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Soluble ICAM-1 and E-selectin levels correlate with clinical and biological aspects of severe ovarian hyperstimulation syndome
FERTILITY AND STERILITY威 VOL. 76, NO. 1, JULY 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
OVULATION INDUCTION
Soluble ICAM-1 and E-selectin levels correlate with clinical and biological aspects of severe ovarian hyperstimulation syndome Yoram Abramov, M.D.,a Joseph G. Schenker, M.D.,a Aby Lewin, M.D.,a Irit Kafka, M.D.,b Haim Jaffe, M.D.,b and Vivian Barak, Ph.D.c Hadassah Hebrew University Medical Center and Bikur-Holim Medical Center, Jerusalem, Israel
Received October 11, 2000; revised and accepted January 10, 2001. Reprint requests: Yoram Abramov, M.D., Department of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Ein-Kerem, Jerusalem, Israel 91120 (FAX: 972-2-6433337; E-mail: abramo@md2. huji.ac.il). a Department of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center. b Department of Obstetrics and Gynecology, BikurHolim Medical Center. c Immunology Laboratory for Tumor Diagnosis, Oncology Department, Hadassah Hebrew University Medical Center. 0015-0282/01/$20.00 PII S0015-0282(01)01820-9
Objective: To assess the potential involvement of the soluble endothelial cell-leukocyte adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1) in the pathophysiology of capillary hyperpermeability in the ovarian hyperstimulation syndrome (OHSS). Design: Controlled clinical study. Setting: Women hospitalized for severe OHSS after ovulation induction for IVF at two tertiary medical centers. Patient(s): Eleven patients with severe OHSS undergoing IVF and 20 controls who had received a similar ovulation induction regimen and did not develop OHSS. Intervention(s): Serial serum samples were obtained from all patients with OHSS from admission until discharge. Ascitic fluid was obtained from all patients by therapeutic paracentesis. Serum was obtained from all controls 4 – 8 days after ET. Main Outcome Measure(s): Samples were assayed for soluble E-selectin and soluble ICAM-1 by using enzyme-linked immunosorbent assay and results were correlated with clinical and biological aspects of OHSS. Result(s): Compared with controls, patients with severe OHSS had higher levels of soluble ICAM-1 and lower levels of soluble E-selectin detected in serum and ascites. Serum levels of soluble ICAM-1 decreased, while soluble E-selectin levels increased along with clinical and biological improvement. Serum soluble ICAM-1 showed significant positive correlation and serum soluble E-selectin showed significant negative correlation with clinical and biological aspects of severe OHSS. Conclusion(s): Soluble ICAM-1 and soluble E-selectin seem to be involved in the pathophysiology of capillary hyperpermeability in severe OHSS. (Fertil Steril威 2001;76:51–7. ©2001 by American Society for Reproductive Medicine.) Key Words: ICAM-1, E-selectin, ovarian hyperstimulation syndrome
It was recently 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 (1). During induction of ovulation for ART, multiple follicles are recruited synchronically and eventually ovulate simultaneously after hCG administration. This process is accompanied by a local inflammatory response within the ovary. In extreme cases, an uncontrolled and selfperpetuated ovarian response may occur, a phe-
nomenon known as the ovarian hyperstimulation syndrome (OHSS). This potentially lifethreatening complication is characterized by release of ovarian-derived offensive mediators to the bloodstream and third space, which cause 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 (2). Our group and others (3) have shown that certain inflammatory cytokines (interleukin-6, tumor necrosis factor-␣, and interleukin-1) (3) and growth factors (vascular endothelial 51
TABLE 1 Patient characteristics. Variable Age (y) IVF E2 level (pg/mL)a Pregnancies
Patients with OHSS (n ⫽ 11)
Controls (n ⫽ 20)
P value
28.5 ⫾ 4.5 11 (100) 2,980 ⫾ 370 8 (72)
28.3 ⫾ 4.1 20 (100) 2,240 ⫾ 280 13 (65)
NS NS NS NS
Note: Values are presented as mean ⫾ SE or no. (%). NS ⫽ not significant; OHSS ⫽ ovarian hyperstimulation syndrome. a On day of hCG administration. Abramov. Soluble ICAM-1 and E-selectin in OHSS. Fertil Steril 2001.
growth factor) (4) may play a major role in the pathophysiology of this systemic acute phase response. However, a rapidly expanding body of data has revealed the importance of endothelial cell adhesion molecules in the evolution and propagation of the inflammatory process in many human tissues (5–11). These molecules support leukocyte adhesion and extravasation through the vessel wall, which are key steps in human response to infection and tissue injury. It was recently reported that some adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), are expressed on human granulosa cells and mediate their binding to lymphoid cells (12). Soluble forms of these molecules can also be detected in follicular fluid after controlled ovarian stimulation for ART (12). Two of the best-examined endothelial-cell adhesion molecules are E-selectin and ICAM-1. The former is a well-recognized member of the selectin family characterized by an amino terminal lectin domain; the latter is a single-chain 90- to 114-kDa sialo glycoprotein that is also a member of the immunoglobulin gene superfamily. Daniel et al. (13) found increased levels of soluble ICAM-1 and soluble vascular cell adhesion molecule-1 (VCAM-1) in ascitic fluid from patients with severe OHSS. Soluble VCAM-1 but not soluble ICAM-1 levels were increased in the serum of these patients. We sought to determine whether soluble E-selectin and soluble ICAM-1 play a role in the acute phase response and vascular hyperpermeability characterizing severe OHSS.
MATERIALS AND METHODS Participants
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The ovulation induction regimen leading to OHSS was similar in all 11 patients. Nafarelin acetate as a nasal solution (Synarel; Syntex Laboratories Inc., Palo Alto, CA), 200 g three times daily, was started in the mid-luteal phase of the cycle. When pituitary suppression was documented (serum E2 level ⬍30 pg/mL [100 pmol/L] and serum P ⬍0.3 ng/mL [1 nmol/L]), intramuscular hMG (Pergonal; Serono Laboratories Inc., Randolph, MA) was added at 300 IU/d. Serum E2 and P, serial pelvic examinations and vaginal sonography were used to monitor patients as required. Doses of hMG were adjusted after 5 days of administration according to patients’ individual responses. Intramuscular hCG (Chorigon; TEVA Pharmaceutical Industries Ltd., Netanya, Israel) was administered 36 hours before oocyte retrieval. Luteal phase support included intramuscular P supplementation (Gestone, Paines & Byrne, Greenford, United Kingdom), 50 mg/d. No patients received hCG for luteal support. All 11 patients were hospitalized at the Hadassah Hebrew University and Bikur-Holim Medical Centers, Jerusalem, Israel, for severe OHSS and were treated with intravenous colloid and crystalloid solutions. Colloid solutions usually
TABLE 2 Manifestations of severe OHSS.
Eleven patients were included in the study group, all of whom were healthy infertile women 28.5 ⫾ 4.5 years of age who had undergone ovulation induction for IVF (Table 1). All presented with severe OHSS according to the classification systems of Golan et al. (2) and Navot et al. (14); criteria included massive ovarian enlargement (mean ovarian size, 12.5 ⫾ 1.3 cm); massive ascites (11 patients) with or without hydrothorax (3 patients); and laboratory evidence of hemoconcentration, as demonstrated by increased hematocrit (mean, 42.7% ⫾ 1.4%) or leukocyte count (mean, 22,100 ⫾ 52
1,800 cells/mm3) (Table 2). Markedly elevated serum E2 levels (mean, 4,990 ⫾ 320 pg/mL) were also recorded in all study patients. Seven patients had moderate to severe gastrointestinal symptoms, including diarrhea or vomiting.
Soluble ICAM-1 and E-selectin in OHSS
Variable
Value 12.5 ⫾ 1.3 11 (100) 3 (27) 42.7 ⫾ 1.4 22,100 ⫾ 1,800 4,990 ⫾ 320 7 (64)
Mean ovarian size (cm) Massive ascites Hydrothorax Hematocrit (%) Leukocyte count (cells/mm3) E2 level (pg/mL) Gastrointestinal symptoms Note: Values are presented as mean ⫾ SE or no. (%).
Abramov. Soluble ICAM-1 and E-Selectin in OHSS. Fertil Steril 2001.
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given were Polygeline, 3.5% (Haemaccel, Behringwerke AG, Marburg, Germany), and human albumin, 25%, (Kamada, Kibbuz Beit Kama, Israel). Crystalloid solutions usually given were normal saline, 0.9%. Solutions were administered daily and titrated according to urine output and hemodynamic variables. Serial blood counts, serum electrolytes measurements, liver function tests, and coagulation screening were performed daily. Ovarian size and the amount of ascitic fluid were assessed by using transvaginal and transabdominal sonography. Pleural effusion was assessed by physical examination and verified sonographically. Four of 11 patients required hospitalization in the intensive care unit for various intervals (2–7 days) for hemodynamic imbalance or deteriorating respiratory status. All 11 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 8 patients. We enrolled a matched control group consisting of 20 women undergoing controlled ovarian stimulation for IVF without developing OHSS. Controls were healthy women 28.3 ⫾ 4.1 years of age who were matched to the study group for age, fertility treatment, and pregnancy rate (Table 1). The ovulation induction regimen was similar to that in the study group. Human chorionic gonadotropin, 10,000 U, was administered 36 hours before oocyte retrieval. Mean E2 levels on the day of hCG administration were 2,240 ⫾ 180 pg/mL. All women underwent oocyte retrieval (mean number of oocytes retrieved, 8 ⫾ 3) and subsequent embryo transfer (mean number of embryos transferred, 2.6 ⫾ 0.6). Luteal support was similar to that described for the study group. Thirteen pregnancies (65%) related to the same ovulation induction treatment course were recorded in the control group. Blood samples from this group were analyzed for soluble ICAM-1 and soluble E-selectin levels by using the assays described below.
Blood and Ascitic Fluid Samples Three blood samples were obtained by venipuncture from each patient in the OHSS group. Sample 1 was obtained on admission for severe OHSS (acute phase; mean time after embryo transfer, 7.5 ⫾ 2.4 days). Sample 2 was obtained when significant clinical and laboratory improvement was apparent, including reduced ovarian size and ascites and decreased hemoconcentration (occurring on hospitalization days 7–13 [mean, 9.6 days]) (convalescence phase). Sample 3 was obtained after resolution of the syndrome, on the first visit to our clinic 7–10 days after discharge (resolution phase). One blood sample was obtained from each patient in the control group 4 to 8 days after embryo transfer. Samples were analyzed for hematocrit; leukocyte count; and levels of E2, soluble ICAM-1, and soluble E-selectin. All patients underwent therapeutic abdominal paracentesis because of increasing dyspnea related to ascites; ascitic fluid was anaFERTILITY & STERILITY威
lyzed for soluble ICAM-1 and soluble E-selectin levels. Informed consent was approved by the institutional human subjects review boards committee and was obtained from all study participants.
Estradiol, Soluble ICAM-1, and Soluble E-selectin Measurements Estradiol was measured by using a commercially available recombinant immunoassay kit (Diagnostic Products, Los Angeles, CA) according to the manufacturer’s recommendations. The intraassay and interassay coefficients of variation were 6% and 11%, respectively. Soluble ICAM-1 and E-selectin levels were measured by using a solid-phase enzyme-linked immunosorbent assay (Quantikine; R&D Systems Inc., Minneapolis, MN). The soluble ICAM-1 and E-selectin kit contains a polyclonal antibody against soluble ICAM-1 and E-selectin and a monoclonal antibody specific to soluble ICAM-1 and E-selectin. The assay uses the quantitative “sandwich” enzyme immunoassay technique, by which a polystyrene microtiter plate is precoated with a monoclonal antibody specific for the soluble ICAM-1 and E-selectin molecule. Standards and samples are introduced into the wells, and soluble ICAM-1 and E-selectin are bound by the immobilized antibody. After unbound proteins are washed away, the second enzymelinked polyclonal or monoclonal antibody specific for the soluble ICAM-1 and E-selectin is added to the wells to “sandwich” the soluble ICAM-1 and E-selectin immobilized during the first incubation. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells, and color develops in proportion to the amount of the soluble ICAM-1 and E-selectin bound in the initial step. The color development is stopped, and the intensity of the color is measured. A curve is prepared by plotting optical density against concentration of soluble ICAM-1 and E-selectin in the standard wells. By comparing the optical density of the samples to this standard curve, the concentration of soluble ICAM-1 and E-selectin in unknown samples is then determined. The intraassay coefficients of variations for the soluble ICAM-1 and E-selectin assays were 4.7% and 4.8%, respectively, and the interassay coeffiecients of varitation were 6.5% and 6.6%, respectively. The soluble ICAM-1 and E-selectin assay recognizes both natural human and recombinant human soluble ICAM-1 and E-selectin. However, no cross-reactivity between soluble ICAM-1 and E-selectin and other recombinant cytokines and growth factors (including interleukins 1–13, tumor necrosis factor, platelet-derived growth factor, epidermal growth factor, granulocyte-macrophage colony-stimulating factor, vascular endothelial growth factor, interferon-␥, and insulin-like growth factor) has been found using this assay.
Statistical Analysis Unpaired t-tests were used to compare group mean values for soluble ICAM-1, E-selectin, hematocrit, and leukocyte 53
FIGURE 1
FIGURE 2
Leukocyte count (WBC) and hematocrit (HCT) in patients with the ovarian hyperstimulation syndrome. Values are means ⫾ SE of three serial blood samples. All differences between the three samples were statistically significant (P⬍.05).
Serum concentrations of soluble (s) intracellular adhesion molecule (ICAM)-1 and soluble E-selectin in patients with the ovarian hyperstimulation syndrome during the acute phase (dark bars) and in controls (white bars). Values are means ⫾ SE. *P⬍.05 between patients and controls.
Abramov. Soluble ICAM-1 and E-Selectin in OHSS. Fertil Steril 2001. Abramov. Soluble ICAM-1 and E-Selectin in OHSS. Fertil Steril 2001.
count in the OHSS and control groups. Correlation between laboratory variables, including hematocrit, leukocyte count, and serum levels of soluble ICAM-1 and E-selectin, was tested by using the Pearson product moment correlation test. A P value ⬍.05 was considered statistically significant for all comparisons. All data are expressed as means ⫾ SE.
RESULTS Hematocrit and leukocyte count in the 11 study patients and 20 controls are shown in Figure 1. On admission, high hematocrit and leukocyte counts were recorded for all patients with OHSS, indicating hemoconcentration. Both hematocrit and leukocyte counts in the 11 patients with OHSS patients decreased significantly in later blood samples, after their clinical condition had improved (P⬍.05 for comparisons between the acute and convalescence phase and between the convalescence and resolution phase for both leukocyte count and hematocrit). Compared with controls, patients with OHSS had significantly higher serum levels of soluble ICAM-1 and significantly lower levels of soluble E-selectin during the acute phase (P⬍ .05 for both comparisons) (Fig. 2). Soluble ICAM-1 levels decreased significantly along with clinical improvement, reaching normal values in the convalescence phase (Fig. 3). In contrast, soluble E-selectin levels increased significantly along with clinical improvement, reaching maximum values during the convalescence phase (P⬍.05 for comparisons between the acute and convalescence phase and 54
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between the convalescence and resolution phase for both soluble ICAM-1 and soluble E-selectin). Statistical correlations between soluble ICAM-1 and soluble E-selectin levels and biological parameters of capillary leakage and hemoconcentration were calculated for the OHSS group by using the Pearson product moment correlation test. A significant correlation was found between soluble ICAM-1 levels and hematocrit (r⫽.62; P⬍.05) and between soluble ICAM-1 levels and leukocyte count (r⫽.63; P⬍.05). An inverse correlation was found between soluble E-selectin and hematocrit (r⫽⫺.63; P⬍.05) as well as leukocyte count (r⫽⫺.61; P⬍.05). Mean levels of soluble ICAM-1 in ascitic fluids aspirated from patients with OHSS were higher than those detected in serum specimens from these patients (430 ⫾ 43 vs. 320 ⫾ 45 pg/mL). However, mean soluble E-selectin levels detected in ascitic fluid did not differ significantly from those found in serum (14 ⫾ 4 vs. 12 ⫾ 3 pg/mL).
DISCUSSION Our results indicate that soluble forms of the adhesion molecules ICAM-1 and E-selectin correlate with biological and clinical aspects of severe OHSS. These endothelial-cell– leukocyte adhesion molecules may therefore be involved in the pathophysiology of this syndrome. Leukocyte-mediated tissue damage is a major component in various human disease processes, including the adult respiratory distress syndrome, autoimmune diseases, graft rejection, and postVol. 76, No. 1, July 2001
FIGURE 3 Kinetics of serum soluble (s) intercellular adhesion molecule (ICAM)-1 and soluble E-selectin in patients with the ovarian hyperstimulation syndrome. Values are means ⫾ SE. All differences between samples obtained during the acute phase and those obtained during convalescence or resolution were statistically significant (P⬍.05).
Abramov. Soluble ICAM-1 and E-Selectin in OHSS. Fertil Steril 2001.
ischemia reperfusion injury. Endothelial-cell–leukocyte adhesion molecules have been implicated as mediators in most of these disorders (15, 16). The ovarian hyperstimulation syndrome may be another entity in which these adhesion molecules are involved in exaggerated leukocyte recruitment and transendothelial migration, causing tissue damage and capillary hyperpermeability. The recent literature on ovarian physiology ascribes a central role to the immune system in regulation of ovarian function and, especially, ovulation. Leukocytes in the ovary may be in situ modulators of ovarian function that act through local secretion of regulatory soluble factors (17). Actual rupture of the follicle during ovulation may depend on tissue remodeling that is characteristic of an acute inflammatory reaction and includes mobilization of thecal fibroblasts, increased leukocyte migration, release of various mediators, and loosening of connective tissue elements in the follicle wall. Both corpus luteum formation and luteal regression involve progressive infiltration of lymphocytes and macrophages, release of chemokines and cytokines, and communication through cell adhesion molecules (17). The ovarian hyperstimulation syndrome is a life-threatening disorder caused by multiple uncontrolled simultaneous ovulations induced by exogenous gonadotropins and hCG. It has long been appreciated that the principal pathophysiologic mechanism behind this syndrome is capillary hyperpermeability, resulting in massive shift of fluids and proteins FERTILITY & STERILITY威
from the intravascular compartment to the third space. This in turn causes intravascular volume depletion, third-space fluid accumulation, and hemoconcentration, which are responsible for most of the morbidity associated with OHSS (2, 14). It is now thought that the above-mentioned capillary hyperpermeability is a result of endothelial damage induced by ovarian-derived inflammatory mediators and growth factors released by resident ovarian leukocytes and granulosa cells. We and others (3) have suggested a cardinal role for inflammatory cytokines (interleukin-1, interleukin-6, interleukin-8, and tumor necrosis factor) and vascular endothelial growth factor (4) in this process. We performed the current study to assess possible involvement of endothelial-cell– leukocyte adhesion molecules in this intraovarian and systemic acute-phase response. The physical interaction between leukocytes and endothelial cells in the microcirculation is crucial in the migration of circulating leukocytes into perivascular tissues (5). This interaction necessitates the presence of certain endothelial–leukocyte adhesion molecules that work in concert with cell-associated and soluble mediators to direct the movement of blood leukocytes. Selectins are a family of such adhesion molecules whose ligands are carbohydrates (6). E-selectin is exclusively produced by the endothelium after cytokine activation. It recognizes carbohydrates and supports the initial rolling of leukocytes to the activated endothelium. In contrast, ICAM-1 is a member of the immunoglobulin superfamily that is also expressed by vascular endothelial cells. It contains five extracellular immunoglobulin domains that can bind to leukocytes through specific integrins on their surface, mostly of the 2 group (7, 8). Like E-selectin, ICAM-1 is expressed in abundance on vascular endothelium after several hours of stimulation by inflammatory cytokines, such as interleukin-1 or tumor necrosis factor-␣ (9, 10). Endothelial cell–surface ICAM-1 appears to contribute to the adhesion and transmigration of most leukocyte types through an interaction with 2 integrins. In addition, neutrophils, monocytes, lymphocytes, and natural killer cells express CD11a/ CD18, which has also been shown to bind to ICAM-1 (8, 11). The pattern of expression of ICAM-1 differs from that of E-selectin in several ways (8). First, a low level of ICAM-1 but not E-selectin is typically found on unstimulated endothelial cells. Second, interleukin-1 or tumor necrosis factor␣–induced expression of ICAM-1 occurs more slowly and is prolonged compared with that of E-selectin. Third, interferon-␥ induces ICAM-1 expression but not E-selectin expression. Intercellular adhesion molecule-1 is also expressed in an inducible manner on various other cell types and may thereby contribute to adhesive events in many settings (8). The involvement of endothelial-cell–leukocyte adhesion molecules in ovarian function has been described. Vigano et 55
al. (18) found that ICAM-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells. Giavazzi et al. (19) later reported increased soluble ICAM-1 levels in serum and ascitic fluid from patients with ovarian cancer. Recently, Daniel et al. (13) reported increased serum levels of soluble ICAM-1 and soluble VCAM-1 in severe OHSS. Here, we report follow-up on 11 patients who developed severe OHSS and clinically improved later on; this improvement was parallel to serum soluble ICAM-1 and soluble E-selectin kinetics. During the acute phase of the syndrome, high levels of soluble ICAM-1 and low levels of soluble E-selectin were observed in the serum of all patients, accompanied by clinical features of capillary leakage and hemoconcentration. Soluble ICAM-1 levels decreased, whereas soluble E-selectin levels increased significantly after clinical convalescence, when extracellular fluid accumulation and hemoconcentration had resolved. This finding suggests possible involvement of these agents in the process of capillary hyperpermeability seen in OHSS. The strong statistical correlation (positive correlation for soluble ICAM-1 and inverse correlation for soluble E-selectin) found between these molecules and certain biological aspects of capillary leakage, such as elevated hematocrit and leukocyte count, further supports this assumption. However, a cause-and-effect relationship between these adhesion molecules and OHSS cannot be established by statistical correlations alone, and further studies are needed. Women who did not develop OHSS after controlled ovarian stimulation had significantly lower serum soluble ICAM-1 levels, thereby precluding the assumption that a larger ovarian mass and multiplicity of luteinized follicles that are surgically ruptured during egg retrieval are sufficient explanations for elevated levels of soluble ICAM-1. Soluble ICAM-1 levels found in ascitic fluid from affected persons exceeded those measured in the serum, suggesting either a direct spill of this compound from the ovaries or mesothelial surfaces (i.e., peritoneum) or active transport through ovarian or mesothelial blood vessels. Hence, soluble ICAM-1 may play a role in transudation of fluid and protein into the peritoneal cavity in OHSS. Although it is difficult to understand why soluble ICAM-1 and soluble E-selectin showed inverse kinetics in our study, two facts bear on this matter. First, levels of the soluble forms of these adhesion molecules do not necessarily reflect the amount of nonsoluble forms present in the affected tissues. In addition, the soluble forms may not have the same effect as the nonsoluble forms. Therefore, the amount of soluble forms detected is an indirect indicator of both the amount and activity of the nonsoluble forms present within the tissues. Under these circumstances, different kinetics for soluble E-selectin and soluble ICAM-1 seem plausible. Such a discordance has also been reported in other disorders of acute inflammatory response, such as systemic lupus erythematosus (20) and the post– cardiopulmonary by56
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pass syndrome (21), in which levels of serum soluble Eselectin were relatively low while those of ICAM-1 were high. Further research is clearly needed to clarify the exact role of each of these adhesion molecules in the pathophysiology of OHSS. An important question is whether biological alterations in OHSS or certain treatment methods may have interfered with evaluation of adhesion molecules in our study. Elevated leukocyte count, for instance, may increase serum cytokine concentrations and induce production of endothelial-cell– leukocyte adhesion molecules. It is hard to tell, however, whether the elevation in serum levels of soluble ICAM-1 is the result or the cause of the increased leukocyte count. It is clear that leukocyte count did not affect the assay for cytokine measurements itself, since the latter is performed after the cellular components have been separated. Coagulation disturbances, often encountered in patients with severe OHSS, probably also did not affect this assay, which is based on specific antibodies. To our knowledge, luteal-phase progesterone supplementation does not affect the assay for adhesion molecules measurements; however, more data are needed to reach firm conclusions on this matter. References 1. Mori T, Takakura K, Fujiwara H, Hayashi K. Immunology of ovarian function. In: Bronson RA, Alexander NJ, Anderson D, Ware Branch D, Kutten WH (eds). Reproductive immunology. London: Blackwell Science, 1996:83– 417. 2. 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. 3. Abramov Y, Schenker JG, Lewin A, Friedler S, Nisman B, Barak V. Plasma inflammatory cytokines correlate to the ovarian hyperstimulation syndrome. Hum Reprod 1996;11:1381– 6. 4. 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. 5. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 1994;7:2068 –101. 6. Bevilacqua MP, Nelson RM. Selectins. J Clin Invest 1993;91:379 – 87. 7. Simmons D, Madgoba MW, Seed B. ICAM-1, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecules NCAM. Nature 1988;331:624 – 6. 8. Springer TA. Adhesion receptors of the immune system. Nature 1990; 346:425–34. 9. Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA. Induction by IL-1 and interferon-␥: tissue distribution, biochemistry, function of a natural adherence molecule (ICAM-1). J Immunol 1986;137: 245–54. 10. Pober JS, Gimbrone MA Jr, Lapierre LA, Mendrick DL, Fiers W, Rothlein R, Springer TA. Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor ␣, and immune interferon. J Immunol 1986;137:1893– 6. 11. Carlos TM, Harlan JM. Membrane proteins involved in phagocyte adherence to endothelium. Immunol Rev 1990;114:5–28. 12. 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. 13. Daniel Y, Geva E, Amit A, Baram A, Englander T, Kupferminc 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. 14. Navot D, Bergh PA, Laufer N. Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil Steril 1992;58:249 – 61. 15. Mulligan MS, Varani J, Dame MK, Lane CL, Smith CW, Anderson, DC et al. Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats. J Clin Invest 1991;1396 – 406. 16. Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L,
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Karin N. Prevention of experimental autoimmune encephalomyelitis by antibodies against ␣41 integrin. Nature 1992;356:63– 6. 17. Adashi EY. The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocrine Rev 1990;11:454 – 64. 18. Vigano P, Gaffuri B, Ragni G, Di Blasio AM, Vignali M. Intercellular adhesion molecule-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells. J Clin Endocrinol Metab 1997;82: 101–5. 19. Giavazzi R, Nicoletti MI, Chirivi RGS, Hemingway I, Bernasconi S, Allavena P, et al. Soluble intercellular adhesion molecule-1 (ICAM-1)
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is released into the serum and ascites of human ovarian carcinoma patients and in nude mice bearing tumor xenografts. Eur J Cancer 1994;30:1865–70. 20. Spronk PE, Bootsma H, Huitema MG, Limburg PC, Kallenberg CGM. Levels of soluble VCAM-1, soluble ICAM-1 and soluble E-selectin during disease exacerbations in patients with systemic lupus erythematosus (SLE): a long term prospective study. Exp Immunol 1994;97:439 – 44. 21. Diago MC, Garcia-Unzueta MT, Marcano G, Merino J, Salas E, Amado JA. Serum soluble selectins in patients undergoing cardiopulmonary bypass. Relationship with circulating blood cells and inflammationrelated cytokines. Acta Anaesthesiol Scand 1997;41:725-30.
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OVULATION INDUCTION
Soluble ICAM-1 and E-selectin levels correlate with clinical and biological aspects of severe ovarian hyperstimulation syndome Yoram Abramov, M.D.,a Joseph G. Schenker, M.D.,a Aby Lewin, M.D.,a Irit Kafka, M.D.,b Haim Jaffe, M.D.,b and Vivian Barak, Ph.D.c Hadassah Hebrew University Medical Center and Bikur-Holim Medical Center, Jerusalem, Israel
Received October 11, 2000; revised and accepted January 10, 2001. Reprint requests: Yoram Abramov, M.D., Department of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Ein-Kerem, Jerusalem, Israel 91120 (FAX: 972-2-6433337; E-mail: abramo@md2. huji.ac.il). a Department of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center. b Department of Obstetrics and Gynecology, BikurHolim Medical Center. c Immunology Laboratory for Tumor Diagnosis, Oncology Department, Hadassah Hebrew University Medical Center. 0015-0282/01/$20.00 PII S0015-0282(01)01820-9
Objective: To assess the potential involvement of the soluble endothelial cell-leukocyte adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1) in the pathophysiology of capillary hyperpermeability in the ovarian hyperstimulation syndrome (OHSS). Design: Controlled clinical study. Setting: Women hospitalized for severe OHSS after ovulation induction for IVF at two tertiary medical centers. Patient(s): Eleven patients with severe OHSS undergoing IVF and 20 controls who had received a similar ovulation induction regimen and did not develop OHSS. Intervention(s): Serial serum samples were obtained from all patients with OHSS from admission until discharge. Ascitic fluid was obtained from all patients by therapeutic paracentesis. Serum was obtained from all controls 4 – 8 days after ET. Main Outcome Measure(s): Samples were assayed for soluble E-selectin and soluble ICAM-1 by using enzyme-linked immunosorbent assay and results were correlated with clinical and biological aspects of OHSS. Result(s): Compared with controls, patients with severe OHSS had higher levels of soluble ICAM-1 and lower levels of soluble E-selectin detected in serum and ascites. Serum levels of soluble ICAM-1 decreased, while soluble E-selectin levels increased along with clinical and biological improvement. Serum soluble ICAM-1 showed significant positive correlation and serum soluble E-selectin showed significant negative correlation with clinical and biological aspects of severe OHSS. Conclusion(s): Soluble ICAM-1 and soluble E-selectin seem to be involved in the pathophysiology of capillary hyperpermeability in severe OHSS. (Fertil Steril威 2001;76:51–7. ©2001 by American Society for Reproductive Medicine.) Key Words: ICAM-1, E-selectin, ovarian hyperstimulation syndrome
It was recently 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 (1). During induction of ovulation for ART, multiple follicles are recruited synchronically and eventually ovulate simultaneously after hCG administration. This process is accompanied by a local inflammatory response within the ovary. In extreme cases, an uncontrolled and selfperpetuated ovarian response may occur, a phe-
nomenon known as the ovarian hyperstimulation syndrome (OHSS). This potentially lifethreatening complication is characterized by release of ovarian-derived offensive mediators to the bloodstream and third space, which cause 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 (2). Our group and others (3) have shown that certain inflammatory cytokines (interleukin-6, tumor necrosis factor-␣, and interleukin-1) (3) and growth factors (vascular endothelial 51
TABLE 1 Patient characteristics. Variable Age (y) IVF E2 level (pg/mL)a Pregnancies
Patients with OHSS (n ⫽ 11)
Controls (n ⫽ 20)
P value
28.5 ⫾ 4.5 11 (100) 2,980 ⫾ 370 8 (72)
28.3 ⫾ 4.1 20 (100) 2,240 ⫾ 280 13 (65)
NS NS NS NS
Note: Values are presented as mean ⫾ SE or no. (%). NS ⫽ not significant; OHSS ⫽ ovarian hyperstimulation syndrome. a On day of hCG administration. Abramov. Soluble ICAM-1 and E-selectin in OHSS. Fertil Steril 2001.
growth factor) (4) may play a major role in the pathophysiology of this systemic acute phase response. However, a rapidly expanding body of data has revealed the importance of endothelial cell adhesion molecules in the evolution and propagation of the inflammatory process in many human tissues (5–11). These molecules support leukocyte adhesion and extravasation through the vessel wall, which are key steps in human response to infection and tissue injury. It was recently reported that some adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), are expressed on human granulosa cells and mediate their binding to lymphoid cells (12). Soluble forms of these molecules can also be detected in follicular fluid after controlled ovarian stimulation for ART (12). Two of the best-examined endothelial-cell adhesion molecules are E-selectin and ICAM-1. The former is a well-recognized member of the selectin family characterized by an amino terminal lectin domain; the latter is a single-chain 90- to 114-kDa sialo glycoprotein that is also a member of the immunoglobulin gene superfamily. Daniel et al. (13) found increased levels of soluble ICAM-1 and soluble vascular cell adhesion molecule-1 (VCAM-1) in ascitic fluid from patients with severe OHSS. Soluble VCAM-1 but not soluble ICAM-1 levels were increased in the serum of these patients. We sought to determine whether soluble E-selectin and soluble ICAM-1 play a role in the acute phase response and vascular hyperpermeability characterizing severe OHSS.
MATERIALS AND METHODS Participants
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The ovulation induction regimen leading to OHSS was similar in all 11 patients. Nafarelin acetate as a nasal solution (Synarel; Syntex Laboratories Inc., Palo Alto, CA), 200 g three times daily, was started in the mid-luteal phase of the cycle. When pituitary suppression was documented (serum E2 level ⬍30 pg/mL [100 pmol/L] and serum P ⬍0.3 ng/mL [1 nmol/L]), intramuscular hMG (Pergonal; Serono Laboratories Inc., Randolph, MA) was added at 300 IU/d. Serum E2 and P, serial pelvic examinations and vaginal sonography were used to monitor patients as required. Doses of hMG were adjusted after 5 days of administration according to patients’ individual responses. Intramuscular hCG (Chorigon; TEVA Pharmaceutical Industries Ltd., Netanya, Israel) was administered 36 hours before oocyte retrieval. Luteal phase support included intramuscular P supplementation (Gestone, Paines & Byrne, Greenford, United Kingdom), 50 mg/d. No patients received hCG for luteal support. All 11 patients were hospitalized at the Hadassah Hebrew University and Bikur-Holim Medical Centers, Jerusalem, Israel, for severe OHSS and were treated with intravenous colloid and crystalloid solutions. Colloid solutions usually
TABLE 2 Manifestations of severe OHSS.
Eleven patients were included in the study group, all of whom were healthy infertile women 28.5 ⫾ 4.5 years of age who had undergone ovulation induction for IVF (Table 1). All presented with severe OHSS according to the classification systems of Golan et al. (2) and Navot et al. (14); criteria included massive ovarian enlargement (mean ovarian size, 12.5 ⫾ 1.3 cm); massive ascites (11 patients) with or without hydrothorax (3 patients); and laboratory evidence of hemoconcentration, as demonstrated by increased hematocrit (mean, 42.7% ⫾ 1.4%) or leukocyte count (mean, 22,100 ⫾ 52
1,800 cells/mm3) (Table 2). Markedly elevated serum E2 levels (mean, 4,990 ⫾ 320 pg/mL) were also recorded in all study patients. Seven patients had moderate to severe gastrointestinal symptoms, including diarrhea or vomiting.
Soluble ICAM-1 and E-selectin in OHSS
Variable
Value 12.5 ⫾ 1.3 11 (100) 3 (27) 42.7 ⫾ 1.4 22,100 ⫾ 1,800 4,990 ⫾ 320 7 (64)
Mean ovarian size (cm) Massive ascites Hydrothorax Hematocrit (%) Leukocyte count (cells/mm3) E2 level (pg/mL) Gastrointestinal symptoms Note: Values are presented as mean ⫾ SE or no. (%).
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given were Polygeline, 3.5% (Haemaccel, Behringwerke AG, Marburg, Germany), and human albumin, 25%, (Kamada, Kibbuz Beit Kama, Israel). Crystalloid solutions usually given were normal saline, 0.9%. Solutions were administered daily and titrated according to urine output and hemodynamic variables. Serial blood counts, serum electrolytes measurements, liver function tests, and coagulation screening were performed daily. Ovarian size and the amount of ascitic fluid were assessed by using transvaginal and transabdominal sonography. Pleural effusion was assessed by physical examination and verified sonographically. Four of 11 patients required hospitalization in the intensive care unit for various intervals (2–7 days) for hemodynamic imbalance or deteriorating respiratory status. All 11 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 8 patients. We enrolled a matched control group consisting of 20 women undergoing controlled ovarian stimulation for IVF without developing OHSS. Controls were healthy women 28.3 ⫾ 4.1 years of age who were matched to the study group for age, fertility treatment, and pregnancy rate (Table 1). The ovulation induction regimen was similar to that in the study group. Human chorionic gonadotropin, 10,000 U, was administered 36 hours before oocyte retrieval. Mean E2 levels on the day of hCG administration were 2,240 ⫾ 180 pg/mL. All women underwent oocyte retrieval (mean number of oocytes retrieved, 8 ⫾ 3) and subsequent embryo transfer (mean number of embryos transferred, 2.6 ⫾ 0.6). Luteal support was similar to that described for the study group. Thirteen pregnancies (65%) related to the same ovulation induction treatment course were recorded in the control group. Blood samples from this group were analyzed for soluble ICAM-1 and soluble E-selectin levels by using the assays described below.
Blood and Ascitic Fluid Samples Three blood samples were obtained by venipuncture from each patient in the OHSS group. Sample 1 was obtained on admission for severe OHSS (acute phase; mean time after embryo transfer, 7.5 ⫾ 2.4 days). Sample 2 was obtained when significant clinical and laboratory improvement was apparent, including reduced ovarian size and ascites and decreased hemoconcentration (occurring on hospitalization days 7–13 [mean, 9.6 days]) (convalescence phase). Sample 3 was obtained after resolution of the syndrome, on the first visit to our clinic 7–10 days after discharge (resolution phase). One blood sample was obtained from each patient in the control group 4 to 8 days after embryo transfer. Samples were analyzed for hematocrit; leukocyte count; and levels of E2, soluble ICAM-1, and soluble E-selectin. All patients underwent therapeutic abdominal paracentesis because of increasing dyspnea related to ascites; ascitic fluid was anaFERTILITY & STERILITY威
lyzed for soluble ICAM-1 and soluble E-selectin levels. Informed consent was approved by the institutional human subjects review boards committee and was obtained from all study participants.
Estradiol, Soluble ICAM-1, and Soluble E-selectin Measurements Estradiol was measured by using a commercially available recombinant immunoassay kit (Diagnostic Products, Los Angeles, CA) according to the manufacturer’s recommendations. The intraassay and interassay coefficients of variation were 6% and 11%, respectively. Soluble ICAM-1 and E-selectin levels were measured by using a solid-phase enzyme-linked immunosorbent assay (Quantikine; R&D Systems Inc., Minneapolis, MN). The soluble ICAM-1 and E-selectin kit contains a polyclonal antibody against soluble ICAM-1 and E-selectin and a monoclonal antibody specific to soluble ICAM-1 and E-selectin. The assay uses the quantitative “sandwich” enzyme immunoassay technique, by which a polystyrene microtiter plate is precoated with a monoclonal antibody specific for the soluble ICAM-1 and E-selectin molecule. Standards and samples are introduced into the wells, and soluble ICAM-1 and E-selectin are bound by the immobilized antibody. After unbound proteins are washed away, the second enzymelinked polyclonal or monoclonal antibody specific for the soluble ICAM-1 and E-selectin is added to the wells to “sandwich” the soluble ICAM-1 and E-selectin immobilized during the first incubation. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells, and color develops in proportion to the amount of the soluble ICAM-1 and E-selectin bound in the initial step. The color development is stopped, and the intensity of the color is measured. A curve is prepared by plotting optical density against concentration of soluble ICAM-1 and E-selectin in the standard wells. By comparing the optical density of the samples to this standard curve, the concentration of soluble ICAM-1 and E-selectin in unknown samples is then determined. The intraassay coefficients of variations for the soluble ICAM-1 and E-selectin assays were 4.7% and 4.8%, respectively, and the interassay coeffiecients of varitation were 6.5% and 6.6%, respectively. The soluble ICAM-1 and E-selectin assay recognizes both natural human and recombinant human soluble ICAM-1 and E-selectin. However, no cross-reactivity between soluble ICAM-1 and E-selectin and other recombinant cytokines and growth factors (including interleukins 1–13, tumor necrosis factor, platelet-derived growth factor, epidermal growth factor, granulocyte-macrophage colony-stimulating factor, vascular endothelial growth factor, interferon-␥, and insulin-like growth factor) has been found using this assay.
Statistical Analysis Unpaired t-tests were used to compare group mean values for soluble ICAM-1, E-selectin, hematocrit, and leukocyte 53
FIGURE 1
FIGURE 2
Leukocyte count (WBC) and hematocrit (HCT) in patients with the ovarian hyperstimulation syndrome. Values are means ⫾ SE of three serial blood samples. All differences between the three samples were statistically significant (P⬍.05).
Serum concentrations of soluble (s) intracellular adhesion molecule (ICAM)-1 and soluble E-selectin in patients with the ovarian hyperstimulation syndrome during the acute phase (dark bars) and in controls (white bars). Values are means ⫾ SE. *P⬍.05 between patients and controls.
Abramov. Soluble ICAM-1 and E-Selectin in OHSS. Fertil Steril 2001. Abramov. Soluble ICAM-1 and E-Selectin in OHSS. Fertil Steril 2001.
count in the OHSS and control groups. Correlation between laboratory variables, including hematocrit, leukocyte count, and serum levels of soluble ICAM-1 and E-selectin, was tested by using the Pearson product moment correlation test. A P value ⬍.05 was considered statistically significant for all comparisons. All data are expressed as means ⫾ SE.
RESULTS Hematocrit and leukocyte count in the 11 study patients and 20 controls are shown in Figure 1. On admission, high hematocrit and leukocyte counts were recorded for all patients with OHSS, indicating hemoconcentration. Both hematocrit and leukocyte counts in the 11 patients with OHSS patients decreased significantly in later blood samples, after their clinical condition had improved (P⬍.05 for comparisons between the acute and convalescence phase and between the convalescence and resolution phase for both leukocyte count and hematocrit). Compared with controls, patients with OHSS had significantly higher serum levels of soluble ICAM-1 and significantly lower levels of soluble E-selectin during the acute phase (P⬍ .05 for both comparisons) (Fig. 2). Soluble ICAM-1 levels decreased significantly along with clinical improvement, reaching normal values in the convalescence phase (Fig. 3). In contrast, soluble E-selectin levels increased significantly along with clinical improvement, reaching maximum values during the convalescence phase (P⬍.05 for comparisons between the acute and convalescence phase and 54
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Soluble ICAM-1 and E-selectin in OHSS
between the convalescence and resolution phase for both soluble ICAM-1 and soluble E-selectin). Statistical correlations between soluble ICAM-1 and soluble E-selectin levels and biological parameters of capillary leakage and hemoconcentration were calculated for the OHSS group by using the Pearson product moment correlation test. A significant correlation was found between soluble ICAM-1 levels and hematocrit (r⫽.62; P⬍.05) and between soluble ICAM-1 levels and leukocyte count (r⫽.63; P⬍.05). An inverse correlation was found between soluble E-selectin and hematocrit (r⫽⫺.63; P⬍.05) as well as leukocyte count (r⫽⫺.61; P⬍.05). Mean levels of soluble ICAM-1 in ascitic fluids aspirated from patients with OHSS were higher than those detected in serum specimens from these patients (430 ⫾ 43 vs. 320 ⫾ 45 pg/mL). However, mean soluble E-selectin levels detected in ascitic fluid did not differ significantly from those found in serum (14 ⫾ 4 vs. 12 ⫾ 3 pg/mL).
DISCUSSION Our results indicate that soluble forms of the adhesion molecules ICAM-1 and E-selectin correlate with biological and clinical aspects of severe OHSS. These endothelial-cell– leukocyte adhesion molecules may therefore be involved in the pathophysiology of this syndrome. Leukocyte-mediated tissue damage is a major component in various human disease processes, including the adult respiratory distress syndrome, autoimmune diseases, graft rejection, and postVol. 76, No. 1, July 2001
FIGURE 3 Kinetics of serum soluble (s) intercellular adhesion molecule (ICAM)-1 and soluble E-selectin in patients with the ovarian hyperstimulation syndrome. Values are means ⫾ SE. All differences between samples obtained during the acute phase and those obtained during convalescence or resolution were statistically significant (P⬍.05).
Abramov. Soluble ICAM-1 and E-Selectin in OHSS. Fertil Steril 2001.
ischemia reperfusion injury. Endothelial-cell–leukocyte adhesion molecules have been implicated as mediators in most of these disorders (15, 16). The ovarian hyperstimulation syndrome may be another entity in which these adhesion molecules are involved in exaggerated leukocyte recruitment and transendothelial migration, causing tissue damage and capillary hyperpermeability. The recent literature on ovarian physiology ascribes a central role to the immune system in regulation of ovarian function and, especially, ovulation. Leukocytes in the ovary may be in situ modulators of ovarian function that act through local secretion of regulatory soluble factors (17). Actual rupture of the follicle during ovulation may depend on tissue remodeling that is characteristic of an acute inflammatory reaction and includes mobilization of thecal fibroblasts, increased leukocyte migration, release of various mediators, and loosening of connective tissue elements in the follicle wall. Both corpus luteum formation and luteal regression involve progressive infiltration of lymphocytes and macrophages, release of chemokines and cytokines, and communication through cell adhesion molecules (17). The ovarian hyperstimulation syndrome is a life-threatening disorder caused by multiple uncontrolled simultaneous ovulations induced by exogenous gonadotropins and hCG. It has long been appreciated that the principal pathophysiologic mechanism behind this syndrome is capillary hyperpermeability, resulting in massive shift of fluids and proteins FERTILITY & STERILITY威
from the intravascular compartment to the third space. This in turn causes intravascular volume depletion, third-space fluid accumulation, and hemoconcentration, which are responsible for most of the morbidity associated with OHSS (2, 14). It is now thought that the above-mentioned capillary hyperpermeability is a result of endothelial damage induced by ovarian-derived inflammatory mediators and growth factors released by resident ovarian leukocytes and granulosa cells. We and others (3) have suggested a cardinal role for inflammatory cytokines (interleukin-1, interleukin-6, interleukin-8, and tumor necrosis factor) and vascular endothelial growth factor (4) in this process. We performed the current study to assess possible involvement of endothelial-cell– leukocyte adhesion molecules in this intraovarian and systemic acute-phase response. The physical interaction between leukocytes and endothelial cells in the microcirculation is crucial in the migration of circulating leukocytes into perivascular tissues (5). This interaction necessitates the presence of certain endothelial–leukocyte adhesion molecules that work in concert with cell-associated and soluble mediators to direct the movement of blood leukocytes. Selectins are a family of such adhesion molecules whose ligands are carbohydrates (6). E-selectin is exclusively produced by the endothelium after cytokine activation. It recognizes carbohydrates and supports the initial rolling of leukocytes to the activated endothelium. In contrast, ICAM-1 is a member of the immunoglobulin superfamily that is also expressed by vascular endothelial cells. It contains five extracellular immunoglobulin domains that can bind to leukocytes through specific integrins on their surface, mostly of the 2 group (7, 8). Like E-selectin, ICAM-1 is expressed in abundance on vascular endothelium after several hours of stimulation by inflammatory cytokines, such as interleukin-1 or tumor necrosis factor-␣ (9, 10). Endothelial cell–surface ICAM-1 appears to contribute to the adhesion and transmigration of most leukocyte types through an interaction with 2 integrins. In addition, neutrophils, monocytes, lymphocytes, and natural killer cells express CD11a/ CD18, which has also been shown to bind to ICAM-1 (8, 11). The pattern of expression of ICAM-1 differs from that of E-selectin in several ways (8). First, a low level of ICAM-1 but not E-selectin is typically found on unstimulated endothelial cells. Second, interleukin-1 or tumor necrosis factor␣–induced expression of ICAM-1 occurs more slowly and is prolonged compared with that of E-selectin. Third, interferon-␥ induces ICAM-1 expression but not E-selectin expression. Intercellular adhesion molecule-1 is also expressed in an inducible manner on various other cell types and may thereby contribute to adhesive events in many settings (8). The involvement of endothelial-cell–leukocyte adhesion molecules in ovarian function has been described. Vigano et 55
al. (18) found that ICAM-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells. Giavazzi et al. (19) later reported increased soluble ICAM-1 levels in serum and ascitic fluid from patients with ovarian cancer. Recently, Daniel et al. (13) reported increased serum levels of soluble ICAM-1 and soluble VCAM-1 in severe OHSS. Here, we report follow-up on 11 patients who developed severe OHSS and clinically improved later on; this improvement was parallel to serum soluble ICAM-1 and soluble E-selectin kinetics. During the acute phase of the syndrome, high levels of soluble ICAM-1 and low levels of soluble E-selectin were observed in the serum of all patients, accompanied by clinical features of capillary leakage and hemoconcentration. Soluble ICAM-1 levels decreased, whereas soluble E-selectin levels increased significantly after clinical convalescence, when extracellular fluid accumulation and hemoconcentration had resolved. This finding suggests possible involvement of these agents in the process of capillary hyperpermeability seen in OHSS. The strong statistical correlation (positive correlation for soluble ICAM-1 and inverse correlation for soluble E-selectin) found between these molecules and certain biological aspects of capillary leakage, such as elevated hematocrit and leukocyte count, further supports this assumption. However, a cause-and-effect relationship between these adhesion molecules and OHSS cannot be established by statistical correlations alone, and further studies are needed. Women who did not develop OHSS after controlled ovarian stimulation had significantly lower serum soluble ICAM-1 levels, thereby precluding the assumption that a larger ovarian mass and multiplicity of luteinized follicles that are surgically ruptured during egg retrieval are sufficient explanations for elevated levels of soluble ICAM-1. Soluble ICAM-1 levels found in ascitic fluid from affected persons exceeded those measured in the serum, suggesting either a direct spill of this compound from the ovaries or mesothelial surfaces (i.e., peritoneum) or active transport through ovarian or mesothelial blood vessels. Hence, soluble ICAM-1 may play a role in transudation of fluid and protein into the peritoneal cavity in OHSS. Although it is difficult to understand why soluble ICAM-1 and soluble E-selectin showed inverse kinetics in our study, two facts bear on this matter. First, levels of the soluble forms of these adhesion molecules do not necessarily reflect the amount of nonsoluble forms present in the affected tissues. In addition, the soluble forms may not have the same effect as the nonsoluble forms. Therefore, the amount of soluble forms detected is an indirect indicator of both the amount and activity of the nonsoluble forms present within the tissues. Under these circumstances, different kinetics for soluble E-selectin and soluble ICAM-1 seem plausible. Such a discordance has also been reported in other disorders of acute inflammatory response, such as systemic lupus erythematosus (20) and the post– cardiopulmonary by56
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pass syndrome (21), in which levels of serum soluble Eselectin were relatively low while those of ICAM-1 were high. Further research is clearly needed to clarify the exact role of each of these adhesion molecules in the pathophysiology of OHSS. An important question is whether biological alterations in OHSS or certain treatment methods may have interfered with evaluation of adhesion molecules in our study. Elevated leukocyte count, for instance, may increase serum cytokine concentrations and induce production of endothelial-cell– leukocyte adhesion molecules. It is hard to tell, however, whether the elevation in serum levels of soluble ICAM-1 is the result or the cause of the increased leukocyte count. It is clear that leukocyte count did not affect the assay for cytokine measurements itself, since the latter is performed after the cellular components have been separated. Coagulation disturbances, often encountered in patients with severe OHSS, probably also did not affect this assay, which is based on specific antibodies. To our knowledge, luteal-phase progesterone supplementation does not affect the assay for adhesion molecules measurements; however, more data are needed to reach firm conclusions on this matter. References 1. Mori T, Takakura K, Fujiwara H, Hayashi K. Immunology of ovarian function. In: Bronson RA, Alexander NJ, Anderson D, Ware Branch D, Kutten WH (eds). Reproductive immunology. London: Blackwell Science, 1996:83– 417. 2. 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. 3. Abramov Y, Schenker JG, Lewin A, Friedler S, Nisman B, Barak V. Plasma inflammatory cytokines correlate to the ovarian hyperstimulation syndrome. Hum Reprod 1996;11:1381– 6. 4. 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. 5. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 1994;7:2068 –101. 6. Bevilacqua MP, Nelson RM. Selectins. J Clin Invest 1993;91:379 – 87. 7. Simmons D, Madgoba MW, Seed B. ICAM-1, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecules NCAM. Nature 1988;331:624 – 6. 8. Springer TA. Adhesion receptors of the immune system. Nature 1990; 346:425–34. 9. Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA. Induction by IL-1 and interferon-␥: tissue distribution, biochemistry, function of a natural adherence molecule (ICAM-1). J Immunol 1986;137: 245–54. 10. Pober JS, Gimbrone MA Jr, Lapierre LA, Mendrick DL, Fiers W, Rothlein R, Springer TA. Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor ␣, and immune interferon. J Immunol 1986;137:1893– 6. 11. Carlos TM, Harlan JM. Membrane proteins involved in phagocyte adherence to endothelium. Immunol Rev 1990;114:5–28. 12. 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. 13. Daniel Y, Geva E, Amit A, Baram A, Englander T, Kupferminc 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. 14. Navot D, Bergh PA, Laufer N. Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil Steril 1992;58:249 – 61. 15. Mulligan MS, Varani J, Dame MK, Lane CL, Smith CW, Anderson, DC et al. Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats. J Clin Invest 1991;1396 – 406. 16. Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L,
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Karin N. Prevention of experimental autoimmune encephalomyelitis by antibodies against ␣41 integrin. Nature 1992;356:63– 6. 17. Adashi EY. The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocrine Rev 1990;11:454 – 64. 18. Vigano P, Gaffuri B, Ragni G, Di Blasio AM, Vignali M. Intercellular adhesion molecule-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells. J Clin Endocrinol Metab 1997;82: 101–5. 19. Giavazzi R, Nicoletti MI, Chirivi RGS, Hemingway I, Bernasconi S, Allavena P, et al. Soluble intercellular adhesion molecule-1 (ICAM-1)
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is released into the serum and ascites of human ovarian carcinoma patients and in nude mice bearing tumor xenografts. Eur J Cancer 1994;30:1865–70. 20. Spronk PE, Bootsma H, Huitema MG, Limburg PC, Kallenberg CGM. Levels of soluble VCAM-1, soluble ICAM-1 and soluble E-selectin during disease exacerbations in patients with systemic lupus erythematosus (SLE): a long term prospective study. Exp Immunol 1994;97:439 – 44. 21. Diago MC, Garcia-Unzueta MT, Marcano G, Merino J, Salas E, Amado JA. Serum soluble selectins in patients undergoing cardiopulmonary bypass. Relationship with circulating blood cells and inflammationrelated cytokines. Acta Anaesthesiol Scand 1997;41:725-30.
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