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Interleukin-18 levels correlate with severe ovarian hyperstimulation syndrome
FERTILITY AND STERILITY威 VOL. 82, NO. 2, AUGUST 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Interleukin-18 levels correlate with severe ovarian hyperstimulation syndrome Vivian Barak, Ph.D.,a,b Uriel Elchalal, M.D.,c Michal Edelstein, M.D.,a,b Inna Kalickman, M.Sc.,a,b Aby Lewin, M.D.,c and Yoram Abramov, M.D.c Hadassah Hebrew University Medical Center, Jerusalem, Israel
Objective: To assess the involvement of interleukin-18 (IL-18) in the pathophysiology of severe ovarian hyperstimulation syndrome (OHSS), and study its use as a marker of disease and its correlation to capillary hyperpermeability. Design: Prospective controlled study. Setting: An IVF unit in a tertiary medical center. Patient(s): Twenty-four patients with OHSS in an IVF program, two control groups: group 1, 40 healthy age-matched women without ovulation-induction treatment; group 2, 19 women who received the same ovulation-induction regimen without experiencing OHSS. Intervention(s): Blood samples were obtained at three times: during acute OHSS, on significant clinical improvement, after complete resolution. Ascitic and pleural fluids were obtained by therapeutic paracentesis. Serum, peritoneal, and pleural fluids were analyzed for IL-18 and IL-6, and blood for hematocrit, white blood cell count, and E2 levels. Main Outcome Measure(s): Hematocrit white blood cell count, serum, peritoneal, pleural fluid levels of IL-18, IL-6, E2 in severe OHSS. Result(s): Significantly higher IL-18 levels were detected in serum, peritoneal, and pleural fluids of patients with severe OHSS as compared with both control groups. Serum IL-18 dropped significantly on transition to the diuretic phase and resolution. A statistically significant correlation between serum IL-18 and hyperpermeability characteristics (white blood cell count, hematocrit), serum E2, and IL-6 levels was recorded. Conclusion(s): This is the first study suggesting a role of IL-18 as a marker of OHSS, with correlation to capillary hyperpermeability parameters. (Fertil Steril威 2004;82:415–20. ©2004 by American Society for Reproductive Medicine.) Key Words: IL-18, inflammatory cytokines, OHSS, ovulation induction, hyperpermeability Received April 26, 2003; revised and accepted March 8, 2004. Reprint requests: Vivian Barak, Ph.D., Immunology and Tumor Diagnosis Laboratory Oncology Department, Hadassah Hebrew University Medical Center, Jerusalem, Israel (FAX: 972-2-6435-308; Email: [email protected]). a Immunology and Tumor Diagnosis Laboratory, Oncology Department. b Israeli Cytokine Standardization Laboratory. c Obstetrics and Gynecology Department. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 03.024
Severe ovarian hyperstimulation syndrome (OHSS) is a complication occurring in approximately 0.5% to 1% of patients undergoing ovulation induction (1, 2). This rare but potentially life-threatening iatrogenic disorder is characterized by release of ovarian-derived offensive mediators to the bloodstream and third space, which causes endothelial damage and increased capillary permeability. This in turn results in massive fluid shifts from the intravascular compartment to the third space, with subsequent hypovolemia and hemoconcentration (3). Our group and others have shown that certain inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-␣ (TNF␣) (4, 5), and growth factors such as vascular endothelial growth fac-
tor (VEGF) (6 –9), and adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin (10, 11) are extremely elevated. According to their characteristic functions, they probably play a major role in the pathophysiology of endothelial damage and capillary hyperpermeability in OHSS. An additional factor that contributes to vasodilatation, neurotransmission, and cytotoxic activities is nitric oxide (NO), which we have previously shown to be decreased in the peritoneal fluid of patients with OHSS (4). Interleukin-18 (IL-18), initially described as an interferon-gamma (IFN␥) inducing factor, is a relatively newly discovered proinflammatory cytokine starting the cascade of additional inflammatory cytokines (12). Interleukin-18 can stimulate responses mediated by both T-helper 415
type 1 (TH1) and T-helper type 2 (TH2) (13, 14). On the basis of the structural homology, receptor family, intracellular transduction pathway, and biological effects, IL-18 is considered a member of the IL-1 family (13) and participates in both innate and acquired immunity. Interleukin-18 is synthesized as an inactive precursor, pro-IL-18, which is cleaved by IL-1-converting enzyme/caspase-1 (13). Although IL-18 exerts some of its proinflammatory effects through induction of IFN␥, recent data suggest also that IL-18 has direct proinflammatory properties in diverse cell types, including the ovary. It has been suggested that biologically active IL-18 production is a feature of the normal ovarian surface epithelium (15). Tsuji et al. (16) found that the expression of IL-18 and its receptor are very low in immature murine ovaries. Ovulation induction with pregnant mare’s serum gonadotropin and hCG caused a dramatic increase in the expression of both compounds from theca cells. In respect to direct effects, IL-18 stimulates activation of the transcription factor NF-kB and induces the cascade of proinflammatory cytokines such as TNF␣, IL-1, IL-6, and IL-2 receptor; it promotes nitric acid (NO) production, which may explain our former results (4, 17–20). In addition, chemokines such as IL-8 and macrophage inflammatory protein 1 alpha (MIP-1␣), and adhesion molecules such as ICAM-1 are also up-regulated (13, 21). An IL-18 binding protein (IL-18 BP) has been identified that serves as a modulator of IL-18 activities and therefore plays a role in the reduction of TH1 cytokine responses (22). Both IL-18 and IL-18 BP have been shown to play protective roles in host defense against a variety of intracellular microbes and a pathological role in some autoimmune diseases such as rheumatoid arthritis, Crohn disease, and multiple sclerosis (23). We have also shown that IL-18 is extremely elevated in acute graft-versus-host disease (GVHD) patients and that anti-IL-18 antibodies decrease GVHD in a mouse model (24). Treatment with an anti-IL-18 receptor monoclonal antibody during ovarian stimulation reduced the number of ovulated ova and inhibited the expansion of cumulus cells surrounding the ovum. Both IL-18 and IL-18 receptor may therefore play a role in the process of folliculogenesis and ovulation (16). Interleukin-18 has been reported also to play an important role in the pathophysiology of vascular hyperpermeability in several inflammatory diseases, including rheumatoid arthritis (23), inflammatory bowel disease (25), and chronic rhinosinusitis (26). Increased levels of IL-18 were also found in patients with minimal change nephrotic syndrome (27) and lupus nephritis (28). These levels correlated with the activity of the disease and with glomerular vascular hyperpermeability. Jordan et al. (29) reported upregulation of IL-18 mRNA in inflamed rat lung. Interleukin-18 was found to induce the production of vascular-cell adhesion molecule-1 (VCAM-1) (30), and was also found to induce secretion of vascular permeability factor in patients with minimal change nephrotic syndrome (27). 416
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IL-18 correlates with severe OHSS
The present study assessed the potential role of IL-18 levels as a marker of severe OHSS and its correlation with capillary hyperpermeability parameters.
MATERIALS AND METHODS Participants Inclusion criteria were massive ovarian enlargement (mean ovarian size: 13.2 ⫾ 1.5 cm); massive ascites (13 patients); hydrothorax (4 patients); and laboratory evidence of hemoconcentration, as demonstrated by increased hematocrit (mean: 44.7 ⫾ 1.4%) or leukocyte count (mean: 26.2 ⫾ 1.8 cell/mm3). Other criteria for severe OHSS included oliguria (8 patients), azothemia (1 patient), liver dysfunction (4 patients), adult respiratory distress syndrome (ARDS; 1 patient), and venous thromboembolism (two patients). Seven patients had moderate to severe gastrointestinal symptoms, including diarrhea or vomiting. The ovulation induction regiment leading to OHSS was similar in all 24 patients. Nafarelin acetate as a nasal spray (Synarel; Syntex Laboratories Inc., Palo Alto, CA) 200 g, three times daily, was started in the midluteal phase of the cycle. When pituitary suppression was documented, as evident by serum estradiol (E2) level ⬍30 pg/mL (110 pmol/L) and serum P ⬍0.3 ng/mL (1 nmol/L), IM hMG (Pergonal; Teva Pharmaceutical, Ltd., Petach-Tikva, Israel) was added at 300 IU/day. Serum E2 and P levels, serial pelvic examinations, and transvaginal sonography were used to monitor patients as required. Doses of hMG were adjusted after 5 days of administration, according to patients’ individual response. Intramuscular hCG (Chorigon; Teva Pharmaceutical, Ltd.) was administered 36 hours before oocyte retrieval. Luteal phase support consisted of IM P (Gestone; Panes & Byrne, Greenford, United Kingdom) supplementation, 50 mg/day. None of the patients received hCG for luteal support. All 24 patients were hospitalized at the Hadassah Hebrew University Medical Center, Jerusalem, Israel, for severe OHSS and were treated with intravenous colloid and crystalloid solutions. Colloid solutions usually given were Polygeline 3.5% (Haemacel; Behringwerke AG, Marburg, Germany) and human albumin 25% (Kamada; Kibutz Beit Kama, Israel). The crystalloid solution used in all OHSS patients was normal saline 0.9%, which was administered daily and titrated according to urine output and hemodynamic variables. Serial blood counts, serum electrolyte measurements, liver and kidney function tests, and coagulation screening were performed daily. Ovarian size and the amount of ascitic fluid were assessed by transvaginal and transabdominal sonography. Pleural effusion was assessed by physical examination and was verified by chest x-ray. Four of the 24 patients required hospitalization in the intensive care unit for various time intervals (2 to 7 days) for hemodynamic imbalance, oliguria, or deteriorating respiraVol. 82, No. 2, August 2004
tory status. All 24 patients required therapeutic abdominal paracentesis for increasing dyspnea related to ascites. Total hospitalization intervals ranged from 10 to 25 days (mean: 17.5 days). Pregnancies related to the same ovulation induction treatment course were recorded in 18 patients. We enrolled two control groups for this study. The first control group consisted of 40 healthy women, 28.3 ⫾ 4.1 years old, who were matched to the study group for age and parity (control group 1). All of these women were nonpregnant, had regular periods, had no history of infertility, and did not use hormonal contraception. The second group consisted of 19 women who had received the same ovulation induction treatment for IVF without developing OHSS (control group 2). The mean E2 level on the day of hCG administration was 2,240 ⫾ 180 pg/mL. All women underwent oocyte retrieval (mean number of oocytes retrieved: 8 ⫾ 3) and subsequent ET (mean number of embryos transferred: 2.6 ⫾ 0.6). Luteal support was similar to that described for the study group. Ten pregnancies related to the same ovulation-induction treatment course were recorded in this control group.
Blood, Peritoneal, and Pleural Fluid Samples Three blood samples were obtained by venipuncture from each patient of the OHSS group. Sample 1 was obtained on admission for severe OHSS (acute phase, mean time after ET: 7.5 ⫾ 2.4 days). Sample 2 was obtained upon transition to the diuretic phase as evident by a negative body fluid balance, substantial weight loss (⬎50% of previous weight gain), and complete resolution of hemoconcentration; this convalescence phase occurred on hospitalization days 7 to 14 (mean: 9.7 days). Sample 3 was obtained on the first visit to our clinic 7 days after discharge from the hospital (resolution phase). One blood sample was obtained from each patient in control group 1, 4 to 8 days after ET. One blood sample was obtained from each woman in control group 2 during the midluteal phase (day 21). Samples were analyzed for hematocrit (HCT %); leukocyte count (WBC ⫻ 103); and levels of E2, IL-6, and IL-18. Therapeutic pleural paracentesis was performed in four patients. In addition, peritoneal and pleural fluids were analyzed for IL-6 and IL-18 levels by ELISA (R&D Systems, Inc., Minneapolis, MN), as we have previously discussed elsewhere (5–7). Informed consent, approved by the institutional human subjects review boards committee, was obtained from all study participants.
Statistical Analysis Unpaired t-tests were used to compare group mean values for IL-6, IL-18, hematocrit, and leukocyte count in the OHSS and control groups. Correlations between IL-18 levels and OHSS variables, including hematocrit, leukocyte count, and serum E2 and IL-6 levels, were tested using the Pearson’s product moment correlation test. P⬍.05 was considered statistically significant for all comparisons. FERTILITY & STERILITY威
FIGURE 1 Interleukin-18 (IL-18) levels in serum, peritoneal, and pleural fluids during the acute phase of OHSS (red colors) as compared to serum of IVF patients not developing OHSS (control group 2) and of healthy controls (control group 1) (green). Values are presented as mean ⫾ SE. Asterisks indicate statistically significant differences, as compared with both control groups (P⬍.05).
Barak. IL-18 correlates with severe OHSS. Fertil Steril 2004.
RESULTS Twenty-four patients were included in the study, all of whom were healthy infertile women, 28.5 ⫾ 4.5 years old, who had undergone ovulation induction for IVF. All presented with severe OHSS according to the classification systems of Golan et al. (1) and Navot et al. (2). Markedly elevated serum E2 levels (mean: 5,600 ⫾ 320 pg/mL) were recorded in all study patients. Number of follicles on the day of oocyte retrieval (22 ⫾ 5 vs. 19 ⫾ 4 in the OHSS and control group 2, respectively) and number of oocytes retrieved (18 ⫾ 4 vs. 14 ⫾ 4) were not statistically different between the two groups. Indeed, 18 (75%) of 24 patients of the OHSS group were pregnant in this study. These findings are consistent with our previously published data on pregnancy rates in severe OHSS (31). The IL-18 levels were not statistically significantly different between pregnant and nonpregnant patients. The IL-18 levels of serum, pleural, and peritoneal fluids during the acute phase of OHSS were all found to be significantly (P⬍.04) elevated as compared with both control groups (Fig. 1). The IL-18 levels were higher in control group 2, as compared with control group 1. In patients with OHSS, IL-18 serum levels decreased significantly along with clinical convalescence, and even more so upon resolution, as shown in Figure 2. Serum IL-6 levels showed similar kinetics to IL-18 levels, with substantially elevated levels during the acute phase that dropped significantly along with clinical convalescence and resolution (Fig. 3). 417
DISCUSSION
FIGURE 2 Serum interleukin-18 (IL-18) levels during the acute (red), convalescence (blue), and resolution (green) phases of OHSS. Values are presented as mean ⫾ SE. Asterisks indicate statistically significant differences between the acute and both convalescence and resolution phases (P⬍.05).
Barak. IL-18 correlates with severe OHSS. Fertil Steril 2004.
The clinical parameters correlated to hyperpermeability evaluated in patients with OHSS, included HCT and WBC. Serum E2 levels were recorded as well. All were substantially elevated during the acute phase, dropping significantly with convalescence and even more so after complete resolution (Fig. 4). Statistically significant correlations (P⬍.05) were found between serum IL-18 and serum IL-6 levels in patients with OHSS. Serum IL-18 levels also significantly correlated with serum E2 levels, hematocrit, and white blood cell count (P⬍.05 for all comparisons).
FIGURE 3 Serum interleukin 6 (IL-6) levels during the acute (red), convalescence (blue), and resolution (green) phases of OHSS. Values are presented as mean ⫾ SE. Asterisks indicate statistically significant differences (P⬍.05).
Barak. IL-18 correlates with severe OHSS. Fertil Steril 2004.
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It has recently been suggested that a complex communication network connects the reproductive and immune systems, and controls ovarian function. The ovulatory process itself has been perceived as a physiologic inflammatory response, characterized by migration of leukocytes into ovulatory follicles (32). Both corpus luteum formation and luteal regression involve progressive infiltration of lymphocytes and macrophages, and communication through chemokines, cytokines, and cell adhesion molecules (32). We and others have shown cytokines to mediate some important ovarian functions in general, and specifically the IL-1 family in the process of ovulation (17, 33). We have previously reported high amounts of various cytokines in the follicular fluid of patients undergoing ovulation induction and studied their correlation to E2 concentrations (18, 19). In extreme cases of ovulation induction, the potentially life-threatening complication of OHSS, characterized by hypovolemia and hemoconcentration, may occur (1, 2). It has been suggested that the principal pathophysiologic mechanism behind OHSS is capillary hyperpermeability, expressed by a massive shift of fluids and proteins from the intravascular compartment to the third space (34 –36), which is similar to our findings (17, 18). This in turn causes intravascular volume depletion, third-space fluid accumulation, and hemoconcentration, as well as endothelial damage induced by various cytokines, which are responsible for most of the morbidity associated with OHSS (1, 2). Interleukin-1, IL-6, IL-8, and TNF␣ have been implicated as mediators of the acute phase response occurring in sepsis, injury, and immunologic challenge (37, 38), characterized by capillary leakage that is very similar to that seen in OHSS. Other studies have found important effects of the cytokine network upon the vessel wall. Endothelial morphologic changes have been shown to occur in cytokine-treated endothelial cells. For instance, TNF␣ increases the permeability of endothelial cell monolayers to macromolecules, via an action on membrane G-proteins or by endothelial cell injury (39). More recently, we and others have suggested a cardinal role for inflammatory cytokines (IL-1, IL-6, IL-8, and TNF␣) in the pathogenesis of OHSS (4, 5). We also reported increased levels of serum ICAM-1 and decreased levels of E-selectin in patients with severe OHSS (10). Both factors showed statistically significant correlations with clinical and biological aspects of this syndrome, thereby suggesting a possible role for these adhesion molecules in the pathogenesis of OHSS (10, 11). We have also suggested a role for inflammatory cytokines in the formation of ascites and hydrothorax in Meig syndrome which, in a similar manner to OHSS, manifested with third-space fluid accumulation associated with a benign ovarian pathology (40). We performed the current study to assess the possible involvement of IL-18 in the pathophysiologic mechanisms of capillary hyperpermeability in the OHSS and study its Vol. 82, No. 2, August 2004
FIGURE 4 Changes in clinical parameters of OHSS during the acute, convalescence, and resolution phases. Hematocrit (HCT) ⫽ green line, white blood cell count (WBC ⫻ 103) ⫽ red line, serum estradiol levels (E2 ⫻ 103) ⫽ blue line.
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potential as a marker of the syndrome. The data presented herein suggest for the first time that IL-18 is probably involved in the pathophysiology of capillary hyperpermeability and third-space fluid accumulation in severe OHSS. In this study, we evaluated 24 patients who developed severe OHSS and clinically improved later in parallel to serum IL-18 kinetics. During the acute phase of OHSS, high serum IL-18 levels were recorded in all patients, accompanied by clinical features of capillary leakage and hemoconcentration (WBC, HCT). The IL-18 levels decreased significantly in parallel to clinical convalescence, after third-space fluid accumulation and hemoconcentration had disappeared (41). These results suggest the involvement of IL-18 in the process of vascular hyperpermeability seen in OHSS. The strong statistical correlation found between IL-18 levels and characteristics of capillary leakage and hemoconcentration, such as elevated hematocrit and WBC count, further supports this assumption. In patients with OHSS, IL-18 levels also correlated with IL-6 levels (shown previously by us and by others to be the best inflammatory marker), suggesting an interplay between these two cytokines in the pathogenesis of OHSS (42, 43). FERTILITY & STERILITY威
Women who did not develop OHSS after controlled ovarian stimulation (control group 2) had significantly lower serum IL-18 levels than the OHSS patients; however, their levels were more elevated than control group 1, as an effect of ovulation induction. We also found extremely high IL-18 levels in ascitic fluid obtained from affected individuals. These levels were substantially higher than the levels in serum obtained simultaneously, suggesting either a direct spill of this cytokine from the ovaries and/or peritoneum or active transport through ovarian and/or peritoneal blood vessels. Hence, IL-18 may play a role in the peritoneal acutephase responses in OHSS. Considering its pivotal role as an inducer of the inflammatory cytokine cascade, these results suggest IL-18 production may be one of the primary inflammatory events leading to capillary hyperpermeability in OHSS. The results presented herein suggest that IL-18 may serve as a possible target for immunologic treatment of severe OHSS. Anti-IL-18 treatment has many beneficial effects in diseases characterized by production of high IL-18 levels. Treatment of mice undergoing ovulation induction with 419
IL-18 receptor monoclonal antibodies during ovarian stimulation reduced the number of ovulated ova and inhibited the expansion of cumulus cells surrounding the ovum (16). It may therefore be postulated that administration of IL-18 BP or anti-IL-18 antibodies can block the IL-18 –induced inflammatory cascade activation and thereby decrease endothelial damage and capillary hyperpermeability in OHSS. This potential clinical application of our study must be tested in animal models before application to humans can be safely performed. In conclusion, the results presented in this study suggest a close association between IL-18, a primary mediator and inducer of the inflammatory cytokine cascade, and parameters of capillary hyperpermeability in severe OHSS. Levels of IL-18 may well be used as markers of disease in OHSS. References 1. Golan A, Ron-el R, Herman A, Soffer Y, Weinraub Z, Caspi E. Ovarian hyperstimulation syndrome: an update review. Obstet Gynecol Surv 1989;44:430 –40. 2. Navot D, Bergh PA, Laufer N. Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil Steril 1992;58:249 –61. 3. Elchalal U, Schenker JG. The pathophysiology of ovarian hyperstimulation syndrome—views and ideas. Hum Reprod 1997;12:1129 –37. 4. Revel A, Barak V, Lavy Y, Anteby E, Abramov Y, Schenker JG, et al. Characterization of intraperitoneal cytokines and nitrites in women with severe OHSS. Fertil Steril 1996;66:66 –71. 5. Abramov Y, Schenker JG, Levin A, Friedler S, Nisman B, Barak V. Plasma inflammatory cytokines correlate to the ovarian hyperstimulation syndrome (OHSS). Hum Reprod 1996;11:1381–6. 6. Abramov Y, Barak V, Nisman B, Schenker JG. Vascular endothelial growth factor plasma levels correlate to the clinical picture in severe ovarian hyperstimulation syndrome. Fertil Steril 1997;67:261–5. 7. Geva E, Amit A, Lossing GB, Lerner-Geva L, Yovel I, Azem F, et al. Follicular fluid levels of VEGF may serve as a predictive marker for the development of ovarian hyperstimulation syndrome. J Reprod Med 1999;44:91–5. 8. McClure N, Healy DL, Rogers PAW, Sullivan J, Beaton L, Haning RV Jr, et al. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome. Lancet 1994;344:235–6. 9. Neulen J, Yan Z, Raczek S, Weindel K, Keck C, Weich HA, et al. Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 1995;80:1967–71. 10. Abramov Y, Schenker JG, Levin A, Kafka I, Jaffe H, Barak V. The role of plasma adhesive molecules (ELAM ⫹ ICAM) and biological aspects of ovarian hyperstimulation syndrome (OHSS). Fertil Steril 2001;76:51–7. 11. Daniel Y, Geva E, Amit A, Baram A, Englandern T, Kupfermine MJ, et al. Levels of soluble vascular cell adhesion molecule-1 and soluble intercellular adhesion molecule-1 are increased in women with ovarian hyperstimulation syndrome. Fertil Steril 1999;71:896 –901. 12. Lebel-Binay S, Berger A, Zinzindohoue F, Cugnenc PH, Thiounn N, Fridman WH, et al. Interleukin-18: biological properties and clinical implications (review). Eur Cytokine Netw 2000;11:15–25. 13. Dinarello CA. IL-18: A Th1 inducing, proinflammatory cytokine and new member of IL-1 family. J Allergy Clin Immunol 1999;103:11–24. 14. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin 18 regulates both Th1 and Th2 responses. Annu Rev Immunol 2001;19: 423–74. 15. Wang ZY, Gaggero A, Rubartelli A, Rosso O, Miotti S, Mezzanzanica D, et al. Expression of interleukin-18 in human ovarian carcinoma and normal ovarian epithelium: evidence for defective processing on tumor cells. Int J Cancer 2002;98:873–8. 16. Tsuji Y, Tamaoki TH, Hasegawa A, Kashiwamura S, Iemoto A, Ueda H, et al. Expression of interleukin-18 and its receptor in mouse ovary. Am J Reprod Immunol 2001;46:349 –57. 17. Barak V, Yanai P, Simon A, Roisman I, Treves AJ, Laufer N. Interleukin 1: local production and modulation of human granulosa luteal cells steroidogenesis. Fertil Steril 1992;58:719 –25. 18. Barak V, Mordel N, Holzer H, Zajicek G, Treves AJ, Laufer N. The correlation of IL-1 and TNF to estradiol, progesterone and testosterone levels in periovulatory follicular fluid (FF) of IVF patients. Hum Reprod 1992;7:462–4.
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Interleukin-18 levels correlate with severe ovarian hyperstimulation syndrome Vivian Barak, Ph.D.,a,b Uriel Elchalal, M.D.,c Michal Edelstein, M.D.,a,b Inna Kalickman, M.Sc.,a,b Aby Lewin, M.D.,c and Yoram Abramov, M.D.c Hadassah Hebrew University Medical Center, Jerusalem, Israel
Objective: To assess the involvement of interleukin-18 (IL-18) in the pathophysiology of severe ovarian hyperstimulation syndrome (OHSS), and study its use as a marker of disease and its correlation to capillary hyperpermeability. Design: Prospective controlled study. Setting: An IVF unit in a tertiary medical center. Patient(s): Twenty-four patients with OHSS in an IVF program, two control groups: group 1, 40 healthy age-matched women without ovulation-induction treatment; group 2, 19 women who received the same ovulation-induction regimen without experiencing OHSS. Intervention(s): Blood samples were obtained at three times: during acute OHSS, on significant clinical improvement, after complete resolution. Ascitic and pleural fluids were obtained by therapeutic paracentesis. Serum, peritoneal, and pleural fluids were analyzed for IL-18 and IL-6, and blood for hematocrit, white blood cell count, and E2 levels. Main Outcome Measure(s): Hematocrit white blood cell count, serum, peritoneal, pleural fluid levels of IL-18, IL-6, E2 in severe OHSS. Result(s): Significantly higher IL-18 levels were detected in serum, peritoneal, and pleural fluids of patients with severe OHSS as compared with both control groups. Serum IL-18 dropped significantly on transition to the diuretic phase and resolution. A statistically significant correlation between serum IL-18 and hyperpermeability characteristics (white blood cell count, hematocrit), serum E2, and IL-6 levels was recorded. Conclusion(s): This is the first study suggesting a role of IL-18 as a marker of OHSS, with correlation to capillary hyperpermeability parameters. (Fertil Steril威 2004;82:415–20. ©2004 by American Society for Reproductive Medicine.) Key Words: IL-18, inflammatory cytokines, OHSS, ovulation induction, hyperpermeability Received April 26, 2003; revised and accepted March 8, 2004. Reprint requests: Vivian Barak, Ph.D., Immunology and Tumor Diagnosis Laboratory Oncology Department, Hadassah Hebrew University Medical Center, Jerusalem, Israel (FAX: 972-2-6435-308; Email: [email protected]). a Immunology and Tumor Diagnosis Laboratory, Oncology Department. b Israeli Cytokine Standardization Laboratory. c Obstetrics and Gynecology Department. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 03.024
Severe ovarian hyperstimulation syndrome (OHSS) is a complication occurring in approximately 0.5% to 1% of patients undergoing ovulation induction (1, 2). This rare but potentially life-threatening iatrogenic disorder is characterized by release of ovarian-derived offensive mediators to the bloodstream and third space, which causes endothelial damage and increased capillary permeability. This in turn results in massive fluid shifts from the intravascular compartment to the third space, with subsequent hypovolemia and hemoconcentration (3). Our group and others have shown that certain inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-␣ (TNF␣) (4, 5), and growth factors such as vascular endothelial growth fac-
tor (VEGF) (6 –9), and adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin (10, 11) are extremely elevated. According to their characteristic functions, they probably play a major role in the pathophysiology of endothelial damage and capillary hyperpermeability in OHSS. An additional factor that contributes to vasodilatation, neurotransmission, and cytotoxic activities is nitric oxide (NO), which we have previously shown to be decreased in the peritoneal fluid of patients with OHSS (4). Interleukin-18 (IL-18), initially described as an interferon-gamma (IFN␥) inducing factor, is a relatively newly discovered proinflammatory cytokine starting the cascade of additional inflammatory cytokines (12). Interleukin-18 can stimulate responses mediated by both T-helper 415
type 1 (TH1) and T-helper type 2 (TH2) (13, 14). On the basis of the structural homology, receptor family, intracellular transduction pathway, and biological effects, IL-18 is considered a member of the IL-1 family (13) and participates in both innate and acquired immunity. Interleukin-18 is synthesized as an inactive precursor, pro-IL-18, which is cleaved by IL-1-converting enzyme/caspase-1 (13). Although IL-18 exerts some of its proinflammatory effects through induction of IFN␥, recent data suggest also that IL-18 has direct proinflammatory properties in diverse cell types, including the ovary. It has been suggested that biologically active IL-18 production is a feature of the normal ovarian surface epithelium (15). Tsuji et al. (16) found that the expression of IL-18 and its receptor are very low in immature murine ovaries. Ovulation induction with pregnant mare’s serum gonadotropin and hCG caused a dramatic increase in the expression of both compounds from theca cells. In respect to direct effects, IL-18 stimulates activation of the transcription factor NF-kB and induces the cascade of proinflammatory cytokines such as TNF␣, IL-1, IL-6, and IL-2 receptor; it promotes nitric acid (NO) production, which may explain our former results (4, 17–20). In addition, chemokines such as IL-8 and macrophage inflammatory protein 1 alpha (MIP-1␣), and adhesion molecules such as ICAM-1 are also up-regulated (13, 21). An IL-18 binding protein (IL-18 BP) has been identified that serves as a modulator of IL-18 activities and therefore plays a role in the reduction of TH1 cytokine responses (22). Both IL-18 and IL-18 BP have been shown to play protective roles in host defense against a variety of intracellular microbes and a pathological role in some autoimmune diseases such as rheumatoid arthritis, Crohn disease, and multiple sclerosis (23). We have also shown that IL-18 is extremely elevated in acute graft-versus-host disease (GVHD) patients and that anti-IL-18 antibodies decrease GVHD in a mouse model (24). Treatment with an anti-IL-18 receptor monoclonal antibody during ovarian stimulation reduced the number of ovulated ova and inhibited the expansion of cumulus cells surrounding the ovum. Both IL-18 and IL-18 receptor may therefore play a role in the process of folliculogenesis and ovulation (16). Interleukin-18 has been reported also to play an important role in the pathophysiology of vascular hyperpermeability in several inflammatory diseases, including rheumatoid arthritis (23), inflammatory bowel disease (25), and chronic rhinosinusitis (26). Increased levels of IL-18 were also found in patients with minimal change nephrotic syndrome (27) and lupus nephritis (28). These levels correlated with the activity of the disease and with glomerular vascular hyperpermeability. Jordan et al. (29) reported upregulation of IL-18 mRNA in inflamed rat lung. Interleukin-18 was found to induce the production of vascular-cell adhesion molecule-1 (VCAM-1) (30), and was also found to induce secretion of vascular permeability factor in patients with minimal change nephrotic syndrome (27). 416
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IL-18 correlates with severe OHSS
The present study assessed the potential role of IL-18 levels as a marker of severe OHSS and its correlation with capillary hyperpermeability parameters.
MATERIALS AND METHODS Participants Inclusion criteria were massive ovarian enlargement (mean ovarian size: 13.2 ⫾ 1.5 cm); massive ascites (13 patients); hydrothorax (4 patients); and laboratory evidence of hemoconcentration, as demonstrated by increased hematocrit (mean: 44.7 ⫾ 1.4%) or leukocyte count (mean: 26.2 ⫾ 1.8 cell/mm3). Other criteria for severe OHSS included oliguria (8 patients), azothemia (1 patient), liver dysfunction (4 patients), adult respiratory distress syndrome (ARDS; 1 patient), and venous thromboembolism (two patients). Seven patients had moderate to severe gastrointestinal symptoms, including diarrhea or vomiting. The ovulation induction regiment leading to OHSS was similar in all 24 patients. Nafarelin acetate as a nasal spray (Synarel; Syntex Laboratories Inc., Palo Alto, CA) 200 g, three times daily, was started in the midluteal phase of the cycle. When pituitary suppression was documented, as evident by serum estradiol (E2) level ⬍30 pg/mL (110 pmol/L) and serum P ⬍0.3 ng/mL (1 nmol/L), IM hMG (Pergonal; Teva Pharmaceutical, Ltd., Petach-Tikva, Israel) was added at 300 IU/day. Serum E2 and P levels, serial pelvic examinations, and transvaginal sonography were used to monitor patients as required. Doses of hMG were adjusted after 5 days of administration, according to patients’ individual response. Intramuscular hCG (Chorigon; Teva Pharmaceutical, Ltd.) was administered 36 hours before oocyte retrieval. Luteal phase support consisted of IM P (Gestone; Panes & Byrne, Greenford, United Kingdom) supplementation, 50 mg/day. None of the patients received hCG for luteal support. All 24 patients were hospitalized at the Hadassah Hebrew University Medical Center, Jerusalem, Israel, for severe OHSS and were treated with intravenous colloid and crystalloid solutions. Colloid solutions usually given were Polygeline 3.5% (Haemacel; Behringwerke AG, Marburg, Germany) and human albumin 25% (Kamada; Kibutz Beit Kama, Israel). The crystalloid solution used in all OHSS patients was normal saline 0.9%, which was administered daily and titrated according to urine output and hemodynamic variables. Serial blood counts, serum electrolyte measurements, liver and kidney function tests, and coagulation screening were performed daily. Ovarian size and the amount of ascitic fluid were assessed by transvaginal and transabdominal sonography. Pleural effusion was assessed by physical examination and was verified by chest x-ray. Four of the 24 patients required hospitalization in the intensive care unit for various time intervals (2 to 7 days) for hemodynamic imbalance, oliguria, or deteriorating respiraVol. 82, No. 2, August 2004
tory status. All 24 patients required therapeutic abdominal paracentesis for increasing dyspnea related to ascites. Total hospitalization intervals ranged from 10 to 25 days (mean: 17.5 days). Pregnancies related to the same ovulation induction treatment course were recorded in 18 patients. We enrolled two control groups for this study. The first control group consisted of 40 healthy women, 28.3 ⫾ 4.1 years old, who were matched to the study group for age and parity (control group 1). All of these women were nonpregnant, had regular periods, had no history of infertility, and did not use hormonal contraception. The second group consisted of 19 women who had received the same ovulation induction treatment for IVF without developing OHSS (control group 2). The mean E2 level on the day of hCG administration was 2,240 ⫾ 180 pg/mL. All women underwent oocyte retrieval (mean number of oocytes retrieved: 8 ⫾ 3) and subsequent ET (mean number of embryos transferred: 2.6 ⫾ 0.6). Luteal support was similar to that described for the study group. Ten pregnancies related to the same ovulation-induction treatment course were recorded in this control group.
Blood, Peritoneal, and Pleural Fluid Samples Three blood samples were obtained by venipuncture from each patient of the OHSS group. Sample 1 was obtained on admission for severe OHSS (acute phase, mean time after ET: 7.5 ⫾ 2.4 days). Sample 2 was obtained upon transition to the diuretic phase as evident by a negative body fluid balance, substantial weight loss (⬎50% of previous weight gain), and complete resolution of hemoconcentration; this convalescence phase occurred on hospitalization days 7 to 14 (mean: 9.7 days). Sample 3 was obtained on the first visit to our clinic 7 days after discharge from the hospital (resolution phase). One blood sample was obtained from each patient in control group 1, 4 to 8 days after ET. One blood sample was obtained from each woman in control group 2 during the midluteal phase (day 21). Samples were analyzed for hematocrit (HCT %); leukocyte count (WBC ⫻ 103); and levels of E2, IL-6, and IL-18. Therapeutic pleural paracentesis was performed in four patients. In addition, peritoneal and pleural fluids were analyzed for IL-6 and IL-18 levels by ELISA (R&D Systems, Inc., Minneapolis, MN), as we have previously discussed elsewhere (5–7). Informed consent, approved by the institutional human subjects review boards committee, was obtained from all study participants.
Statistical Analysis Unpaired t-tests were used to compare group mean values for IL-6, IL-18, hematocrit, and leukocyte count in the OHSS and control groups. Correlations between IL-18 levels and OHSS variables, including hematocrit, leukocyte count, and serum E2 and IL-6 levels, were tested using the Pearson’s product moment correlation test. P⬍.05 was considered statistically significant for all comparisons. FERTILITY & STERILITY威
FIGURE 1 Interleukin-18 (IL-18) levels in serum, peritoneal, and pleural fluids during the acute phase of OHSS (red colors) as compared to serum of IVF patients not developing OHSS (control group 2) and of healthy controls (control group 1) (green). Values are presented as mean ⫾ SE. Asterisks indicate statistically significant differences, as compared with both control groups (P⬍.05).
Barak. IL-18 correlates with severe OHSS. Fertil Steril 2004.
RESULTS Twenty-four patients were included in the study, all of whom were healthy infertile women, 28.5 ⫾ 4.5 years old, who had undergone ovulation induction for IVF. All presented with severe OHSS according to the classification systems of Golan et al. (1) and Navot et al. (2). Markedly elevated serum E2 levels (mean: 5,600 ⫾ 320 pg/mL) were recorded in all study patients. Number of follicles on the day of oocyte retrieval (22 ⫾ 5 vs. 19 ⫾ 4 in the OHSS and control group 2, respectively) and number of oocytes retrieved (18 ⫾ 4 vs. 14 ⫾ 4) were not statistically different between the two groups. Indeed, 18 (75%) of 24 patients of the OHSS group were pregnant in this study. These findings are consistent with our previously published data on pregnancy rates in severe OHSS (31). The IL-18 levels were not statistically significantly different between pregnant and nonpregnant patients. The IL-18 levels of serum, pleural, and peritoneal fluids during the acute phase of OHSS were all found to be significantly (P⬍.04) elevated as compared with both control groups (Fig. 1). The IL-18 levels were higher in control group 2, as compared with control group 1. In patients with OHSS, IL-18 serum levels decreased significantly along with clinical convalescence, and even more so upon resolution, as shown in Figure 2. Serum IL-6 levels showed similar kinetics to IL-18 levels, with substantially elevated levels during the acute phase that dropped significantly along with clinical convalescence and resolution (Fig. 3). 417
DISCUSSION
FIGURE 2 Serum interleukin-18 (IL-18) levels during the acute (red), convalescence (blue), and resolution (green) phases of OHSS. Values are presented as mean ⫾ SE. Asterisks indicate statistically significant differences between the acute and both convalescence and resolution phases (P⬍.05).
Barak. IL-18 correlates with severe OHSS. Fertil Steril 2004.
The clinical parameters correlated to hyperpermeability evaluated in patients with OHSS, included HCT and WBC. Serum E2 levels were recorded as well. All were substantially elevated during the acute phase, dropping significantly with convalescence and even more so after complete resolution (Fig. 4). Statistically significant correlations (P⬍.05) were found between serum IL-18 and serum IL-6 levels in patients with OHSS. Serum IL-18 levels also significantly correlated with serum E2 levels, hematocrit, and white blood cell count (P⬍.05 for all comparisons).
FIGURE 3 Serum interleukin 6 (IL-6) levels during the acute (red), convalescence (blue), and resolution (green) phases of OHSS. Values are presented as mean ⫾ SE. Asterisks indicate statistically significant differences (P⬍.05).
Barak. IL-18 correlates with severe OHSS. Fertil Steril 2004.
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It has recently been suggested that a complex communication network connects the reproductive and immune systems, and controls ovarian function. The ovulatory process itself has been perceived as a physiologic inflammatory response, characterized by migration of leukocytes into ovulatory follicles (32). Both corpus luteum formation and luteal regression involve progressive infiltration of lymphocytes and macrophages, and communication through chemokines, cytokines, and cell adhesion molecules (32). We and others have shown cytokines to mediate some important ovarian functions in general, and specifically the IL-1 family in the process of ovulation (17, 33). We have previously reported high amounts of various cytokines in the follicular fluid of patients undergoing ovulation induction and studied their correlation to E2 concentrations (18, 19). In extreme cases of ovulation induction, the potentially life-threatening complication of OHSS, characterized by hypovolemia and hemoconcentration, may occur (1, 2). It has been suggested that the principal pathophysiologic mechanism behind OHSS is capillary hyperpermeability, expressed by a massive shift of fluids and proteins from the intravascular compartment to the third space (34 –36), which is similar to our findings (17, 18). This in turn causes intravascular volume depletion, third-space fluid accumulation, and hemoconcentration, as well as endothelial damage induced by various cytokines, which are responsible for most of the morbidity associated with OHSS (1, 2). Interleukin-1, IL-6, IL-8, and TNF␣ have been implicated as mediators of the acute phase response occurring in sepsis, injury, and immunologic challenge (37, 38), characterized by capillary leakage that is very similar to that seen in OHSS. Other studies have found important effects of the cytokine network upon the vessel wall. Endothelial morphologic changes have been shown to occur in cytokine-treated endothelial cells. For instance, TNF␣ increases the permeability of endothelial cell monolayers to macromolecules, via an action on membrane G-proteins or by endothelial cell injury (39). More recently, we and others have suggested a cardinal role for inflammatory cytokines (IL-1, IL-6, IL-8, and TNF␣) in the pathogenesis of OHSS (4, 5). We also reported increased levels of serum ICAM-1 and decreased levels of E-selectin in patients with severe OHSS (10). Both factors showed statistically significant correlations with clinical and biological aspects of this syndrome, thereby suggesting a possible role for these adhesion molecules in the pathogenesis of OHSS (10, 11). We have also suggested a role for inflammatory cytokines in the formation of ascites and hydrothorax in Meig syndrome which, in a similar manner to OHSS, manifested with third-space fluid accumulation associated with a benign ovarian pathology (40). We performed the current study to assess the possible involvement of IL-18 in the pathophysiologic mechanisms of capillary hyperpermeability in the OHSS and study its Vol. 82, No. 2, August 2004
FIGURE 4 Changes in clinical parameters of OHSS during the acute, convalescence, and resolution phases. Hematocrit (HCT) ⫽ green line, white blood cell count (WBC ⫻ 103) ⫽ red line, serum estradiol levels (E2 ⫻ 103) ⫽ blue line.
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potential as a marker of the syndrome. The data presented herein suggest for the first time that IL-18 is probably involved in the pathophysiology of capillary hyperpermeability and third-space fluid accumulation in severe OHSS. In this study, we evaluated 24 patients who developed severe OHSS and clinically improved later in parallel to serum IL-18 kinetics. During the acute phase of OHSS, high serum IL-18 levels were recorded in all patients, accompanied by clinical features of capillary leakage and hemoconcentration (WBC, HCT). The IL-18 levels decreased significantly in parallel to clinical convalescence, after third-space fluid accumulation and hemoconcentration had disappeared (41). These results suggest the involvement of IL-18 in the process of vascular hyperpermeability seen in OHSS. The strong statistical correlation found between IL-18 levels and characteristics of capillary leakage and hemoconcentration, such as elevated hematocrit and WBC count, further supports this assumption. In patients with OHSS, IL-18 levels also correlated with IL-6 levels (shown previously by us and by others to be the best inflammatory marker), suggesting an interplay between these two cytokines in the pathogenesis of OHSS (42, 43). FERTILITY & STERILITY威
Women who did not develop OHSS after controlled ovarian stimulation (control group 2) had significantly lower serum IL-18 levels than the OHSS patients; however, their levels were more elevated than control group 1, as an effect of ovulation induction. We also found extremely high IL-18 levels in ascitic fluid obtained from affected individuals. These levels were substantially higher than the levels in serum obtained simultaneously, suggesting either a direct spill of this cytokine from the ovaries and/or peritoneum or active transport through ovarian and/or peritoneal blood vessels. Hence, IL-18 may play a role in the peritoneal acutephase responses in OHSS. Considering its pivotal role as an inducer of the inflammatory cytokine cascade, these results suggest IL-18 production may be one of the primary inflammatory events leading to capillary hyperpermeability in OHSS. The results presented herein suggest that IL-18 may serve as a possible target for immunologic treatment of severe OHSS. Anti-IL-18 treatment has many beneficial effects in diseases characterized by production of high IL-18 levels. Treatment of mice undergoing ovulation induction with 419
IL-18 receptor monoclonal antibodies during ovarian stimulation reduced the number of ovulated ova and inhibited the expansion of cumulus cells surrounding the ovum (16). It may therefore be postulated that administration of IL-18 BP or anti-IL-18 antibodies can block the IL-18 –induced inflammatory cascade activation and thereby decrease endothelial damage and capillary hyperpermeability in OHSS. This potential clinical application of our study must be tested in animal models before application to humans can be safely performed. In conclusion, the results presented in this study suggest a close association between IL-18, a primary mediator and inducer of the inflammatory cytokine cascade, and parameters of capillary hyperpermeability in severe OHSS. Levels of IL-18 may well be used as markers of disease in OHSS. References 1. Golan A, Ron-el R, Herman A, Soffer Y, Weinraub Z, Caspi E. Ovarian hyperstimulation syndrome: an update review. Obstet Gynecol Surv 1989;44:430 –40. 2. Navot D, Bergh PA, Laufer N. Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil Steril 1992;58:249 –61. 3. Elchalal U, Schenker JG. The pathophysiology of ovarian hyperstimulation syndrome—views and ideas. Hum Reprod 1997;12:1129 –37. 4. Revel A, Barak V, Lavy Y, Anteby E, Abramov Y, Schenker JG, et al. Characterization of intraperitoneal cytokines and nitrites in women with severe OHSS. Fertil Steril 1996;66:66 –71. 5. Abramov Y, Schenker JG, Levin A, Friedler S, Nisman B, Barak V. Plasma inflammatory cytokines correlate to the ovarian hyperstimulation syndrome (OHSS). Hum Reprod 1996;11:1381–6. 6. Abramov Y, Barak V, Nisman B, Schenker JG. Vascular endothelial growth factor plasma levels correlate to the clinical picture in severe ovarian hyperstimulation syndrome. Fertil Steril 1997;67:261–5. 7. Geva E, Amit A, Lossing GB, Lerner-Geva L, Yovel I, Azem F, et al. Follicular fluid levels of VEGF may serve as a predictive marker for the development of ovarian hyperstimulation syndrome. J Reprod Med 1999;44:91–5. 8. McClure N, Healy DL, Rogers PAW, Sullivan J, Beaton L, Haning RV Jr, et al. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome. Lancet 1994;344:235–6. 9. Neulen J, Yan Z, Raczek S, Weindel K, Keck C, Weich HA, et al. Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 1995;80:1967–71. 10. Abramov Y, Schenker JG, Levin A, Kafka I, Jaffe H, Barak V. The role of plasma adhesive molecules (ELAM ⫹ ICAM) and biological aspects of ovarian hyperstimulation syndrome (OHSS). Fertil Steril 2001;76:51–7. 11. Daniel Y, Geva E, Amit A, Baram A, Englandern T, Kupfermine MJ, et al. Levels of soluble vascular cell adhesion molecule-1 and soluble intercellular adhesion molecule-1 are increased in women with ovarian hyperstimulation syndrome. Fertil Steril 1999;71:896 –901. 12. Lebel-Binay S, Berger A, Zinzindohoue F, Cugnenc PH, Thiounn N, Fridman WH, et al. Interleukin-18: biological properties and clinical implications (review). Eur Cytokine Netw 2000;11:15–25. 13. Dinarello CA. IL-18: A Th1 inducing, proinflammatory cytokine and new member of IL-1 family. J Allergy Clin Immunol 1999;103:11–24. 14. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin 18 regulates both Th1 and Th2 responses. Annu Rev Immunol 2001;19: 423–74. 15. Wang ZY, Gaggero A, Rubartelli A, Rosso O, Miotti S, Mezzanzanica D, et al. Expression of interleukin-18 in human ovarian carcinoma and normal ovarian epithelium: evidence for defective processing on tumor cells. Int J Cancer 2002;98:873–8. 16. Tsuji Y, Tamaoki TH, Hasegawa A, Kashiwamura S, Iemoto A, Ueda H, et al. Expression of interleukin-18 and its receptor in mouse ovary. Am J Reprod Immunol 2001;46:349 –57. 17. Barak V, Yanai P, Simon A, Roisman I, Treves AJ, Laufer N. Interleukin 1: local production and modulation of human granulosa luteal cells steroidogenesis. Fertil Steril 1992;58:719 –25. 18. Barak V, Mordel N, Holzer H, Zajicek G, Treves AJ, Laufer N. The correlation of IL-1 and TNF to estradiol, progesterone and testosterone levels in periovulatory follicular fluid (FF) of IVF patients. Hum Reprod 1992;7:462–4.
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