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Levels of soluble vascular cell adhesion molecule-1 and soluble intercellular adhesion molecule-1 are increased in women with ovarian hyperstimulation syndrome
REPRODUCTIVE BIOLOGY
FERTILITY AND STERILITYt VOL. 71, NO. 5, MAY 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Levels of soluble vascular cell adhesion molecule-1 and soluble intercellular adhesion molecule-1 are increased in women with ovarian hyperstimulation syndrome Yair Daniel, M.D., Eli Geva, M.D., Ami Amit, M.D., Amiram Baram, M.D., Talma Englander, Ph.D., Michael J. Kupferminc, M.D., Gideon Fait, M.D., and Joseph B. Lessing, M.D. Department of Obstetrics and Gynecology, and the Sara Racine In Vitro Fertilization Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Received August 24, 1998; revised and accepted December 6, 1998. Supported by a grant from the W. Shriber Fund, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. Reprint requests: Joseph B. Lessing, M.D., Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel (FAX: 972-36925721; E-mail: lessing @inter.net.il). 0015-0282/99/$20.00 PII S0015-0282(99)00015-1
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Objective: To determine whether plasma and peritoneal fluid levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) are altered in women with ovarian hyperstimulation syndrome (OHSS). Design: Prospective, case-control study. Setting: Lis Maternity Hospital and the Sara Racine IVF Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. Patient(s): The study group comprised 16 women with severe OHSS. The control groups comprised 10 women treated with controlled ovarian hyperstimulation and 8 women with normal findings at diagnostic laparoscopy. Intervention(s): Plasma samples were obtained from the study group and the first control group. Peritoneal fluid samples were obtained during paracentesis from the study group and during diagnostic laparoscopy from the second control group. Main Outcome Measure(s): Samples were assayed by specific ELISA for sVCAM-1 and sICAM-1. Result(s): The mean peritoneal fluid levels of sVCAM-1 and sICAM-1 and the mean plasma levels of sVCAM-1 were significantly higher in the women with OHSS than in the control groups. However, the mean plasma levels of sICAM-1 were comparable. A positive correlation was demonstrated between the levels of sVCAM-1 and plasma E2 at the time of hCG administration and between the levels of sICAM-1 and number of ova retrieved. Conclusion(s): Our findings suggest that soluble cell adhesion molecules may have a role in the pathogenesis and progression of OHSS. (Fertil Sterilt 1999;71:896 –901. ©1999 by American Society for Reproductive Medicine.) Key Words: Soluble ICAM-1, soluble VCAM-1, OHSS, inflammation, ovulation induction, cell adhesion molecules
Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic condition caused by intense ovulation induction (1–3). This syndrome occurs with an incidence of 0.1%– 4% of all ovulation induction treatments and ranges in severity from a mild disorder to a life-threatening condition (1, 2). The clinical characteristic of the syndrome evolves from an intense inflammatory reaction that results in massive accumulation of extracellular exudate that mani-
fests as ascites, pleural effusion, and pericardial effusion (1, 2). The massive shift of fluid to “third spaces” causes profound intravascular depletion, which may result in oliguria and renal insufficiency (1, 2). Other severe clinical manifestations that may accompany the syndrome include a state of hypercoagulability, thromboembolic phenomena, adult respiratory distress syndrome,
and death (1, 2). Risk factors for the development of OHSS include the following: polycystic ovary syndrome, ovulation induction with a GnRH agonist (GnRH-a), high serum E2 levels, multiple immature and intermediate follicles, luteal support with hCG, and pregnancy (1, 2). However, the pathophysiology of the syndrome is not completely understood, and no specific therapy or prevention is available (2). Many cytokines, vasoactive factors (1, 3–5), and angiogenic factors, such as vascular endothelial growth factor (6, 7), have been implicated in the pathogenesis of OHSS. The role of cell adhesion molecules, which play a major role in the mediation of inflammatory and immune reactions, has never been investigated in OHSS. Further, their role in ovarian physiology has been investigated only recently (8 –11). The cell adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), are transmembrane glycoproteins that are members of the immunoglobulin superfamily (12–17). This group of molecules is a major mediator of white blood cell adhesion, interaction, and extravasation during inflammatory and immune reactions (12–17). Endothelial and dendritic cells, such as macrophages and the placenta trophoblasts, synthesize and express VCAM-1 (12–17). After cell activation, the expression of VCAM-1 is induced by various cytokines, and it is released in an active soluble form (sVCAM-1) during inflammatory and immune reactions (12–17). The b1 integrin, VLA4 (b1a4), serves as a VCAM-1 counter-receptor (13–17). In contrast to VCAM-1, ICAM-1 is constitutively expressed by the vascular endothelium (12–17). However, during inflammation, its expression is markedly unregulated by various cytokines, and it is released in an active soluble form (sICAM-1) (12–17). Intercellular adhesion molecule-1 mediates its functions through its counter-receptor, the b2 integrin (13–17). Because inflammation seems to play a crucial role in ovarian physiology and OHSS pathophysiology, the present study was undertaken to evaluate whether altered levels of sVCAM-1 and sICAM-1 are involved in the pathogenesis and progression of OHSS.
MATERIALS AND METHODS The study group comprised 16 women with severe OHSS. Ovarian hyperstimulation syndrome was diagnosed and graded according to the classification proposed by Golan et al. (1) and Navot et al. (2). In its severe form, OHSS includes clinical evidence of breathing difficulties, ascites, hydrothorax, oliguria, anuria, renal failure, thromboembolic phenomena, and adult respiratory distress syndrome, and laboratory evidence of hemoconcentration, coagulation abnormalities, leukocytosis, and abnormal kidney function. Plasma samples were obtained from all women at the time of diagnosis, before the initiation of any treatment. PeritoFERTILITY & STERILITYt
neal fluid samples also were obtained from 14 women during paracentesis. All patients were treated with ovulation induction protocols that included hMG (75 IU of FSH and 75 IU of LH per ampule) and 10,000 IU of hCG. In addition, a GnRH-a (buserelin acetate in a nasal spray) was administered to 14 patients according to the protocol described later. The first control group comprised 10 women who underwent controlled ovarian hyperstimulation before IVF-ET, which was performed using hMG in combination with a GnRH-a. The GnRH-a was administered on the first day of the menstrual cycle in the form of 900 mg/d of buserelin acetate in a nasal spray. Treatment with hMG (75 IU of FSH and 75 IU of LH per ampule) was started on the third day of the menstrual cycle with the administration of three ampules per day. The dosage was adjusted individually according to the serum E2 levels. When the follicles reached a mean diameter of 18 mm, 10,000 IU of hCG was administered. Oocyte retrieval was scheduled for 34 hours after hCG administration, using an ultrasound-guided approach. In all cases, 2–3 embryos were transferred. Ovarian hyperstimulation syndrome did not develop in any of the women. Blood samples were drawn 5–7 days after the administration of hCG. Control peritoneal fluid samples were obtained from eight healthy women during an uneventful normal diagnostic laparoscopy. Seven of these women underwent laparoscopy as part of an infertility evaluation, and one for suspected endometriosis, which was not confirmed. The institutional review board at our center approved the study, and we obtained informed consent from each patient. All the specimens were centrifuged at 1,000 3 g for 10 minutes, and the plasma and peritoneal fluid were stored in aliquots at 270°C until assayed collectively by an investigator who was blinded to patient assignment. Soluble VCAM-1 and sICAM-1 were measured in duplicate by ELISAs (human soluble VCAM-1 and ICAM-1; R&D Systems, Minneapolis, MN) specific for each molecule. These assays use the quantitative sandwich immunoassay technique that involves the simultaneous reaction of the measured molecules to two murine monoclonal antibodies specifically directed against two different epitopes on human sVCAM-1 and sICAM-1. One of these antibodies is conjugated to the enzyme horseradish peroxidase. After the removal of unbound material by aspiration and washing, the amount bound to the well is detected by reaction with a substrate specific for the enzyme that yields a colored product proportional to the amount of conjugate. The colored product is quantified photometrically, and the concentration is calculated by analyzing standards of known concentration. The monoclonal antibodies were specific, and cross-reactivity with other cell adhesion molecules was not observed. 897
TABLE 1 Clinical and demographic data of the study and control groups. Group
Variable Mean (6SD) age (y) No. (%) of patients with indicated cause of infertility Male factor Unexplained Polycystic ovaries Mechanical factor No. (%) of patients undergoing IVF-ET Mean (6SD) no. of ova retrieved Mean (6SD) E2 level at hCG administration (pg/mL) Mean (6SD) no. of ETs
sICAM-1 (n 5 16)
sVCAM-1 (n 5 10)
Plasma control (n 5 10)
P value
29.4 6 4.9
29.9 6 5.0
32.3 6 4.4
NS
8 (50) 2 (13) 3 (19) 3 (19) 14 (88) 25 6 6 3,139 6 1,654 3 6 1.5
5 (50) 2 (20) 2 (20) 1 (10) 9 (90) 25 6 5 3,307 6 1,920 2.3 6 1.3
4 (40) 3 (30) 2 (20) 1 (10) 10 (100) 763 1,501 6 287 2.5 6 0.5
NS NS NS NS NS ,.01 ,.01 NS
Note: NS 5 not significant; sICAM-1 5 soluble intercellular adhesion molecule-1; sVCAM-1 5 soluble vascular cell adhesion molecule-1.
The interassay and intra-assay coefficients of variation for sVCAM-1 were ,10.3% and ,6%, respectively, and those for sICAM-1 were ,10.1% and ,4.9%, respectively. The sensitivities of the assays were ,2 ng/mL for sVCAM-1 and ,0.35 ng/mL for sICAM-1. Because specimens were insufficient for all the women, sVCAM-1 was analyzed in the plasma and peritoneal fluid of 10 women with OHSS. Demographic and clinical data were compared using the Student’s t-test and the x2 test. Soluble VCAM-1 and sICAM-1 concentrations were compared using the Student’s t-test. Associations between sVCAM-1 and sICAM-1 levels and other clinical and laboratory parameters were analyzed by the Pearson correlation test. Statistical significance was assumed at a level of P#.05.
RESULTS Relevant clinical data for the study and control groups are shown in Table 1. Clinical and laboratory characteristics of the study groups are presented in Table 2. Both sVCAM-1 and sICAM-1 were detected in the plasma and peritoneal fluid of all the women studied. The mean (6SD) plasma and peritoneal fluid concentrations of sVCAM-1 were significantly higher in the women with OHSS than in the women in the control groups (1,003.6 6 395.7 ng/mL versus 696.6 6 142.6 ng/mL, P 5 .01; and 743.8 6 256.7 ng/mL versus 350.2 6 168.6 ng/mL, P 5 .01, respectively) (Fig. 1 and 2). The mean (6SD) peritoneal fluid level of sICAM-1 was significantly higher in the women with OHSS than in the women in the control group (180.4 6 86.3 ng/mL versus 105.7 6 61.6 ng/mL; P,.05) (Fig. 3). However, the mean (6SD) plasma level was comparable in the study and control groups (255.5 6 161.1 ng/mL versus 226.4 6 72.2 ng/mL) (Fig. 4). Plasma and ascites fluid levels of sVCAM-1 were posi898
Daniel et al.
Soluble adhesion molecules in OHSS
tively correlated with plasma E2 levels at the time of hCG administration (r 5 .47, P 5 .03 and r 5 .67, P 5 .03, respectively). Further, there was a trend toward a positive correlation with the number of ova retrieved (r 5 .4, P 5 .08). Nevertheless, no significant correlation was observed between the plasma and peritoneal fluid levels of VCAM-1 and other laboratory or clinical parameters of OHSS. Levels of sICAM-1 in the ascites fluid were positively correlated with the number of ova retrieved (r 5 .69, P 5 .03). However, no significant correlation was demonstrated between the plasma or peritoneal fluid levels of sICAM-1 and other laboratory or clinical parameters of OHSS.
DISCUSSION To our knowledge, this is the first report of increased sVCAM-1 and sICAM-1 levels in women with OHSS. Our
TABLE 2 Clinical data of the study groups. Variable
sICAM-1 group (n 5 16)
sVCAM-1 group (n 5 10)
Hemoglobin level (g/dL) Hematocrit (%) No. of leukocytes per microliter Blood urea nitrogen level (mg/dL) Creatinine level (mg/dL) Paracentesis volume (mL) Prothrombin time (s) Partial thromboplastin time (s)
14.3 6 1.8 42.6 6 4.9 16,107 6 5,335 14.3 6 5.6 0.82 6 0.07 4,804 6 3,558 14.2 6 1.9 27.3 6 2.9
14.5 6 1.9 43.0 6 5.2 16,940 6 5,740 14.2 6 5.5 0.84 6 0.05 5,425 6 3,830 14.4 6 2.1 27.6 6 2.7
Note: All values are means 6 SD. No statistically significant differences in any of the variables were observed between the groups. sICAM-1 5 soluble intercellular adhesion molecule-1; sVCAM-1 5 soluble vascular cell adhesion molecule-1.
Vol. 71, No. 5, May 1999
FIGURE 1 Plasma levels of soluble vascular cell adhesion molecule-1 (VCAM-1) in women with ovarian hyperstimulation syndrome (OHSS) and in women in the control group. The horizontal lines indicate the mean levels of the two groups.
findings suggest that these soluble cell adhesion molecules may play a role in the pathogenesis and progression of the disease. Further, we found a positive correlation between the plasma and peritoneal fluid concentrations of these molecules and both the plasma E2 concentration at the time of hCG administration and the number of ova retrieved, both of which are known to be risk factors for the development of OHSS (1, 2). Our results are in accordance with several observations that suggest the involvement of cell adhesion molecules in ovarian physiology (8 –11). The oocyte and early embryo express both ICAM-1 and VCAM-1 (10), and human granulosa cells express both surface ICAM-1 and its messenger RNA (9). Further, sICAM-1 was demonstrated in the follicular fluid of women undergoing ovulation induction, and its concentrations were positively correlated with the numbers of oocytes and follicles with a diameter of .15 mm (8). Mantzavinos et al. (11) reported that serum sICAM-1 levels were significantly higher in women undergoing IVF-ET than in women with normal cycles, probably because of multiple ovulation. These data suggest that cell adhesion molecules may play a role in physiologic ovarian processes such as folliculogenesis, ovulation, corpus luteum formation, and luteolysis, and as suggested by our findings, in the pathophysiology of OHSS that results from the exaggeration of these processes (1–5). The participation of cell adhesion molecules, which are major mediators of inflammation (12– FERTILITY & STERILITYt
FIGURE 2 Peritoneal fluid levels of soluble vascular cell adhesion molecule-1 (VCAM-1) in women with ovarian hyperstimulation syndrome (OHSS) and in women in the control group. The horizontal lines indicate the mean levels of the two groups.
17), in ovarian physiology and pathophysiology is not surprising in view of the many indications suggesting that inflammation may be a major physiologic phenomenon in the reproductive processes of the ovary (18 –20). Unregulated expression and increased shedding of VCAM-1 and ICAM-1 from the granulosa cell as a result of multiple ovulation and corpus luteum formation may result in the development of OHSS. These cell adhesion molecules may contribute to the evolution and progression of OHSS as a result of their inflammatory endowment (13–17). Both VCAM-1 and ICAM-1, together with the selectins, another group of adhesion molecules, participate in the “rolling” movement, adhesion, extravasation, recruitment, and activation of leukocytes and endothelial cells at sites of inflammation (13–17). Thus, the increased levels of cell adhesion molecules in the plasma and peritoneal fluid of women with OHSS may contribute to the initiation and propagation of the intense local and systemic inflammatory reactions that result from leukocyte recruitment and activation. Further, VCAM-1 is an angiogenic factor that may contribute to the angiogenesis and increased vascular permeability that characterize OHSS (21). Another source of the increased levels of sVCAM-1 and sICAM-1 in patients with OHSS is the formation of multiple 899
FIGURE 3
FIGURE 4
Peritoneal fluid levels of soluble intercellular adhesion molecule-1 (ICAM-1) in women with ovarian hyperstimulation syndrome (OHSS) and in women in the control group. The horizontal lines indicate the mean levels of the two groups.
Plasma levels of soluble intercellular adhesion molecule-1 (ICAM-1) in women with ovarian hyperstimulation syndrome (OHSS) and in women in the control group. The horizontal lines indicate the mean levels of the two groups.
corpora lutea. This process is accompanied by intense angiogenesis and endothelial and granulosa cell formation. Because ICAM-1 is constitutively expressed by the endothelium (13–17) and granulosa cells (8), this also may contribute to its increased levels (13–17).
E2 at the time of hCG administration and with the number of ova retrieved, both of which are known to be risk factors for the development of OHSS (1, 2). This observation suggests that concentrations of sVCAM-1 may serve as a marker for the development of OHSS, although a different study design should be used to explore this possibility.
Further, the expression and shedding of VCAM-1 and ICAM-1 are up-regulated by inflammatory cytokines (13– 17) that are ubiquitous in patients with OHSS (3–5). In accordance with this hypothesis, it was reported that interleukin-1b stimulated the secretion of sICAM-1 from granulosa cells in a dose-dependent manner (8). Moreover, it was reported recently (22) that vascular endothelial growth factor, levels of which are elevated in patients with OHSS (6, 7), induces the expression of VCAM-1. Trophoblast formation and invasion is accompanied by the expression of VCAM-1 and ICAM-1 (23–25). As a result, pregnancy, a known risk factor for OHSS progression (2), may result in increased levels of sVCAM-1 and sICAM-1, accounting in part for the disease progression observed in cycles that result in conception. In support of this assumption, Mantzavinos et al. (11) reported that serum sICAM-1 concentrations were significantly higher in cycles that resulted in conception than in those that did not. Plasma levels of sVCAM-1 were correlated with the disease activity of several inflammatory conditions (13–17). In the present study, we found that plasma levels of sVCAM-1 were positively correlated with plasma levels of 900
Daniel et al.
Soluble adhesion molecules in OHSS
Before we arrive at any conclusions, several points regarding the soluble form of cell adhesion molecules should be emphasized: First, this is the first report of increased levels of soluble cell adhesion molecules in patients with OHSS, and further studies are required to corroborate our findings. Further, because it is not possible to obtain peritoneal fluid samples from superovulatory women without OHSS, we used peritoneal fluid samples obtained during diagnostic laparoscopy from non-superovulatory patients as controls. The mean levels of soluble cell adhesion molecules in these samples represent only the basal levels of these molecules, which are higher in patients with OHSS. However, it is unknown whether the peritoneal fluid levels of these molecules are altered in superovulatory women in whom OHSS does not develop or whether the levels fluctuate during the menstrual cycle. Second, the measurement of these soluble molecules in different body fluids gauges only the freely available molecules and may miss a significant proportion of the amount shed because of its binding to adhesion molecule counterreceptors in the tissues. This may account for our failure to Vol. 71, No. 5, May 1999
demonstrate increased plasma levels of sICAM-1 in women with OHSS. Third, the physiologic significance of elevated levels of these soluble cell adhesion molecules is unknown. Because the mechanism of clearance of cell adhesion molecules is vague, these soluble isoforms may reflect merely a means of clearance. However, these soluble isoforms retain their binding capacity in vivo and thus can function in two ways, by triggering the biologic response on binding to the counterreceptor, and by competing in cell-cell interaction, functioning as inhibitors of cell adhesion (13–17). By this mechanism, soluble cell adhesion molecules may be considered as inflammation regulatory factors, assisting in the control of the inflammatory process. With these issues in mind, our findings may support a role for sVCAM-1 and sICAM-1 in the pathogenesis and progression of 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. Geva E, Amit A, Lerner-Geva L, Lessing JB. Ovarian hyperstimulation syndrome: the role of cytokines. Ass Reprod Rev 1997;7:168 –73. 4. Orvieto R, Ben-Rafael Z. Ovarian hyperstimulation syndrome: a new insight into an old enigma. J Soc Gynecol Invest 1998;5:110 –3. 5. Abramov Y, Schenker JG, Lewin A, Friedlender S, Nisman B, Barak V. Plasma inflammatory cytokines correlate to the ovarian hyperstimulation syndrome. Hum Reprod 1996;11:1381– 6. 6. McClure N, Healy DL, Rogers PA, Sullivan J, Beaton L, Haning RV, et al. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome. Lancet 1994;344:235– 6. 7. 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;68:133–7. 8. Vigano P, Fusi F, Gaffuri B, Bonzi V, Ferrari A, Vignali M. Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid. Fertil Steril 1998; 69:774 –9. 9. Vigano P, Gaffuri B, Ragni G, Di Blasio AM, Vignali M. Intercellular adhesion molecule-1 is expressed on human granulosa cells and medi-
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FERTILITY AND STERILITYt VOL. 71, NO. 5, MAY 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Levels of soluble vascular cell adhesion molecule-1 and soluble intercellular adhesion molecule-1 are increased in women with ovarian hyperstimulation syndrome Yair Daniel, M.D., Eli Geva, M.D., Ami Amit, M.D., Amiram Baram, M.D., Talma Englander, Ph.D., Michael J. Kupferminc, M.D., Gideon Fait, M.D., and Joseph B. Lessing, M.D. Department of Obstetrics and Gynecology, and the Sara Racine In Vitro Fertilization Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Received August 24, 1998; revised and accepted December 6, 1998. Supported by a grant from the W. Shriber Fund, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. Reprint requests: Joseph B. Lessing, M.D., Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel (FAX: 972-36925721; E-mail: lessing @inter.net.il). 0015-0282/99/$20.00 PII S0015-0282(99)00015-1
896
Objective: To determine whether plasma and peritoneal fluid levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) are altered in women with ovarian hyperstimulation syndrome (OHSS). Design: Prospective, case-control study. Setting: Lis Maternity Hospital and the Sara Racine IVF Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. Patient(s): The study group comprised 16 women with severe OHSS. The control groups comprised 10 women treated with controlled ovarian hyperstimulation and 8 women with normal findings at diagnostic laparoscopy. Intervention(s): Plasma samples were obtained from the study group and the first control group. Peritoneal fluid samples were obtained during paracentesis from the study group and during diagnostic laparoscopy from the second control group. Main Outcome Measure(s): Samples were assayed by specific ELISA for sVCAM-1 and sICAM-1. Result(s): The mean peritoneal fluid levels of sVCAM-1 and sICAM-1 and the mean plasma levels of sVCAM-1 were significantly higher in the women with OHSS than in the control groups. However, the mean plasma levels of sICAM-1 were comparable. A positive correlation was demonstrated between the levels of sVCAM-1 and plasma E2 at the time of hCG administration and between the levels of sICAM-1 and number of ova retrieved. Conclusion(s): Our findings suggest that soluble cell adhesion molecules may have a role in the pathogenesis and progression of OHSS. (Fertil Sterilt 1999;71:896 –901. ©1999 by American Society for Reproductive Medicine.) Key Words: Soluble ICAM-1, soluble VCAM-1, OHSS, inflammation, ovulation induction, cell adhesion molecules
Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic condition caused by intense ovulation induction (1–3). This syndrome occurs with an incidence of 0.1%– 4% of all ovulation induction treatments and ranges in severity from a mild disorder to a life-threatening condition (1, 2). The clinical characteristic of the syndrome evolves from an intense inflammatory reaction that results in massive accumulation of extracellular exudate that mani-
fests as ascites, pleural effusion, and pericardial effusion (1, 2). The massive shift of fluid to “third spaces” causes profound intravascular depletion, which may result in oliguria and renal insufficiency (1, 2). Other severe clinical manifestations that may accompany the syndrome include a state of hypercoagulability, thromboembolic phenomena, adult respiratory distress syndrome,
and death (1, 2). Risk factors for the development of OHSS include the following: polycystic ovary syndrome, ovulation induction with a GnRH agonist (GnRH-a), high serum E2 levels, multiple immature and intermediate follicles, luteal support with hCG, and pregnancy (1, 2). However, the pathophysiology of the syndrome is not completely understood, and no specific therapy or prevention is available (2). Many cytokines, vasoactive factors (1, 3–5), and angiogenic factors, such as vascular endothelial growth factor (6, 7), have been implicated in the pathogenesis of OHSS. The role of cell adhesion molecules, which play a major role in the mediation of inflammatory and immune reactions, has never been investigated in OHSS. Further, their role in ovarian physiology has been investigated only recently (8 –11). The cell adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), are transmembrane glycoproteins that are members of the immunoglobulin superfamily (12–17). This group of molecules is a major mediator of white blood cell adhesion, interaction, and extravasation during inflammatory and immune reactions (12–17). Endothelial and dendritic cells, such as macrophages and the placenta trophoblasts, synthesize and express VCAM-1 (12–17). After cell activation, the expression of VCAM-1 is induced by various cytokines, and it is released in an active soluble form (sVCAM-1) during inflammatory and immune reactions (12–17). The b1 integrin, VLA4 (b1a4), serves as a VCAM-1 counter-receptor (13–17). In contrast to VCAM-1, ICAM-1 is constitutively expressed by the vascular endothelium (12–17). However, during inflammation, its expression is markedly unregulated by various cytokines, and it is released in an active soluble form (sICAM-1) (12–17). Intercellular adhesion molecule-1 mediates its functions through its counter-receptor, the b2 integrin (13–17). Because inflammation seems to play a crucial role in ovarian physiology and OHSS pathophysiology, the present study was undertaken to evaluate whether altered levels of sVCAM-1 and sICAM-1 are involved in the pathogenesis and progression of OHSS.
MATERIALS AND METHODS The study group comprised 16 women with severe OHSS. Ovarian hyperstimulation syndrome was diagnosed and graded according to the classification proposed by Golan et al. (1) and Navot et al. (2). In its severe form, OHSS includes clinical evidence of breathing difficulties, ascites, hydrothorax, oliguria, anuria, renal failure, thromboembolic phenomena, and adult respiratory distress syndrome, and laboratory evidence of hemoconcentration, coagulation abnormalities, leukocytosis, and abnormal kidney function. Plasma samples were obtained from all women at the time of diagnosis, before the initiation of any treatment. PeritoFERTILITY & STERILITYt
neal fluid samples also were obtained from 14 women during paracentesis. All patients were treated with ovulation induction protocols that included hMG (75 IU of FSH and 75 IU of LH per ampule) and 10,000 IU of hCG. In addition, a GnRH-a (buserelin acetate in a nasal spray) was administered to 14 patients according to the protocol described later. The first control group comprised 10 women who underwent controlled ovarian hyperstimulation before IVF-ET, which was performed using hMG in combination with a GnRH-a. The GnRH-a was administered on the first day of the menstrual cycle in the form of 900 mg/d of buserelin acetate in a nasal spray. Treatment with hMG (75 IU of FSH and 75 IU of LH per ampule) was started on the third day of the menstrual cycle with the administration of three ampules per day. The dosage was adjusted individually according to the serum E2 levels. When the follicles reached a mean diameter of 18 mm, 10,000 IU of hCG was administered. Oocyte retrieval was scheduled for 34 hours after hCG administration, using an ultrasound-guided approach. In all cases, 2–3 embryos were transferred. Ovarian hyperstimulation syndrome did not develop in any of the women. Blood samples were drawn 5–7 days after the administration of hCG. Control peritoneal fluid samples were obtained from eight healthy women during an uneventful normal diagnostic laparoscopy. Seven of these women underwent laparoscopy as part of an infertility evaluation, and one for suspected endometriosis, which was not confirmed. The institutional review board at our center approved the study, and we obtained informed consent from each patient. All the specimens were centrifuged at 1,000 3 g for 10 minutes, and the plasma and peritoneal fluid were stored in aliquots at 270°C until assayed collectively by an investigator who was blinded to patient assignment. Soluble VCAM-1 and sICAM-1 were measured in duplicate by ELISAs (human soluble VCAM-1 and ICAM-1; R&D Systems, Minneapolis, MN) specific for each molecule. These assays use the quantitative sandwich immunoassay technique that involves the simultaneous reaction of the measured molecules to two murine monoclonal antibodies specifically directed against two different epitopes on human sVCAM-1 and sICAM-1. One of these antibodies is conjugated to the enzyme horseradish peroxidase. After the removal of unbound material by aspiration and washing, the amount bound to the well is detected by reaction with a substrate specific for the enzyme that yields a colored product proportional to the amount of conjugate. The colored product is quantified photometrically, and the concentration is calculated by analyzing standards of known concentration. The monoclonal antibodies were specific, and cross-reactivity with other cell adhesion molecules was not observed. 897
TABLE 1 Clinical and demographic data of the study and control groups. Group
Variable Mean (6SD) age (y) No. (%) of patients with indicated cause of infertility Male factor Unexplained Polycystic ovaries Mechanical factor No. (%) of patients undergoing IVF-ET Mean (6SD) no. of ova retrieved Mean (6SD) E2 level at hCG administration (pg/mL) Mean (6SD) no. of ETs
sICAM-1 (n 5 16)
sVCAM-1 (n 5 10)
Plasma control (n 5 10)
P value
29.4 6 4.9
29.9 6 5.0
32.3 6 4.4
NS
8 (50) 2 (13) 3 (19) 3 (19) 14 (88) 25 6 6 3,139 6 1,654 3 6 1.5
5 (50) 2 (20) 2 (20) 1 (10) 9 (90) 25 6 5 3,307 6 1,920 2.3 6 1.3
4 (40) 3 (30) 2 (20) 1 (10) 10 (100) 763 1,501 6 287 2.5 6 0.5
NS NS NS NS NS ,.01 ,.01 NS
Note: NS 5 not significant; sICAM-1 5 soluble intercellular adhesion molecule-1; sVCAM-1 5 soluble vascular cell adhesion molecule-1.
The interassay and intra-assay coefficients of variation for sVCAM-1 were ,10.3% and ,6%, respectively, and those for sICAM-1 were ,10.1% and ,4.9%, respectively. The sensitivities of the assays were ,2 ng/mL for sVCAM-1 and ,0.35 ng/mL for sICAM-1. Because specimens were insufficient for all the women, sVCAM-1 was analyzed in the plasma and peritoneal fluid of 10 women with OHSS. Demographic and clinical data were compared using the Student’s t-test and the x2 test. Soluble VCAM-1 and sICAM-1 concentrations were compared using the Student’s t-test. Associations between sVCAM-1 and sICAM-1 levels and other clinical and laboratory parameters were analyzed by the Pearson correlation test. Statistical significance was assumed at a level of P#.05.
RESULTS Relevant clinical data for the study and control groups are shown in Table 1. Clinical and laboratory characteristics of the study groups are presented in Table 2. Both sVCAM-1 and sICAM-1 were detected in the plasma and peritoneal fluid of all the women studied. The mean (6SD) plasma and peritoneal fluid concentrations of sVCAM-1 were significantly higher in the women with OHSS than in the women in the control groups (1,003.6 6 395.7 ng/mL versus 696.6 6 142.6 ng/mL, P 5 .01; and 743.8 6 256.7 ng/mL versus 350.2 6 168.6 ng/mL, P 5 .01, respectively) (Fig. 1 and 2). The mean (6SD) peritoneal fluid level of sICAM-1 was significantly higher in the women with OHSS than in the women in the control group (180.4 6 86.3 ng/mL versus 105.7 6 61.6 ng/mL; P,.05) (Fig. 3). However, the mean (6SD) plasma level was comparable in the study and control groups (255.5 6 161.1 ng/mL versus 226.4 6 72.2 ng/mL) (Fig. 4). Plasma and ascites fluid levels of sVCAM-1 were posi898
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tively correlated with plasma E2 levels at the time of hCG administration (r 5 .47, P 5 .03 and r 5 .67, P 5 .03, respectively). Further, there was a trend toward a positive correlation with the number of ova retrieved (r 5 .4, P 5 .08). Nevertheless, no significant correlation was observed between the plasma and peritoneal fluid levels of VCAM-1 and other laboratory or clinical parameters of OHSS. Levels of sICAM-1 in the ascites fluid were positively correlated with the number of ova retrieved (r 5 .69, P 5 .03). However, no significant correlation was demonstrated between the plasma or peritoneal fluid levels of sICAM-1 and other laboratory or clinical parameters of OHSS.
DISCUSSION To our knowledge, this is the first report of increased sVCAM-1 and sICAM-1 levels in women with OHSS. Our
TABLE 2 Clinical data of the study groups. Variable
sICAM-1 group (n 5 16)
sVCAM-1 group (n 5 10)
Hemoglobin level (g/dL) Hematocrit (%) No. of leukocytes per microliter Blood urea nitrogen level (mg/dL) Creatinine level (mg/dL) Paracentesis volume (mL) Prothrombin time (s) Partial thromboplastin time (s)
14.3 6 1.8 42.6 6 4.9 16,107 6 5,335 14.3 6 5.6 0.82 6 0.07 4,804 6 3,558 14.2 6 1.9 27.3 6 2.9
14.5 6 1.9 43.0 6 5.2 16,940 6 5,740 14.2 6 5.5 0.84 6 0.05 5,425 6 3,830 14.4 6 2.1 27.6 6 2.7
Note: All values are means 6 SD. No statistically significant differences in any of the variables were observed between the groups. sICAM-1 5 soluble intercellular adhesion molecule-1; sVCAM-1 5 soluble vascular cell adhesion molecule-1.
Vol. 71, No. 5, May 1999
FIGURE 1 Plasma levels of soluble vascular cell adhesion molecule-1 (VCAM-1) in women with ovarian hyperstimulation syndrome (OHSS) and in women in the control group. The horizontal lines indicate the mean levels of the two groups.
findings suggest that these soluble cell adhesion molecules may play a role in the pathogenesis and progression of the disease. Further, we found a positive correlation between the plasma and peritoneal fluid concentrations of these molecules and both the plasma E2 concentration at the time of hCG administration and the number of ova retrieved, both of which are known to be risk factors for the development of OHSS (1, 2). Our results are in accordance with several observations that suggest the involvement of cell adhesion molecules in ovarian physiology (8 –11). The oocyte and early embryo express both ICAM-1 and VCAM-1 (10), and human granulosa cells express both surface ICAM-1 and its messenger RNA (9). Further, sICAM-1 was demonstrated in the follicular fluid of women undergoing ovulation induction, and its concentrations were positively correlated with the numbers of oocytes and follicles with a diameter of .15 mm (8). Mantzavinos et al. (11) reported that serum sICAM-1 levels were significantly higher in women undergoing IVF-ET than in women with normal cycles, probably because of multiple ovulation. These data suggest that cell adhesion molecules may play a role in physiologic ovarian processes such as folliculogenesis, ovulation, corpus luteum formation, and luteolysis, and as suggested by our findings, in the pathophysiology of OHSS that results from the exaggeration of these processes (1–5). The participation of cell adhesion molecules, which are major mediators of inflammation (12– FERTILITY & STERILITYt
FIGURE 2 Peritoneal fluid levels of soluble vascular cell adhesion molecule-1 (VCAM-1) in women with ovarian hyperstimulation syndrome (OHSS) and in women in the control group. The horizontal lines indicate the mean levels of the two groups.
17), in ovarian physiology and pathophysiology is not surprising in view of the many indications suggesting that inflammation may be a major physiologic phenomenon in the reproductive processes of the ovary (18 –20). Unregulated expression and increased shedding of VCAM-1 and ICAM-1 from the granulosa cell as a result of multiple ovulation and corpus luteum formation may result in the development of OHSS. These cell adhesion molecules may contribute to the evolution and progression of OHSS as a result of their inflammatory endowment (13–17). Both VCAM-1 and ICAM-1, together with the selectins, another group of adhesion molecules, participate in the “rolling” movement, adhesion, extravasation, recruitment, and activation of leukocytes and endothelial cells at sites of inflammation (13–17). Thus, the increased levels of cell adhesion molecules in the plasma and peritoneal fluid of women with OHSS may contribute to the initiation and propagation of the intense local and systemic inflammatory reactions that result from leukocyte recruitment and activation. Further, VCAM-1 is an angiogenic factor that may contribute to the angiogenesis and increased vascular permeability that characterize OHSS (21). Another source of the increased levels of sVCAM-1 and sICAM-1 in patients with OHSS is the formation of multiple 899
FIGURE 3
FIGURE 4
Peritoneal fluid levels of soluble intercellular adhesion molecule-1 (ICAM-1) in women with ovarian hyperstimulation syndrome (OHSS) and in women in the control group. The horizontal lines indicate the mean levels of the two groups.
Plasma levels of soluble intercellular adhesion molecule-1 (ICAM-1) in women with ovarian hyperstimulation syndrome (OHSS) and in women in the control group. The horizontal lines indicate the mean levels of the two groups.
corpora lutea. This process is accompanied by intense angiogenesis and endothelial and granulosa cell formation. Because ICAM-1 is constitutively expressed by the endothelium (13–17) and granulosa cells (8), this also may contribute to its increased levels (13–17).
E2 at the time of hCG administration and with the number of ova retrieved, both of which are known to be risk factors for the development of OHSS (1, 2). This observation suggests that concentrations of sVCAM-1 may serve as a marker for the development of OHSS, although a different study design should be used to explore this possibility.
Further, the expression and shedding of VCAM-1 and ICAM-1 are up-regulated by inflammatory cytokines (13– 17) that are ubiquitous in patients with OHSS (3–5). In accordance with this hypothesis, it was reported that interleukin-1b stimulated the secretion of sICAM-1 from granulosa cells in a dose-dependent manner (8). Moreover, it was reported recently (22) that vascular endothelial growth factor, levels of which are elevated in patients with OHSS (6, 7), induces the expression of VCAM-1. Trophoblast formation and invasion is accompanied by the expression of VCAM-1 and ICAM-1 (23–25). As a result, pregnancy, a known risk factor for OHSS progression (2), may result in increased levels of sVCAM-1 and sICAM-1, accounting in part for the disease progression observed in cycles that result in conception. In support of this assumption, Mantzavinos et al. (11) reported that serum sICAM-1 concentrations were significantly higher in cycles that resulted in conception than in those that did not. Plasma levels of sVCAM-1 were correlated with the disease activity of several inflammatory conditions (13–17). In the present study, we found that plasma levels of sVCAM-1 were positively correlated with plasma levels of 900
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Before we arrive at any conclusions, several points regarding the soluble form of cell adhesion molecules should be emphasized: First, this is the first report of increased levels of soluble cell adhesion molecules in patients with OHSS, and further studies are required to corroborate our findings. Further, because it is not possible to obtain peritoneal fluid samples from superovulatory women without OHSS, we used peritoneal fluid samples obtained during diagnostic laparoscopy from non-superovulatory patients as controls. The mean levels of soluble cell adhesion molecules in these samples represent only the basal levels of these molecules, which are higher in patients with OHSS. However, it is unknown whether the peritoneal fluid levels of these molecules are altered in superovulatory women in whom OHSS does not develop or whether the levels fluctuate during the menstrual cycle. Second, the measurement of these soluble molecules in different body fluids gauges only the freely available molecules and may miss a significant proportion of the amount shed because of its binding to adhesion molecule counterreceptors in the tissues. This may account for our failure to Vol. 71, No. 5, May 1999
demonstrate increased plasma levels of sICAM-1 in women with OHSS. Third, the physiologic significance of elevated levels of these soluble cell adhesion molecules is unknown. Because the mechanism of clearance of cell adhesion molecules is vague, these soluble isoforms may reflect merely a means of clearance. However, these soluble isoforms retain their binding capacity in vivo and thus can function in two ways, by triggering the biologic response on binding to the counterreceptor, and by competing in cell-cell interaction, functioning as inhibitors of cell adhesion (13–17). By this mechanism, soluble cell adhesion molecules may be considered as inflammation regulatory factors, assisting in the control of the inflammatory process. With these issues in mind, our findings may support a role for sVCAM-1 and sICAM-1 in the pathogenesis and progression of 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. Geva E, Amit A, Lerner-Geva L, Lessing JB. Ovarian hyperstimulation syndrome: the role of cytokines. Ass Reprod Rev 1997;7:168 –73. 4. Orvieto R, Ben-Rafael Z. Ovarian hyperstimulation syndrome: a new insight into an old enigma. J Soc Gynecol Invest 1998;5:110 –3. 5. Abramov Y, Schenker JG, Lewin A, Friedlender S, Nisman B, Barak V. Plasma inflammatory cytokines correlate to the ovarian hyperstimulation syndrome. Hum Reprod 1996;11:1381– 6. 6. McClure N, Healy DL, Rogers PA, Sullivan J, Beaton L, Haning RV, et al. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome. Lancet 1994;344:235– 6. 7. 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;68:133–7. 8. Vigano P, Fusi F, Gaffuri B, Bonzi V, Ferrari A, Vignali M. Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid. Fertil Steril 1998; 69:774 –9. 9. Vigano P, Gaffuri B, Ragni G, Di Blasio AM, Vignali M. Intercellular adhesion molecule-1 is expressed on human granulosa cells and medi-
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