Interleukin-1 and interleukin-1 may affect the implantation rate of patients undergoing in vitro fertilization embryo transfer

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1 FERTILITY AND STERILITY VOL. 70, NO. 3, SEPTEMBER 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Interleukin-1 and interleukin-1 may affect the implantation rate of patients undergoing in vitro fertilization embryo transfer Evdokia E. Karagouni, Ph.D.,* Athanasios Chryssikopoulos, M.D., Themis Mantzavinos, M.D., Nikos Kanakas, M.D., and Eleni N. Dotsika, Ph.D.* Hellenic Pasteur Institute, Second Department of Obstetrics and Gynecology, Medical School, University of Athens-Areteion Hospital, and Euromedica High Technology Laboratory, Athens, Greece Received December 11, 1997; revised and accepted April 7, Reprint requests: Evdokia E. Karagouni, Ph.D., Department of Cellular Immunology, Hellenic Pasteur Institute, 127 Vass. Sofias Avenue, Athens, Greece (FAX: ; * Hellenic Pasteur Institute. 2nd Department of Obstetrics and Gynecology, Medical School, University of Athens-Areteion Hospital. Euromedica High Technology Laboratory /98/$19.00 PII S (98)00243-X Objective: To investigate whether interleukin-1 (IL-1 ) and interleukin-1 (IL-1 ) affect the implantation rate of patients undergoing IVF-ET. Design: Follicular fluid and serum were obtained on the day of hcg administration, the day of oocyte retrieval, and the day of embryo transfer. Setting: Cellular immunology laboratory in a research institute, a high technology IVF unit in a medical center, and a university hospital. Patient(s): Thirty-three women who were undergoing IVF-ET. Main Outcome Measure(s): IL-1 and IL-1 were measured by specific ELISA and their levels were correlated with the implantation rate. Result(s): Classification of IVF-ET patients according to their implantation rate revealed significantly higher amounts of follicular fluid IL-1 in the implantation versus nonimplantation cycles ( pg/ml versus pg/ml); The difference between the level of IL-1 in the two groups was not statistically significant ( pg/ml versus pg/ml). In parallel, systemic FSH/hMG-dependent IL-1 and IL-1 production was observed in implantation cycles but not in nonimplantation cycles. Statistically significant IL-1 and IL-1 production was observed after administration of hcg. Conclusion(s): Gonadotropins used during IVF-ET induce local and systemic production of IL-1 and IL-1. In addition, the implantation rate for IVF-ET patients who have detectable serum concentrations of IL-1 and IL-1 on the day of hcg administration could be higher than the rate for IVF-ET patients who do not have detectable concentrations of these cytokines. (Fertil Steril 1998;70: by American Society for Reproductive Medicine.) Key Words: In vitro fertilization embryo transfer, follicular fluid, interleukin-1, interleukin-1 In recent years, evidence has accumulated to suggest that the immune system is an additional local regulator of ovarian function, involving reciprocity among soluble polypeptides of immunologic origin, the cytokines, and the reproductive system (1). Because changes observed in the ovary during ovulation may constitute an inflammatory-like reaction (2), and because interleukin-1 (IL-1), an established mediator of inflammation (3), is present in both animal (4) and human (5 9) ovarian follicular fluid, IL-1 has become the focus of intense research. The IL-1 system consists of three structurally related polypeptides. The first two are IL-1 and IL-1, each of which has a broad spectrum of beneficial and harmful biologic actions, and the third is the IL-1-receptor antagonist (IL-1RA), which inhibits the activities of IL-1 (10). The two forms of IL-1, and, are the translation products of distinct genes. They are primarily released from activated monocytes and macrophages and act through common cellsurface receptors that are not restrictive to leukocytes but rather to nearly all tissue (3, 10). Previous studies with several experimental animal models have demonstrated that ovarian theca-interstitial cells are a site of IL-1 gene expression (11). In addition, the ability of IL-1 to modulate the expression of gonado- 553

2 tropin receptor on ovarian granulosa cells (12) as well as progesterone accumulation and prostaglandin secretion (13) is receptor mediated (14). Recent findings have revealed the existence of a complete, highly compartmentalized, gonadotropin-dependent human intraovarian IL-1 system replete with ligands, a receptor, and a receptor antagonist (15). These findings suggest that the IL-1 system might regulate some ovarian functions. However, the physiologic and pathologic roles of IL-1 and IL-1 in ovarian function are not well understood. To investigate the role(s) of IL-1 and IL-1 in ovarian function, we determined and compared systemic and local production of both forms of IL-1 in the serum and follicular fluid of patients who were undergoing IVF-ET, with special reference to E 2 secretion at the time of hcg administration, oocyte maturation, oocyte fertilization, embryo quality, and the pregnancy rate. MATERIALS AND METHODS Treatment From January 1996 through November 1996, 33 patients who were undergoing IVF-ET at the IVF unit of the Euromedica Medical Institute of Athens participated in our study, which was approved by the Human Reproduction Committee of the University of Athens Medical School. The mean ( SD) age of the patients who were undergoing IVF-ET was years (range, years). It was not necessary to modify our routine human IVF protocols for this study. Briefly, all patients were treated with a short protocol of intranasal GnRH agonist (buserelin acetate; 1 mg/d; Hoechst, Frankfurt, Germany) starting from day 2 of the treatment cycle until the day of hcg administration. Pure FSH (150 IU/d; Serono Laboratories, Aubonne, Switzerland) and hmg (150 IU/d; Organon, Oss, the Netherlands) were injected IM from days 3 to 5 of the cycle in the morning and in the evening. Thereafter, FSH was discontinued, whereas hmg ( IU/d) was continued until the day before the administration of 10,000 IU of hcg (Organon, Oss, the Netherlands). Follicular development was monitored from day 6 of the cycle by daily determination of serum E 2 levels and by transvaginal ultrasonographic measurements of follicle diameter. Human chorionic gonadotropin was injected IM when the two leading follicles were at least 16 mm in diameter. Transvaginal ultrasound-guided oocyte retrieval was performed hours later. All patients who were undergoing IVF-ET received P (50 mg/d) beginning on the day of ET for luteal support and continued to receive this medication until serum -hcg levels were determined (14 days after ET). Collection and preparation of samples The contents of large follicles ( 16 mm in diameter) were aspirated in ml of endotoxin-free phosphatebuffered saline (GibcoBRL/Life Technologies, Paisley, UK). Oocytes were identified and separated, and the follicular fluid was centrifuged at 900 g for 10 minutes at 4 C. The supernatant was harvested, aliquoted, and stored at 70 C until the assays were performed. Two to nine pooled autologous follicular fluids were examined for each patient. Follicular fluid contaminated with blood cells (red blood cells, /ml) was excluded. Three blood samples were also obtained from each IVF-ET patient on the day of hcg administration, oocyte retrieval, and ET. The IVF-ET patients were divided into two groups according to their implantation rate: group I consisted of 14 women with a clinical pregnancy, and group II consisted of 19 patients whose oocytes had been fertilized and who had undergone ET but whose embryos had not implanted. Detection of IL-1, IL-1, and E 2 Follicular fluid and serum samples from patients undergoing IVF-ET patients as well as control sera from 11 healthy adult volunteers were tested for IL-1 and IL-1. Commercial ELISA kits were used to quantitatively determine the levels of IL-1 (Genzyme Co., Cambridge, MA) and IL-1 (Immunotech Int., Marseille, France) according to the manufacturer s specifications. Quantitation was performed with use of a standard dose-dependent curve, and the cytokine concentrations detected in the samples are given in pg/ml. The intra-assay and interassay coefficients of variation were 5.1% and 10.9% for the IL-1 assay and 20.5% and 12.4% for the IL-1 assay, respectively. The detection limits were 3 pg/ml for IL-1 and 5 pg/ml for IL-1. E 2 values in the serum samples of IVF-ET patients on the day of hcg administration were also detected by fluorescence immunoassay (Wallac Oy, Turku, Finland). Intra-assay and interassay variations were 7% and 8%, respectively. Statistical analysis Results are expressed as means SD. The cytokines (IL-1 and IL-1 ) and E 2 levels as well as the number of aspirated, fertilized oocytes and the number of transferred embryos were compared in groups I and II. The correlation between serum or follicular IL-1 and IL-1 as well as between serum E 2 levels and the number of oocytes aspirated was also evaluated. Statistical analysis was performed with use of Student s t-test and Pearson s linear regression and correlation analysis. P.05 was considered statistically significant. RESULTS Laboratory values Serum samples as well as follicular fluids were obtained on the day of hcg administration, oocyte retrieval, or ET from all IVF-ET patients and were examined for their IL-1 and IL-1 content (Table 1). The results demonstrated that a mean SD of 58.6% 6.3% and 35.4% 12.6% of sera from patients undergoing IVF-ET contained detectable 554 Karagouni et al. IL-1 and IL-1 in IVF-ET patients Vol. 70, No. 3, September 1998

3 TABLE 1 Level of IL-1 and IL-1 in the serum and follicular fluid of 33 patients undergoing IVF-ET. Type and timing of sample Mean ( SD) level of IL-1 (pg/ml)* Percentage of samples positive for IL-1 Mean ( SD) level of IL-1 (pg/ml)* Percentage of samples positive for IL-1 Serum level on the day of hcg administration Serum level on the day of oocyte retrieval Serum level on the day of embryo transfer Follicular fluid * The detection limit of IL-1 and IL-1 assays was 3 pg/ml and 5 pg/ml, respectively. P.05 (vs. the serum level of IL-1 on the day of oocyte retrieval or on the day of embryo transfer). P.001 (vs. the level of IL-1 in the follicular fluid). amounts of IL-1 and IL-1, respectively, during the course of the study. The serum IL-1 concentration was significantly higher on the day of hcg administration than on the day of oocyte retrieval and on the day of ET (mean SD, pg/ml vs pg/ml and vs pg/ml, respectively; P.05). On the other hand, the levels of IL-1 were constant during all time points of the cycle when IL-1 was also detected. A mean SD of 50.0% 2.1% of follicular fluid from all IVF-ET patients was positive for both forms of IL-1. Follicular IL-1 and IL-1 concentrations were equal to serum IL-1 and IL-1 concentrations on the day of oocyte retrieval, but the concentration of IL-1 was significantly lower in follicular fluid than in serum on the day of hcg administration (mean SD, pg/ml vs pg/ml, P.001). The mean serum level of E 2 measured on the day of hcg administration as well as the mean number of aspirated or fertilized oocytes and transferred preembryos were determined for IVF-ET patients who were classified according to their implantation rate (group I: IVF-ET patients who conceived; group II: IVF-ET patients who failed to conceive). Although no statistically significant differences were observed between the two groups of patients, a positive correlation (r 0.74, P.01) was observed between the E 2 level and the number of aspirated oocytes in group I (data not shown). Serum IL-1 levels Analysis of IL-1 levels in the sera of both groups of IVF-ET patients revealed characteristic patterns. All serum samples of IVF-ET patients who conceived (Group I) contained significant amounts of IL-1 on the day of hcg administration, which ranged from 53.3 pg/ml to pg/ml (mean SD, pg/ml; Fig. 1). These results are in contrast with the age-matched control sera from healthy individuals in which IL-1 was undetectable (P.001, data not shown). A gradual decrease in IL-1 levels was observed 36 and 72 hours after hcg administration on the days of oocyte retrieval and ET (mean SD, and , respectively), whereas approximately 50% of the samples were negative. A significant correlation was observed between the IL-1 serum level on the day of FIGURE 1 Concentration of IL-1 in the serum of 14 IVF-ET patients who conceived (F) and 19 patients who failed to conceive (E) on the days of hcg administration, oocyte retrieval (OR), and ET. The detection limit of the IL-1 assay was 3 pg/ml. Boxes represent the means SD (range) of values over the detection limit. * indicates a statistically significant difference (P.001) between the two groups of IVF-ET patients. Oocyte maturity was assessed by microscopic morphologic examination (16), and a statistically significant difference was observed between the two groups of IVF-ET patients in the rate of oocyte maturation. IVF-ET patients who failed to conceive had a higher percentage of oocytes in the metaphase I stage (nearly mature) than IVF-ET patients who conceived (mean SD, [21.5%] vs [6.6%]). Moreover, evaluation of embryo quality according to the size of blastomeres and cytoplasmic fragmentation (16) did not reveal a significant difference between the two groups (data not shown). FERTILITY & STERILITY 555

4 FIGURE 2 Concentration of IL-1 in the serum of 14 IVF-ET patients who conceived (F) or 19 patients who failed to conceive (E) on the day of hcg administration, oocyte retrieval (OR), and ET. The detection limit of the IL-1 assay was 5 pg/ml. Boxes represent the means SD (range) of values over the detection limit. * indicates a statistically significant difference (P.05) between the two groups of IVF-ET patients. hcg administration and the rate of oocyte maturation or the E 2 level in group I (r 0.70 and r 0.74, P.01 respectively). In contrast, with regard to the IL-1 levels detected in the sera of IVF-ET patients who failed to conceive, this pattern was reversed at the three time points of the cycle where IL-1 was measured. The concentrations of IL-1 above the detection limit of the assay were determined in 31.6% of patient sera on the day of hcg administration (mean SD, pg/ml) and in 47.4% of patient sera on the day of oocyte retrieval (mean SD, pg/ml), whereas the proportion of positive serum samples among Group II patients (73.7%) was higher on the day of ET. Although the serum levels of IL-1 in group II patients on the day of ET were significantly higher than those in control patients (mean SD, pg/ml, P.001), the level remained significantly lower in comparison with the IL-1 serum levels of group I patients on the day of hcg administration (P.001). Serum IL-1 levels The IL-1 serum levels in IVF-ET patients who conceived (group I) or failed to conceive (group II) at the same time points in the cycle are shown in Figure 2. The IL-1 levels were elevated in 78.6% of the sera from IVF-ET patients who conceived on the day of hcg administration (mean SD, pg/ml). On the days of oocyte retrieval and ET, only 15% of the serum samples remained positive, although their mean values bordered on the detection limit of the assay (mean SD, and pg/ml, respectively). Statistical analysis revealed a significant correlation between the IL-1 serum level on the day of hcg administration and E 2 secretion in group I (r 0.59, P.05). On the other hand, 21.0% and 31.6% of serum samples from group II patients on the days of hcg administration and oocyte retrieval had low amounts of IL-1 (mean SD, pg/ml and pg/ml, respectively). There was a moderate increase in the proportion of positive samples (42.1%) as well as in the IL-1 levels (mean SD, pg/ml) among the serum samples of group II patients on the day of ET. IL-1 was not detected in control sera (assay detection limit, 5 pg/ml). A positive correlation was observed between the amount of IL-1 and IL-1 secreted in the sera of group II patients on the day of ET (r 0.73, P.001). A statistically significant correlation was also observed between the number of aspirated oocytes and the IL-1 serum level on the day of ET in group II (r 0.59, P.01). Follicular fluid levels of IL-1 and IL-1 The levels of IL-1 and IL-1 in the follicular fluid of both groups of IVF-ET patients are shown in Table 2. IL-1 was detected in 78.6% and 36.8% of follicular fluid samples from group I and group II, respectively. The levels of IL-1 were significantly higher in follicular fluid samples from group I than from group II (mean SD, vs. TABLE 2 Level of IL-1 and IL-1 in the follicular fluid of 33 patients undergoing IVF-ET. Follicular fluid Patient group Mean ( SD) level of IL-1 (range) (pg/ml)* Percentage of samples positive for IL-1 Mean ( SD) level of IL-1 (range) (pg/ml)* Percentage of samples positive for IL-1 Group I (n 14) ( ) ( ) 57.2 Group II (n 19) ( ) ( ) 42.1 Note: Group I patients who conceived after undergoing IVF-ET; Group II patients who failed to conceive after undergoing IVF-ET. * The detection limit of the IL-1 and IL-1 assays was 3 pg/ml and 5 pg/ml, respectively. P.001 (vs. group II). 556 Karagouni et al. IL-1 and IL-1 in IVF-ET patients Vol. 70, No. 3, September 1998

5 pg/ml, P.001). Similarly, IL-1 was detected in 57.2% and 42.1% of follicular samples from group I and group II, respectively, although no statistically significant differences in IL-1 levels were observed between the two groups (mean SD, pg/ml and pg/ml). A positive correlation was observed between the follicular fluid concentrations of IL-1 and IL-1 in both groups of IVF-ET patients (r 0.67, P.05, and r 0.65, P.01, respectively). The IL-1 levels in the follicular fluid of group II patients were also positively correlated with IL-1 secretion in the serum of these patients on the day of ET (r 0.89, P.001). DISCUSSION In general, our results confirm and extend previous observations that demonstrate the existence of IL-1 and IL-1 in human preovulatory follicular fluid and in serum during follicular maturation, ovulation, and corpus luteum formation in FSH/LH stimulated cycles of women undergoing IVF-ET (Table 1). Our results demonstrated that approximately 50% of sera from IVF-ET patients contained detectable amounts of IL-1 and IL-1 during the periovulatory phase. In contrast, both forms of IL-1 were undetectable in the serum of unstimulated women in the proliferative phase of their menstrual cycle. It has also been shown that IL-1 transcripts were not detected in whole ovarian material from day 4 or 12 of an unstimulated menstrual cycle (15), whereas plasma IL-1 activity is enhanced in postovulatory women during the luteal phase (17), reflecting the relationship between IL-1 and the ovulatory process. Concentrations of IL-1 and IL-1 in follicular fluid were equal to or lower than those in the serum. On the other hand, concentrations of IL-1 in the follicular fluid or serum of IVF-ET patients were lower than those of IL-1. Although the two forms of IL-1 are structurally related at the three-dimensional level and share common biologic activities (3), there is evidence that most IL-1 remains in the cytosol in its precursor form, whereas a considerable amount of IL-1 is released by the cell into the extracellular space and into the circulation (10). These findings are in agreement with those in earlier reports by Hurwitz et al. (15) in which IL-1 transcripts were significantly more abundant than their IL-1 counterparts in preovulatory follicular aspirates of a stimulated IVF-ET cycle. The possibility that both forms of follicular IL-1 were derived from direct blood contamination during follicle aspiration can be excluded because the red blood cell count in these fluids was either negative or very low. The cellular origin of IL-1 and IL-1 present in follicular fluid during IVF-ET procedures remains a matter for speculation. These cytokines could arise from the plasma ultrafiltrate, as occurs for some other follicular fluid substances, or alternatively, they could be produced by ovarian macrophages or somatic ovarian cells. Although in this study we were unable to identify the cell type responsible for IL-1 production, other investigators have noted that granulosa cells, thecal ovarian cells, stromal ovarian cells, as well as resident macrophages and monocytes are capable of secreting IL-1 (15, 18 23). In this study there is evidence that supports local IL-1 production by cellular ovarian compounds because a greater percentage of IVF-ET patients had detectable amounts of IL-1 or IL-1 in follicular fluid than in serum obtained on the day of oocyte retrieval. These findings relate to previous observations in which peripheral monocytes detected on the day of oocyte retrieval do not express IL-1 whereas resident ovarian macrophages do (15). To investigate the role of IL-1 production during IVF-ET, we categorized IVF-ET patients according to their implantation rate. It is surprising that follicular fluid levels of IL-1 were significantly higher in the implantation cycles than in the nonimplantation cycles; in contrast, IL-1 levels in both cycles were comparable (Table 2). In parallel, systemic IL-1 and IL-1 production was observed after FSH/LH treatment in implantation cycles but not after nonimplantation cycles. In the latter case, significant IL-1 and IL-1 production was observed after administration of hcg (Figs. 1 and 2). There is evidence from experiments in animal models that IL-1 as well as other cytokines are dependent on gonadotropins at various stages of the cycle. For instance, LH and hcg induce the formation of large amounts of IL-1 mrna in the thecal layer within a few hours of injection into the preovulatory rat (11), whereas in the perfused rat ovary, the action of LH is augmented by a variety of cytokines including IL-1 and tumor necrosis factor (21). Moreover, experiments in human ovaries revealed that treatment of cell cultures with forskolin, a potent activator of adenylate cyclase, induced IL-1 transcripts in granulosa cells but not in thecal cells (15). Although the conclusion that IL-1 and IL-1 secretion in both groups of IVF-ET patients is affected by FSH/LH or hcg administration may be viewed as theoretical, one must consider the possibility of direct gonadotropin action as well as the possibility of indirect modulation via the intermediacy of ovarian steroids. The positive correlation between serum concentrations of both forms of IL-1 and E 2 secretion during follicular maturation suggests that these cytokines have a regulatory effect on oocyte maturation. This hypothesis is further supported by the direct correlation between the levels of serum IL-1 and the rate of oocyte maturation in the implantation cycles. In contrast with these results, previous studies in humans have shown that there is no obvious relationship between IL-1 and E 2 or P levels in follicular fluid or plasma (6, 9). FERTILITY & STERILITY 557

6 However, it has been reported in animal studies that IL-1 suppresses basal as well as FSH- and LH-stimulated P production in rat and porcine granulosa cells (12) and that products from stimulated leukocytes significantly influence steroid secretion from the ovaries (21, 24). Watanabe et al. (9) found that IL-1 concentrations increased in association with oocyte maturation, and a significantly positive correlation was observed between IL-1 and prostaglandin E 2 and prostaglandin in preovulatory follicular fluid, suggesting that IL-1 affects prostaglandin-induced oocyte maturation and ovulation. Our results demonstrated that the maximal secretion of IL-1 occurred at a different time in the two groups of IVF-ET patients; a time lag of at least 72 hours was observed in nonimplantation cycles. In addition, the systemic IL-1 and IL-1 overproduction that was observed in the implantation cycles declined within 72 hours after the day of hcg administration (Figs. 1 and 2). Although the precise reason(s) underlying these differences in IL-1 production between the two groups of IVF-ET patients remain unknown, the induction of autocrine/paracrine factors regulating the IL-1 hormonal action must be considered. In several disease models, as well as during experimentally induced endotoxemia in humans, levels of IL-1 reach a maximal concentration after 3-4 hours and then decrease rapidly. In these same individuals, the peak levels of IL-1 result in production of IL-1RA after 4 hours (3). On the other hand, in autocrine stimulation of IL-1 gene expression, steady state levels are obtained more slowly and are sustained for up to 30 hours (3, 10). In earlier reports, IL-1RA transcripts were detected in whole human ovarian material as well as in macrophage-free follicular aspirates, suggesting the involvement of IL-1RA in the regulation of IL-1 hormonal action at the ovarian level (15). Moreover, the action of IL-1 was reversed by a naturally occurring IL-1RA in the rat ovary (14). Assuming that high IL-1 and IL-1 levels in the serum of IVF-ET patients can activate the feedback mechanism of IL-1 production, substances such as IL-RA could be produced and control the cytokine overproduction. According to the above hypothesis, maximal IL-1RA production should have occurred during the oocyte retrieval and before the ET in the implantation cycles. In contrast, in the nonimplantation cycles it should have occurred after ET and during early embryo implantation. The low implantation rate may in large part be due to the occurrence of maximal IL-1RA production at this time since Simon et al. (25) recently demonstrated that blockade of maternal endometrial IL-1 receptor type I with IL-1RA prevents implantation in the mouse by interfering with embryonic attachment. Moreover, the increased IL-1 production during early embryo implantation in the nonimplantation cycles, which may suppress FSH- and LH-stimulated P production in granulosa cells (12), could explain the implantation failure. We are currently studying the existence of IL-1RA in the follicular fluid and serum of IVF-ET patients and its correlation with IL-1 and IL-1 to elucidate the key role of the IL-1 system in the reproductive mechanism. In conclusion, our findings reveal that gonadotropins used during IVF (or at least steroid hormones produced during IVF-ET) induce local and systemic production of IL-1 and IL-1, which may reach maximal serum levels some hours before or after the day of oocyte retrieval. The implantation rate for patients undergoing IVF-ET who have detectable serum concentrations of IL-1 and IL-1 on the day of hcg administration may be higher than the rate for IVF-ET patients who do not have detectable concentrations of these cytokines. Although these observations are promising, in vivo studies in a greater number of patients who are undergoing several different IVF-ET protocols are required to validate the positive correlation between elevated serum IL-1 levels on the day of hcg administration and success in IVF-ET cycles. Acknowledgments: We thank Katerina Arvaniti for collecting follicular fluid samples and Georgia Papadopoulou for skillful technical assistance. References 1. Adashi EY. Cytokine-mediated regulation of ovarian function: encounters of a third kind. Endocrinology 1989;124: Espey LL. Ovulation as an inflammatory process a hypothesis. Biol Reprod 1980;22: Dinarello CA. The biology of interleukin-1. In: Kishimoto T, editor. Molecular biology and immunology. Basel: Karger, 1992; Takakura K, Taii S, Fukuoka M, Yasuda K, Tagaya Y, Yodoi J, et al. Interleukin-1 receptor/p55(tac) inducing activity in porcine follicular fluid. Endocrinology 1989;125: Khan SA, Schmidt K, Hallin P, Di Pauli R, De Geyter C, Neischlag E. Human testis cytosol and ovarian follicular fluid contain high amounts of interleukin-1-like factor(s). Mol Cell Endocrinol 1988;58: Wang LJ, Norman RJ. Concentrations of immunoreactive interleukin-1 and interleukin-2 in human preovulatory follicular fluid. Hum Reprod 1992;7: Barak V, Mordel N, Holzer H, Zajicek G, Treves AJ, Laufer N. The correlation of interleukin 1 and tumour necrosis factor to oestradiol, progesterone and testosterone levels in periovulatory follicular fluid of in-vitro fertilization patients. Hum Reprod 1992;7: Huyser C, Fourie FL, Bosmans E, Levay PF. Interleukin-1 beta, interleukin-6 and growth hormone levels in human follicular fluid. J Assist Reprod Genet 1994;11: Watanabe H, Nagai K, Yamaguchi M, Ikenoue T, Mori N. Concentration of interleukin-1 correlates with prostaglandin E 2 and F 2 in human pre-ovulatory follicular fluid. Hum Reprod 1994;9: Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood 1991; 77: Hurwitz A, Ricciarelli E, Botero L, Rohan RM, Hernandez ER, Adashi EY. Endocrine- and autocrine-mediated regulation of rat ovarian (theca-interstitial) interleukin-1 gene expression: gonadotropin-dependent preovulatory acquisition. Endocrinology 1991;129: Gottschall PE, Katsura G, Hoffman ST, Arimura A. Interleukin-1: an inhibitor of luteinizing hormone receptor formation in cultured granulosa cells. FASEB J 1988;2: Brannstrom M, Wang L, Norman RJ. Ovulatory effect of interleukin-1 on the perfused rat ovary. Endocrinology 1993;132: Kokia E, Ben-Shlomo I, Adashi EY. The ovarian action of interleukin-1 is receptor mediated: reversal by a naturally occurring interleukin-1 receptor antagonist. Fertil Steril 1995;63: Hurwitz A, Loukides J, Ricciarelli E, Botero L, Katz E, McAllister JM, et al. Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation of the genes encoding 558 Karagouni et al. IL-1 and IL-1 in IVF-ET patients Vol. 70, No. 3, September 1998

7 IL-1, its receptor, and its receptor antagonist. J Clin Invest 1992;89: Veeck LL. Atlas of the human oocyte and early conceptus. Baltimore: Williams & Wilkins, Cannon JG, Dinarello CA. Increased plasma interleukin-1 activity in women after ovulation. Science 1985;227: Brannstrom M, Pscoe V, Norman RJ, McClure N. Localization of leukocyte subsets in the follicle wall and in the corpus luteum throughout the human menstrual cycle. Fertil Steril 1994;61: Baranao RI, Dain L, defried EP, Rumi LS. Human granulosa cells express HLA-DR antigen and are capable of synthesizing interleukin-1. Horm Metab Res 1995;27: Machelon V, Nome F, Durand-Gasselin I, Emilie D. Macrophage and granulosa interleukin-1 beta mrna in human ovulatory follicles. Hum Reprod 1995;10: Brannstrom M, Norman RJ. Involvement of leukocytes and cytokines in the ovulatory process and corpus luteum function. Hum Reprod 1993;8: Jasper M, Norman RJ. Immunoactive interleukin-1 and tumour necrosis factor-a in thecal, stromal and granulosa cell cultures from normal and polycystic ovaries. Hum Reprod 1995;10: Loukides JI, Loy RA, Edwards R, Honing J, Visintin I, Polan ML. Human follicular fluids contain tissue macrophages. J Clin Endocrinol Metab 1990;71: Wang LJ, Robertson S, Seamark RF, Norman RJ. Lymphokines, including interleukin-2, alter gonadotropin-stimulated progesterone production and proliferation of human granulosa-luteal cells in vitro. J Clin Endocrinol Metab 1991;72: Simon C, Frances A, Piquette GN, El Danasouri I, Zurawski G, Dang W, et al. Embryonic implantation in mice is blocked by interleukin-1 receptor antagonist. Endocrinology 1994;134: FERTILITY & STERILITY 559