Follicular fluid concentrations of interleukin-12 and interleukin-8 in IVF cycles

FERTILITY AND STERILITYt VOL. 74, NO. 5, NOVEMBER 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Follicular fluid concentrations of interleukin-12 and interleukin-8 in IVF cycles M. Rafet Gazvani, M.R.C.O.G.,a Michelle Bates, M.Sc.,b Gillian Vince, Ph.D.,b Stephen Christmas, Ph.D.,b D. Iwan Lewis-Jones, M.D.,a and Charles Kingsland, M.R.C.O.G.a Liverpool Women’s Hospital and University of Liverpool, Liverpool, United Kingdom Objective: To investigate the role of interleukin-12 (IL-12) and IL-8 in the periovulatory follicular fluid during in vitro fertilization cycles. Design: A prospective study. Setting: Reproductive Medicine Unit, Liverpool Women’s Hospital, United Kingdom. Patient(s): Women undergoing in vitro fertilization treatment. Intervention(s): IL-8 and IL-12 concentrations in follicular fluid samples that had been collected during transvaginal oocyte retrieval were measured using an enzyme-linked immunosorbent assay (ELISA). Cytokine concentrations were correlated to fertilization rates and treatment outcome. Main Outcome Measure(s): Fertilization rates and ultrasonographic evidence of intrauterine pregnancy by 4 weeks after embryo transfer. Result(s): Failed fertilization in women with detectable IL-12 was significantly higher (45.5%) than in the IL-12 negative group (6.1%), P5.01. None of the women with detectable IL-12 achieved a pregnancy at the end of the treatment (P5.01). IL-8 was present in the follicular fluid of all women, and no difference in its concentrations was found between the pregnant and nonpregnant groups. No correlation was found between the follicular fluid concentrations of IL-8 and fertilization rates. Conclusion(s): The presence of IL-12 in the follicular fluid appears to be associated with a negative outcome in IVF treatment. Interleukin-8 appears to be an essential part of folliculogenesis, although its concentration is not associated with fertilization or implantation rates. (Fertil Sterilt 2000;74:953– 8. ©2000 by American Society for Reproductive Medicine.) Key words: Follicular fluid, interleukin-12, interleukin-8, in vitro fertilization Received February 22, 2000; accepted May 10, 2000. Reprint requests: Dr. M. R. Gazvani, M.R.C.O.G., University of Aberdeen, Department of Obstetrics and Gynecology, Aberdeen Maternity Hospital, Aberdeen AB25 2ZD, United Kingdom (FAX: 441224 680 880; E-mail: m.r.gazvani@abdn.ac.uk). a Reproductive Medicine Unit, Liverpool Women’s Hospital. b Department of Immunology, University of Liverpool. 0015-0282/00/$20.00 PII S0015-0282(00)01538-7 Immune cells and immunomodulatory cytokines have been shown to be active in several physiological processes in the reproductive tract (1). In the ovary, the immune system seems to play an integral part in physiological processes such as ovulation and luteolysis (2, 3). Ovulation has many features in common with an inflammatory reaction, including the participation of leukocytes and classic inflammatory mediators, such as eicosanoids, histamine, and bradykinin (4). Several lines of evidence now support a role for a regulated cytokine network in the ovulatory process, where leukocyte infiltration and activation may be involved in site-specific tissue degradation and subsequent tissue reorga- nization (5). Cytokines such as interleukin-1 (IL-1), IL-2, IL-6, TNF-a, IFN-g, and GMCSF have been shown to play important roles in ovulation (3). In humans, it has also been shown that follicular fluid exerts chemotactic activity toward neutrophilic granulocytes and that the concentration of this activity is related to the outcome of in vitro fertilization (IVF) treatment (6). Interleukin-12 is a disulphide-linked heterodimeric cytokine composed of a 35-kDa light chain (p35) and a 40-kDa heavy chain (p40) (7), produced by B-cells, phagocytic cells, and other antigen-presenting cells (8). Interleukin-12 acts at different stages of the immune response and is involved in both its 953 induction and maintenance (9). In an immune response, effector functions are controlled by distinct helper T-cell subsets, known as Th-1 and Th-2 cells, which are characterized by the patterns of cytokines they produce. Th-1 cells are primarily responsible for cell-mediated immune responses, whereas Th-2 cells are predisposed to a humoral response. Interleukin-12 has been shown to be a regulatory molecule in the immune system as the key switch cytokine for the induction of Th-1 responses. It has profound effects on cellular immunity, in particular on T-cells and natural killer (NK) cells. In addition, IL-12 increases the production of cytokines, particularly INF-g, another biological response modifier, which in turn augments the killing ability of these key cells of the immune system (10). Interleukin-12 has also been reported to inhibit angiogenesis in animal models (11, 12). Its key role in immune regulation and its antiangiogenic/antitumor effects has suggested its use in patients with tumors, allergy, or immunodeficiencies (13–15). To date, no studies have been conducted on the role of IL-12 in folliculogenesis, ovulation, fertilization, or implantation. Interleukin-8 is a potent angiogenic, proinflammatory, growth-promoting cytokine (16). Three studies to date (5, 17, 18) have investigated IL-8 levels in the follicular fluid and plasma; these investigators found increased levels of IL-8 in the periovulatory follicular fluid. A positive correlation was also found between follicular fluid and plasma concentrations of IL-8 (5). However, these studies made no reference to fertilization or implantation rates with respect to IL-8 concentrations in the follicular fluid. Ample evidence exists regarding the similarities between inflammatory reactions and ovulation. Interleukin-12 and IL-8 both are potent immunomodulatory cytokines involved in inflammatory processes. The high intrafollicular concentrations of IL-8 further highlight the intraovarian cyclic events as physiological inflammatory reactions. We therefore organized a prospective study to measure IL-12 and IL-8 concentrations in the periovulatory follicular fluid of women who were undergoing ovarian stimulation during in vitro fertilization cycles. The findings were correlated to the outcome of treatment with respect to fertilization and pregnancy rates. cluded in the analysis. Patients who had visually bloodstained follicular fluid throughout the collection were excluded from the study (n 5 8). In the 44 women who participated, the indication for IVF was unexplained infertility in 48% (n 5 21), tubal disease in 30% (n 5 13), endometriosis in 18% (n 5 8), and anovulation in 4% (n 5 2). Ovarian stimulation was facilitated by the standard “long protocol.” Women were initially treated with intranasal nafarelin acetate (Synarel, Syntex, Berks, UK) twice daily starting from the 23rd day of the preceding cycle. After confirmation of down-regulation, folliculogenesis was subsequently stimulated by daily subcutaneous injections of gonadotropins (Humegon, Organon, Cambridge, UK) for 9 –10 days. Adequate stimulation was confirmed by ultrasound examination and assessment of serum estradiol concentrations on the 9th or 10th day of stimulation. Follicular puncture and aspiration of follicular fluid, via the vaginal route, from follicles .15 mm in diameter were performed under ultrasound guidance. These procedures took place 36 –37 hours after i.m. administration of human chorionic gonadotropin (hCG) (5000 IU) (Profasi, Serono, Herts, UK). Standard insemination was used for all oocytes, and successful fertilization was defined as the presence of two pronuclei and two polar bodies 16 –20 hours after insemination. Embryos were transferred 48 hours after oocyte retrieval. The embryos were assessed morphologically, and two to three (usually two) embryos with the highest grades were transferred. Following the transfer, luteal support was provided by vaginal progesterone pessaries (Cyclogest, Hoechst, Middlesex, UK). The end point of the study was ultrasonographic evidence of an intrauterine pregnancy and the presence of a fetal heartbeat within 6 weeks following the embryo transfer. Four to six vials of visually blood-free samples of follicular fluid were selected from each patient. The samples were then centrifuged at 400g for 10 minutes, and the supernatants were combined and frozen at –20°C until they could be analyzed. Fifty-two women who were undergoing IVF treatment between February and April 1998 at the Reproductive Medicine Unit, Liverpool Women’s Hospital, United Kingdom were recruited to take part in the study. None of the patients’ partners had any history of male factor infertility. Approval from the local Ethics Committee was obtained before the study began. Interleukin-8 concentrations in follicular fluid samples were measured using an enzyme-linked immunosorbent assay (ELISA) (Innotest for IL-8; Innogenetics N.V., Zwijndrecht, Belgium). The ELISA was designed specifically to measure immunoreactive IL-8 in body fluids. Concentrations were measured according to the manufacturer’s instructions. The standard was recombinant human IL-8 (Innotest for IL-8; Innogenetics N.V., Zwijndrecht, Belgium), and the concentration range used in the standard curve was 15–1500 pg/mL. This ELISA recognizes both the 72 and 77 amino acid forms of IL-8 and has a detection limit of around 10 pg/mL. Forty-four consecutive women with an adequate amount of visually blood-free samples of follicular fluid were in- Interleukin-12 concentrations were measured using an ELISA (Monoclonal Anti-human IL-12 p70 Antibody, R&D MATERIALS AND METHODS 954 Gazvani et al. Interleukin-12 and interleukin-8 in follicular fluid Vol. 74, No. 5, November 2000 Systems, Abingdon, UK) designed specifically to measure IL-12 in body fluids. Concentrations were measured according to the manufacturer’s instructions. The standard was recombinant human IL-12 (R&D Systems, Abingdon, UK), and the concentration range used in the standard curve was 7.8 –1000 pg/mL. The detection limit was around 2 pg/mL. Patient demographics and follicular fluid cytokine levels. Samples were analyzed in duplicate, both neat and diluted 1:10, and OD values falling within the linear portion of the standard curve were used to estimate sample IL-8 and IL-12 concentrations by interpolation. Mean (6SD) age (y) 34.7 6 4.2 Median parity (IQR) 0 (0–2) Median IL-12 concentration (pg/mL) (IQR) ,2 Mean (6SD) IL-8 concentration (pg/mL) 256.9 6 336 Statistical Analysis Gazvani. Interleukin -12 and -8 in follicular fluid. Fertil Steril 2000. Nonparametric data were described as median (interquartile range [IQR]) and parametric data as mean (standard deviation [SD]). An unpaired Student’s t-test was used to compare means, and a Mann-Whitney U test was used for medians. The Pearson correlation test was used to assess the relationship between two variables for parametric data; for nonparametric data, Spearman’s test was used. and 45.1% (SD: 28.2) in the IL-12-positive group, suggesting no difference (Table 2). No correlation existed between IL-12 concentrations in follicular fluid and fertilization rates, r 5 0.02 (Fig. 1). RESULTS The mean (6SD) age of women undergoing treatment was 34 years (3.6), and median parity was 0 (0 –2). After one treatment cycle, oocytes were successfully retrieved in all cases (n 5 44). Oocyte retrieval was accomplished 36 –37 hours after i.m. administration of hCG (5000 IU) (Profasi; Serono, Rome, Italy). There were no cases of ovarian hyperstimulation. In 37 women (84%), successful fertilization occurred, with a mean (6SD) fertilization rate of 51.9% (SD: 26.6), while seven women (16%) experienced “failed” fertilization. The mean (6SD) fertilization rate for women with unexplained infertility was 46.5% (SD: 28.6), whereas it was 56.1% (SD: 22.7) in women with tubal disease, 47.2% (SD: 35.5) in women with endometriosis, and 58% (SD: 16) in women with anovulation. Therefore, 37 women (84%) had a transfer of two to three embryos (two embryos, 82% and three embryos, 18%). Eleven women (25%) had a positive pregnancy test and ultrasonographic evidence of an intrauterine pregnancy within the first 6 weeks following the embryo transfer. No age difference existed between women who conceived and those who did not; mean (6SD) age was 34.7 (SD: 4.2) and 33.1 (SD: 3.2), respectively (Table 1). Interleukin-12 was detectable in the follicular fluid of 11 women (25%) and not detectable in the remaining 33 (75%); median, ,2 pg/mL (,2– 4.5). Mean (6SD) number of oocytes obtained was 7.6 (SD: 2.9) and 6.9 (SD: 2.9), respectively. Failed fertilization occurred in five women (45.5%) with detectable IL-12 and in two women (6.1%) with no detectable IL-12 in follicular fluid, P5.01. In those women who were successfully fertilized, the mean (6SD) fertilization rates were 53.1% (SD: 24.5) in the IL-12-negative group FERTILITY & STERILITYt TABLE 1 Variable Pregnant (n 5 11) Not pregnant (n 5 33) 33.1 6 3.2 0 (0–2) ,2 (,2–17) 321 6 333 Note: IQR 5 Interquartile range. None of the women who had detectable IL-12 in their follicular fluid (n 5 11) achieved a pregnancy; five of these women had failed fertilization, and six experienced failed implantation. Of 33 women with no detectable IL-12 in the follicular fluid, 11 (33%) had ultrasound evidence of an intrauterine pregnancy within 6 weeks following treatment (P5.01) (Table 2). This result confirms that an association exists between the undetectable levels of IL-12 and the outcome of treatment (Fig. 2). Interleukin-8 could be detected in the follicular fluid of all women. No correlation was found between the fertilization rates and follicular fluid concentrations of IL-8, r 5 – 0.2 (Fig. 3). No difference was found between the mean (6SD) IL-8 concentrations of pregnant and nonpregnant women, which were 256.9 pg/mL (SD: 336) and 321 pg/mL (SD: 333), respectively (Fig. 4). No correlation was found between the follicular fluid concentrations of IL-12 and IL-8, r 5 – 0.07. Furthermore, IL-12 and IL-8 concentrations did not show a significant difference in women who had endometriosis (n 5 8) compared to women without endometriosis (n 5 34). TABLE 2 IL-12 in follicular fluid and outcome of treatment. Variable Total number of oocytes obtained Mean (6SD) number of oocytes Number of oocytes fertilized Number of cases of failed fertilization (%) Mean (6SD) percentage fertilization ratea Pregnancy a IL-12 detectable (n 5 11) IL-12 not detectable (n 5 33) P 84 228 7.6 6 2.9 6.9 6 2.9 25 115 5 (45.5) 2 (6.1) .01 45.1 6 28.2 53.1 6 24.5 0 11 .01 In cases where fertilization occurred. Gazvani. Interleukin -12 and -8 in follicular fluid. Fertil Steril 2000. 955 FIGURE 1 FIGURE 2 Follicular fluid concentration of IL-12 and fertilization rate. Follicular fluid concentration of IL-12 and outcome of treatment. Gazvani. Interleukin -12 and -8 in follicular fluid. Fertil Steril 2000. Gazvani. Interleukin -12 and -8 in follicular fluid. Fertil Steril 2000. DISCUSSION In this study we showed that IL-12 was present in the follicular fluid of around 25% of women undergoing IVF treatment. The incidence of fertilization failure was higher in women who had IL-12 in the follicular fluid. When fertilization occurred, on the other hand, rates of fertilization did not seem to be significantly affected by the presence or the concentration of IL-12. None of the women who had a positive IL-12 measurement achieved a pregnancy at the end of treatment. Because all samples stained with blood were excluded from the study, the presence of IL-12 or IL-8 in the follicular fluid cannot be attributed to contamination with peripheral blood. of IL-12, together with its down-regulating effects on Th-2 response, are the underlying cause of impaired implantation. Our findings suggest that the presence of IL-12 in follicular fluid has a strong association with the negative outcome of IVF treatment. IL-12, when present in follicular fluid, FIGURE 3 Follicular fluid concentration of IL-8 and fertilization rate. Interleukin-12 has recently been identified in human preovulatory follicular fluid, and it was noted that levels were much lower in preovulatory than in immature follicles (19). It is difficult to suggest that in our study the negative treatment outcome for all women with detectable IL-12 in the follicular fluid was due solely to the immaturity of all follicles. It is, therefore, plausible to suggest that the negative effect of IL-12 on fertilization may be related to its presence in at least some of the follicles collected. Alternatively, high levels of IL-12 may indicate an ongoing inflammatory response. Interleukin-12 is important in the initiation of a Th-1 response and, indirectly, in down-regulation of a Th-2 response. The latter response is thought to be compatible with successful pregnancy, whereas a Th-1 may be detrimental to implantation. It is possible that the antiangiogenic properties 956 Gazvani et al. Gazvani. Interleukin -12 and -8 in follicular fluid. Fertil Steril 2000. Interleukin-12 and interleukin-8 in follicular fluid Vol. 74, No. 5, November 2000 FIGURE 4 Follicular fluid concentration of IL-8 and outcome of treatment. induced ovulation as well as neutrophil infiltration into the ovaries (20). Our findings suggest that IL-8 is present during folliculogenesis and ovulation, which is in agreement with previously published data. Its concentration, however, does not appear to be important regarding the outcome of treatment. This finding is somewhat contrary to previously published data which implied a direct relationship between IL-8 concentrations and follicular development. Larger studies with data on individual follicles may help identify the physiological role of IL-8 in folliculogenesis, fertilization, and implantation. We conclude that both IL-12 and IL-8 are powerful cytokines that appear to influence folliculogenesis. The presence of IL-12 in follicular fluid is associated with a negative outcome in IVF treatment, which may be an evidence of the immaturity of follicles as well as negative effects of IL-12 on folliculogenesis, oocyte quality, and/or implantation. Gazvani. Interleukin -12 and -8 in follicular fluid. Fertil Steril 2000. could affect folliculogenesis and/or implantation and, hence, the outcome of the cycle. Coskun et al. (19) also suggested that no correlation existed between IL-12 and cycle outcome. They conducted a relatively small study involving 24 cases of preovulatory follicular fluid collection; IL-12 was not detectable in 12 (50%) of the participants. No statistical difference was found between pregnant and nonpregnant groups regarding the mean (6SD) concentrations of IL-12, which is in agreement with our results (Table 1). Information regarding the incidence of pregnancy in the groups with and without detectable IL-12, however, was not provided. Interleukin-8 was present in the follicular fluid in all cases. We found no correlation between the follicular fluid concentrations of IL-8 and fertilization rates or the treatment outcomes. To our knowledge, this is the first study that correlates follicular fluid cytokine immunoreactivity to the outcome of IVF treatment. In a previous study, Arici et al. (17) showed that follicular fluid IL-8 concentrations were 14-fold higher than serum IL-8 levels with a positive correlation between them. They also showed that both granulosa–lutein and ovarian stromal cells expressed the mRNA for IL-8 and produced the protein. Modulation of IL-8 in these cell cultures by steroid and trophic hormones suggested that IL-8 may play an important role in the physiology of ovulation, such as aiding follicular rupture and corpus luteum neovascularization. In a study of ovulation in the rabbit, neutralization of IL-8 inhibited hCGFERTILITY & STERILITYt Interleukin-8 appears to be involved in the natural process of folliculogenesis, yet its concentration does not have a demonstrable effect on the outcome. This new information may bring indications for the manipulation of these molecules toward therapeutic use in assisted reproduction to improve fertilization and pregnancy rates. Our results should be considered in the light of the fact that pooled fluid from four to six individual follicles from each woman was used in the analysis. More detailed information can be collected if fluid from each individual follicle is tested separately. This separate testing is technically difficult to achieve, however, as there is always fluid left within the collection tubes even after a follicle is emptied. Future studies should aim to determine the exact role of IL-8 and IL-12 on folliculogenesis and implantation in humans. 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Cytokines in the follicular fluid of stimulated and non-stimulated human ovaries; is ovulation a suppressed inflammatory reaction? Hum Reprod 1999;14:162– 6. 19. Coskun S, Uzumcu M, Jaroudi K, Hollanders JM, Parhar RS, al-Sedairy ST. Presence of leukemia inhibitory factor and interleukin-12 in human follicular fluid during follicular growth. Am J Reprod Immunol 1998; 40:13– 8. 20. Ujioka T, Matsukawa A, Tanaka N, Matsuura K, Yoshinaga M, Okamura H. Interleukin-8 as an essential factor in human chorionic gonadotrophin-induced rabbit ovulatory process: interleukin-8 induces neutrophil accumulation and activation in ovulation. Biol Reprod 1998;58: 526 –30. Interleukin-12 and interleukin-8 in follicular fluid Vol. 74, No. 5, November 2000