarly, the FF viscosity (centipoise) was not different between groups (1.28 ± 0.28; 1.30 ± 0.19; p < 0.05, respectively). Independent of BMI, no correlation was found between FF levels of oxidative markers and the number of oocytes retrieved or the fertilization rates. CONCLUSIONS: In our study, we found no difference in the levels of follicular oxidative and anti-oxidative markers or the follicular fluid viscosity with changing BMI values. We also demonstrated that the levels of oxidative stress markers and the viscosity of follicular fluid did not affect clinical outcomes.

Differential Expression Profile of Immunological Cytokines in Local Ovary in Patients with Polycystic Ovarian Syndrome: analysis by Flow Cytometry.

OBJECTIVE: Immune dysregulation may play an important role in the pathogenesis of polycystic ovary syndrome (PCOS). The purpose of this study was to investigate the Th1 and Th2-related cytokine profile in local ovary of women with PCOS. STUDY DESIGN: The T lymphocytes of follicular fluid (FF) were obtained at the time of oocyte retrieval before in-vitro fertilization (IVF) in woman with or without PCOS. After culturing with PMA, Ionomycin and Golgi stop agent, cells were detected for the intracellular cytokine production by flow cytometry. The profile of Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10) cytokines of CD3(+) CD4(+)T lymphocyte subsets were analyzed through invert gating. These cytokines in FF were also evaluated by ELISA. RESULTS: Flow cytometry analysis showed that the production of Th1 (IFN-γ, IL-2) cytokines in FF lymphocytes in PCOS patients were significantly higher than those in controls; ELISA result also demonstrated that the concentration of Th1 cytokines (IFN-γ, IL-2) in FF in PCOS patients is significantly increased compared with those in controls. CONCLUSION: It is concluded that the immune dominance of Th1 may be the immunological feature of the ovary in PCOS patients. It might participate in the immune pathogenesis in the ovary of PCOS patients. These results suggest that chronic inflammation maybe one of the underlying mechanism for the pathogenesis of PCOS.