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Serum vascular endothelial growth factor concentrations in in vitro fertilization cycles predict the risk of ovarian hyperstimulation syndrome
FERTILITY AND STERILITYt VOL. 71, NO. 2, FEBRUARY 1999 Copyright © 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Serum vascular endothelial growth factor concentrations in in vitro fertilization cycles predict the risk of ovarian hyperstimulation syndrome Rina Agrawal, M.D.,*† Seang Ling Tan, M.D.,†‡ Sarah Wild, M.D.,§ Povilas Sladkevicius, M.D.,†\ Lawrence Engmann, M.D.,† Nadia Payne, M.Sc.,* Jinan Bekir, M.D.,† Stuart Campbell, M.D.,†\ Gerard Conway, M.D.,* and Howard Jacobs, M.D.*† University College London Medical School, The Middlesex Hospital and London Women’s Clinic, London, United Kingdom
Received May 26, 1998; revised and accepted September 23, 1998. Reprint requests: Rina Agrawal, M.D., Department of Reproductive Endocrinology, Cobbold Laboratories, The Middlesex Hospital, Mortimer Street, London W1N 8AA United Kingdom (FAX: 0171-4875850; E-mail: ganapaty@dircon. co.uk). * University College London Medical School, Department of Reproductive Endocrinology, Cobbold Laboratories, The Middlesex Hospital, London. † London Women’s Clinic, London. ‡ Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada. § Epidemiology Unit, London School of Hygiene and Tropical Medicine, London. \ Department of Obstetrics and Gynecology, St. George’s Hospital Medical School, London. 0015-0282/99/$20.00 PII S0015-0282(98)00447-6
Objective: To explore the value of serum vascular endothelial growth factor (VEGF) concentrations during IVF cycles in predicting the risk of ovarian hyperstimulation syndrome (OHSS). Design: Prospective study. Setting: London Women’s Clinic. Patient(s): One hundred seven women undergoing IVF. Mild OHSS developed in 10 women, moderate OHSS in 7, and severe OHSS in 3. Intervention(s): Serum VEGF concentrations were measured before treatment, after pituitary desensitization, and on the days of hCG administration, oocyte collection, and ET. Main Outcome Measure(s): Serum VEGF concentrations. Result(s): Serum VEGF concentrations were higher in women in whom OHSS developed. The increase in the VEGF concentration that occurred between the day of hCG administration and the day of oocyte collection (the “VEGF rise”) was an important marker of OHSS. The VEGF rise was higher in women in whom OHSS developed. A higher VEGF rise predicted all cases of OHSS and moderate/severe cases of OHSS with a sensitivity of 100% and a specificity of 60%. A likelihood ratio test showed that adding the VEGF rise or the VEGF concentration on the day of oocyte collection to a regression model as a continuous variable to the number of follicles, the E2 concentration, and the presence of polycystic ovaries significantly contributed to predicting the risk of OHSS. Conclusion(s): The results support the role of VEGF as an important nonsteroidal index of ovarian response. The VEGF rise may have an advantage over the E2 concentration, the number of follicles, and the number of oocytes, which individually predict only 15%–25% of cases of OHSS. (Fertil Sterilt 1999;71:287–93. ©1999 by American Society for Reproductive Medicine.) Key Words: Risk prediction, likelihood ratio, OHSS, VEGF, E2, follicle numbers, oocytes, PCO
Predicting the development of ovarian hyperstimulation syndrome (OHSS) traditionally has involved indexing the endocrine response and ultrasound (US) findings after ovarian stimulation. Numerous reports attest to the value of measuring E2 concentrations in blood or urine; based on these concentrations, criteria have been developed for withholding hCG administration in protocols of ovarian stimulation (1). Although it is known that high E2 concentrations are an immediate precursor of OHSS,
E2 itself does not cause OHSS (2). Moreover, elevated serum E2 concentrations predict the development of OHSS in only one-fourth of all cases, and severe OHSS has developed in patients with partial 17, 20-desmolase deficiency despite low serum E2 concentrations (2). Other criteria for predicting the development of OHSS, such as the number of follicles on the day of hCG administration, the presence of polycystic ovaries (PCO) on US examination, and the number of oocytes retrieved, may 287
be subject to interobserver variations during US examination and interoperator differences during oocyte retrieval (3, 4). As individual markers of OHSS, both an increased number of follicles and an increased number of oocytes predict only one fourth of all cases of OHSS (6). Although the ovarian prorenin-renin-angiotensin system (7) and the ovarian cytokines (8) may be involved in the pathogenesis of OHSS, currently available measurements of these systems do not predict the risk of developing OHSS (5, 9). Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and multifunctional cytokine with potent angiogenic properties. Vascular endothelial growth factor is recognized as an important vasoactive substance and is the favored candidate as a mediator of OHSS (10 –12). Vascular endothelial growth factor messenger RNA expression becomes prominent in granulosa cells in the immediate preovulatory stage. After exposure to LH or hCG, the predominant site of VEGF production is the luteinized granulosa cell (13). Studies using bioassays (10) suggest that the release of VEGF mediates OHSS, and this relation has been confirmed with studies using immunologic assays performed in our own (14) and other laboratories (12). Therefore, VEGF provides a nonsteroidal index of the ovarian response to gonadotropin stimulation. The purpose of this study was to explore the value of the measurement of serum VEGF concentrations during IVF cycles compared with the value of the measurement of serum E2 concentrations and follicle numbers on the day of hCG administration, the number of oocytes retrieved on the day of oocyte collection, and the presence of PCO on US examination as a method of predicting ovarian overresponse and preventing the development of OHSS.
MATERIALS AND METHODS We recruited 107 consecutively seen women who underwent IVF at our institution between March 1996 and November 1996. Our institution’s ethics committee approved the study. According to established US and clinical criteria (15), 65 women had normal ovaries, 27 had PCO, and 15 had polycystic ovary syndrome (PCOS). The patients’ ages ranged between 27 and 47 years. Patients with concomitant pelvic pathology such as endometriosis, uterine fibroids, ovarian cysts, or pelvic inflammatory disease were excluded from the study. The causes of infertility were a male factor (56.8%), tubal disease (13.7%), unexplained causes (12.5%), mixed causes (16%), and other causes (2.6%). The serum and follicular fluid VEGF concentrations in the same group of patients in relation to OHSS and PCO have been described previously (16). According to the classification proposed by Golan et al. in 288
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1989 (17), 10 (9.3%) patients had mild OHSS, 7 (6.5%) had moderate OHSS, and 3 (2.8%) had severe OHSS. The women were divided into a “no OHSS” group (n 5 87) and an “OHSS” group (mild, moderate, or severe; n 5 20). In a further (post hoc) analysis, the women were divided into a “no/mild OHSS” group (n 5 97) and a “moderate/severe OHSS” group (n 5 10). This analysis was performed to facilitate prediction of the clinically important form of OHSS (i.e., moderate/severe) because it is this form of OHSS that is responsible for the considerable morbidity and occasional mortality associated with ovarian stimulation.
Treatment Protocol All patients underwent our standard “long” protocol of pretreatment with a GnRH analogue followed by ovarian stimulation with hMG (Pergonal; Serono Laboratories Ltd., Welwyn Garden City, Herts, United Kingdom, or Humegon; Organon Laboratories Ltd., Cambridgeshire, United Kingdom), FSH (Metrodin-HP; Serono Laboratories, or Orgafol, Organon Laboratories), or Normegon (Organon Laboratories) (18).
Pelvic US Examination and Doppler Blood Flow Velocity Measurements Ultrasound examination was performed with a 5-MHz transvaginal transducer with color and pulsed Doppler capabilities (128XP/10 OB; Acuson, Mountain View, CA). The peak systolic velocity and time-averaged maximal velocity (measurements of blood flow velocity), and the pulsatility index and the resistance index (measurements of the resistance to blood flow) were measured within both the ovarian blood vessels and the uterine arteries according to previously described methods (19). Observations were made in the early follicular phase of the menstrual cycle (day 2 or day 3) before the initiation of IVF treatment, after pituitary desensitization with buserelin acetate, and on the day of hCG administration for all patients. For 18 patients, Doppler blood flow measurements also were recorded on the day of oocyte retrieval (i.e., 36 hours after the administration of hCG and on the day of ET).
Vascular Endothelial Growth Factor Assay Blood samples for serum VEGF and hormone measurements were obtained between 8 AM and 12 PM immediately after the US examination for all patients. In addition, 20 women in whom OHSS developed and 10 women in whom it did not had blood samples taken 3, 5, and 7 days after ET. Serum was stored at 270°C for subsequent analysis. Vascular endothelial growth factor concentrations were measured with the use of an enzyme immunoassay (Cytokit Red EIA Kits; Peninsula Laboratories, Inc., College Park, MD). The sensitivity of the assay was 0.2 ng/mL (intra-assay coefficient of variation 5 7.8%, interassay coefficient of variation 5 12.2%). The cross-reactivities of the assay were ,0.5% against World Health Organization cytokine standards. The VEGF measured was the “total” circulating
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TABLE 1 Variables used for predicting the risk of ovarian hyperstimulation syndrome. Study group
Variable
Patients without OHSS (n 5 87)
Patients with OHSS (n 5 20)
Patients with moderate/severe OHSS (n 5 10)
P value
Serum No. of No. of No. of Serum Serum
5,759 6 3,285 15.9 6 9.4 10.6 6 5.2 26 (29.8) 0.87 6 0.30 4.42 6 0.80
9,762 6 5,844 29.2 6 8.9 21.6 6 7.8 17 (85) 1.56 6 0.50 5.37 6 0.90
12,525 6 7,141 33.1 6 6.7 23.6 6 7.5 10 (100) 1.58 6 0.60 5.68 6 1.10
,.001 ,.01 ,.005 ,.01 ,.0001 ,.01
E2 level (pmol/L) follicles oocytes obtained patients with PCO (%) VEGF rise (ng/mL) VEGF level on day of oocyte collection (ng/mL)
Note: All values are means 6 SD unless otherwise indicated. OHSS 5 ovarian hyperstimulation syndrome; PCO 5 polycystic ovaries. VEGF 5 vascular endothelial growth factor.
VEGF bound to plasma proteins (e.g., a2 macroglobulin) (19). The major circulating forms of VEGF are VEGF165 and VEGF121, whereas VEGF189 and VEGF206 are largely bound to cell membranes and do not represent measurable VEGF (20).
Hormone Assays Follicle-stimulating hormone and LH levels were measured with a microparticle enzyme immunoassay (Abbott AxSYM reagent Pack; Abbott Laboratories, Chicago, IL) and E2, progesterone, and testosterone levels were measured with chemiluminescent assays (Immulite Kits; Diagnostic Products Corporation, Los Angeles, CA). The intra-assay and interassay coefficients of variation were 2.9% and 3.7% for FSH, 1.8% and 4.1% for LH, 9.5% and 15% for E2, 8.6% and 12% for progesterone, and 6.6% and 9.2% for testosterone, respectively.
Statistical Analysis Data are expressed as means 6 SD. Hormone concentrations were logarithmically transformed to normalize the distribution of data. Mean values were compared across the categories of OHSS by analysis of variance. Post hoc analysis was performed with the use of Scheffe´’s test. Student’s t-test was used to compare continuous variables, and P values of ,.05 were considered statistically significant. A likelihood ratio test, based on logistic regression modeling using unit change in each predictive criterion of OHSS, was used to test whether VEGF concentrations made a significant contribution to predicting the development of OHSS. The sensitivity, specificity, and positive predictive value of various markers for OHSS in this study were investigated and compared. The optimum cutoff values for risk prediction for various variables were identified from receiver operating curves. FERTILITY & STERILITYt
RESULTS There were no statistically significant differences between women with and without OHSS with respect to age, duration of infertility, parity, previous IVF attempts, treatment received (IVF or intracytoplasmic sperm injection), cause of infertility, or basal serum FSH, LH, or E2 concentration. The mean (6SD) basal serum testosterone concentration was higher (1.54 6 1.1 nmol/L vs. 0.87 6 0.5 nmol/L, P ,.05) in the women in whom OHSS developed, probably because there was a higher proportion of women with PCOS in this group. Thus, women in whom OHSS developed were more likely to have PCO and PCOS (85%) than to have normal ovaries (29.8%; P ,.005) (Table 1). The mean (6SD) serum E2 concentration on the day of hCG administration in women who received FSH was lower (5,692 6 2,735 pmol/L) than in women who received hMG (8,006 6 5,860 pmol/L; P ,.01). This difference is not accounted for in predicting the risk of developing OHSS because there was no difference between these two groups in the proportion of patients in whom OHSS developed. The mean serum E2 concentration, mean number of follicles on the day of hCG administration, and mean number of oocytes retrieved in the various groups are shown in Table 1. The sensitivity, specificity, and positive predictive values of each of the variables as a marker of OHSS are shown in Table 2. The mean (6SD) serum VEGF concentration rose significantly in all the women during ovarian stimulation and was higher on the day of hCG administration (3.59 6 0.91 ng/mL) than in the early follicular phase (i.e., at the beginning of the IVF cycle) (2.93 6 0.77 ng/mL; P ,.0001). The mean (6SD) serum VEGF concentration rose further after hCG administration to 4.59 6 0.93 ng/mL on the day of oocyte retrieval and to 4.88 6 1.02 ng/mL on the day of ET. Women in whom OHSS developed had consistently 289
TABLE 2 Sensitivity, specificity, and positive predictive values of criteria used for estimating the risk of ovarian hyperstimulation syndrome. All cases of OHSS (n 5 20)
Variable Serum E2 level No. of follicles No. of oocytes Presence of PCO VEGF rise VEGF rise 1 follicles VEGF rise 1 follicles 1 PCO VEGF rise 1 follicles 1 PCO 1 E2 level VEGF rise 1 oocytes E2 1 follicles 1 VEGF rise E2 1 follicles 1 PCO
Moderate/severe cases of OHSS (n 5 10)
Sensitivity (%)
Specificity (%)
PPV (%)
Sensitivity (%)
Specificity (%)
PPV (%)
75 95 80 85 100 90 85 75 80 70 92
49.0 52.0 79.3 70.0 60.0 93.0 96.5 97.7 91.9 94.2 87.0
25.0 31.0 47.0 40.0 40.0 75.2 84.2 88.0 66.6 66.6 54.0
90 100 90 100 100 100 100 90 90 90 90
52.0 56.0 74.2 66.0 60.0 85.5 90.7 93.8 85.5 90.0 85.0
15.0 19.0 26.4 23.2 40.3 43.4 52.6 60.0 39.1 50.0 38.0
Note: OHSS 5 ovarian hyperstimulation syndrome; PCO 5 polycystic ovaries; PPV 5 positive predictive value; VEGF 5 vascular endothelial growth factor. Cut-off values for variables were established by receiver operating curves and were as follows: 6,500 pmol/L for serum E2 concentrations, 25 for number of follicles, 15 for number of oocytes retrieved, and 1 ng/mL for VEGF rise.
higher serum VEGF concentrations before and throughout their IVF cycles, except after pituitary desensitization (Fig. 1). The rise in the serum VEGF concentration that occurred between the day of hCG administration and the day of oocyte collection (subsequently referred to as the “VEGF rise”) proved to be a good marker of the development of OHSS (Table 2). The VEGF rise was higher in women in
whom OHSS developed than in those in whom it did not (Table 1). Although serum VEGF concentrations were higher in women in whom OHSS developed throughout the IVF cycle, including the early follicular phase (before the initiation of the IVF cycle), on the days of hCG administration, oocyte collection, and ET, serum VEGF concentrations as single markers did not predict the development of OHSS (statistical analysis not shown).
FIGURE 1 Differences in serum vascular endothelial growth factor (VEGF) concentrations in IVF cycles in women in whom mild ovarian hyperstimulation syndrome (OHSS) developed (n 5 10), moderate or severe OHSS developed (n 5 10), and OHSS did not develop (n 5 87). All differences were statistically significant (P ,.0001). GnRHa 5 GnRH agonist.
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follicles on the day of hCG administration also predicted the development of OHSS effectively (Table 2).
TABLE 3 Odds ratios and confidence intervals for variables that served as markers of ovarian response. Model number
Marker
Odds ratio
95% confidence interval
1
Log E2 level No. of follicles Presence of PCO VEGF rise Log E2 level No. of follicles Presence of PCO VEGF level on day of oocyte collection
5.32 1.08 5.75 11.30 4.80 1.10 2.57
1.10–24.60 1.00–1.10 0.67–49.00 1.40–90.40 1.10–20.20 1.02–1.19 0.35–18.40
3.01
1.01–8.90
2
Note: All values were determined by regression analysis and by the likelihood ratio test. PCO 5 polycystic ovaries; VEGF 5 vascular endothelial growth factor.
The sensitivity, specificity, and predictive values of various markers and a combination of markers showed that the VEGF rise, the number of follicles on the day of hCG administration, and a pretreatment diagnosis of PCO were the best markers of the development of OHSS (Table 2). The combination of these three factors gave a positive predictive value of 84.2% for all cases of OHSS (sensitivity 5 85%, specificity 5 96.5%) and of 52.6% for moderate and severe cases of OHSS (sensitivity 5 100%, specificity 5 90.7%). The combination of the VEGF rise and the number of
A likelihood ratio was calculated, which compared the log likelihood of two logistic regression models, one of which included all four continuous variables (i.e., number of follicles, log-transformed E2 concentration, presence of PCO, and VEGF rise) and the other of which excluded the VEGF rise. The result showed that adding the VEGF rise as a continuous variable significantly improved the model for calculating the odds of developing moderate and severe OHSS (P 5 .015) (Table 3). Similarly, adding the serum VEGF concentration on the day of oocyte collection as a continuous variable significantly improved the model (P 5 .02). The odds ratios and confidence intervals for the various markers are shown in Table 3. Within the ovarian stromal blood vessels, Doppler blood flow velocities (peak systolic velocity and time-averaged maximal velocity) were higher in women in whom OHSS developed than in those in whom it did not develop (Fig. 2). However, there were no differences in the pulsatility index and the resistance index within the ovarian stromal blood vessels and no differences in the blood flow velocities within the uterine arteries between the two groups of women. Although ovarian stromal blood flow velocities were higher in the women in whom OHSS developed than in those in whom it did not develop (probably because there were more patients with PCO in the group in whom OHSS developed), blood flow velocities on the day of hCG administration did not predict the risk of developing OHSS (statistical analysis not shown).
FIGURE 2 Changes in serum vascular endothelial growth factor (VEGF) concentrations and Doppler blood flow velocity (peak systolic velocity [PSV]) in IVF cycles in women in whom ovarian hyperstimulation syndrome (OHSS) did (n 5 20) and did not (n 5 87) develop. All differences were statistically significant (P ,.001). GnRHa 5 GnRH agonist.
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DISCUSSION Vascular endothelial growth factor is produced by the ovary after ovulation or after hCG administration (11). Therefore, the serum VEGF concentration before hCG administration does not predict the risk of OHSS. Although serum VEGF concentrations were higher in the women in whom OHSS developed than in those in whom it did not, the serum VEGF concentration on the day of hCG administration or earlier did not predict the risk of developing OHSS. The results of our study show that the rise in the serum VEGF concentrations that occurs between the day of hCG administration and the day of oocyte collection (the VEGF rise) may be an important nonsteroidal marker of OHSS. The VEGF rise predicted 40% of all cases of OHSS and 40.3% of cases of moderate and severe OHSS, with no false-negative results. However, the serum VEGF concentration as a single marker during the early follicular phase, on the day of hCG administration, on the day of oocyte collection, or on the day of ET did not predict the development of OHSS. These prediction rates for all cases of OHSS and for cases of moderate and severe OHSS were only marginally better than those of serum E2 concentrations on the day of hCG administration (Table 2). However, there were no falsenegative results with the use of the VEGF rise. It may be more important to have fewer false-negative results than to have higher prediction rates, because IVF is never a vital necessity but OHSS can be life-threatening (4). The biologic relation of OHSS with VEGF has been established in several studies (10 –12). However, serial measurement of the serum VEGF concentration before and after hCG administration has not been performed and its role in the prediction of the development of OHSS has not been studied. Ludwig et al. (21) did not find the serum VEGF concentration to be predictive of the clinical outcome of OHSS in IVF cycles. However, they did not evaluate its role in the prediction of risk. The diagnosis of PCO (synonymous with “necklace pattern of ovaries”) (22, 23) is a crucial prestimulation marker of OHSS, because OHSS occurs more commonly in women with PCO (22). With the use of immunohistochemical analyses, Kamat et al. (24) described increased expression of VEGF messenger RNA in the theca cells in PCO. Our recent work indicated that women with PCO have higher basal (prestimulation) serum concentrations of VEGF than women with normal ovaries on US examination (19). In the present study, we observed higher serum VEGF concentrations at all stages of ovarian stimulation in women with PCO compared with women with normal ovaries. Subjects in whom OHSS developed (85% of whom had PCO) had the highest VEGF concentrations compared with subjects who did not have PCO (29% of whom had OHSS). In the present study, as in our earlier report (18), the mean 292
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E2 concentration was significantly lower in patients who received FSH preparations than in those who received hMG; however, there was no difference in the risk of OHSS in relation to the type of gonadotropins used. Further, using an elevated serum E2 concentration on the day of hCG administration as a marker of OHSS, we could predict OHSS in only 25% of the cases. These results are similar to those published previously (25, 26). A clear cutoff value for the serum E2 concentration above which OHSS is likely to occur has not been established, in part because of differences in IVF techniques and variability in E2 assays. Thus, the serum E2 concentration on the day of hCG administration as a criteria for withholding hCG injection has varied from 800 pg/mL to 6,000 pg/mL (3, 4, 23, 25). Further, one would expect lower E2 concentrations as regimens of ovarian stimulation shift from the use of combinations of gonadotropins to preparations that contain FSH alone (18), making the use of E2 as a marker of OHSS increasingly unreliable. The number of follicles observed on the day of hCG administration and the number of oocytes retrieved are subject to variations among centers, US equipment, and operator techniques (3, 4). Previous reports have shown that an increased number of follicles on the day of hCG administration predicts the development of OHSS in only one-fourth of all cases (25). Using the same criteria, we were able to predict only 31% of all cases of OHSS and 19% of moderate and severe cases of OHSS. Again, there are no identified cutoff levels for the number of follicles (range, 10 –35) or the number of oocytes retrieved (range, 14 –30) above which the risk of developing OHSS is increased (3, 4, 25). Although several investigators have recommended the use of US examination and determination of the E2 concentration on the day of hCG administration for predicting the risk of OHSS (23), we found that the combination of a pretreatment diagnosis of PCO, the number of follicles on the day of hCG administration, and the VEGF rise gave the highest prediction rate for the development of OHSS (Table 2). The addition of the serum E2 concentration on the day of hCG administration improved the prediction rate marginally, but the rate of false-negative results increased from 15%– 25% for all cases of OHSS and from 0 –10% for moderate and severe cases of OHSS (Table 2). Further, a likelihood ratio test showed that adding the VEGF rise or the serum VEGF concentration on the day of oocyte collection as a continuous variable to the number of follicles, the serum E2 concentration on the day of hCG administration, and the presence of PCO during a regression analysis made a significant contribution to the model in predicting OHSS. No individual criteria predicted the risk of developing OHSS reliably. Delvigne et al. (22) also reported the lack of predictive power of individual criteria and suggested a step-
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wise discriminant analysis of several variables (log serum E2 rise, hMG dose, number of oocytes obtained, LH/FSH ratio, necklace sign of the ovaries) to provide a predictive mathematic function for evaluating the risk of OHSS before and after hCG administration. They obtained prediction rates of 76.1% and 78.5%, with a mean rate of false-negative results of 18.1% for all cases of OHSS. This result was superior to an evaluation based on the E2 concentration and/or the number of follicles or oocytes obtained.
9. 10.
11.
In clinical terms, it appears that VEGF may provide an important nonsteroidal index of ovarian response; this is biologically plausible because VEGF is also a mediator of OHSS. It is independent of the other markers of OHSS. We believe that the serum VEGF concentration on the day of hCG administration and on the day of oocyte collection, along with the total number of follicles on the day of hCG administration and a pretreatment diagnosis of PCO, may be more accurate than the serum E2 concentration in predicting the development of OHSS. The serum E2 concentration is becoming progressively less reliable as regimens of ovarian stimulation move from combined gonadotropin preparations to preparations containing FSH alone, because the serum E2 concentration may underrepresent the degree of follicular response to ovarian stimulation. Further prospective studies will be required to define the most reliable cutoff points for predicting and, therefore, ultimately preventing, OHSS.
12.
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Serum vascular endothelial growth factor concentrations in in vitro fertilization cycles predict the risk of ovarian hyperstimulation syndrome Rina Agrawal, M.D.,*† Seang Ling Tan, M.D.,†‡ Sarah Wild, M.D.,§ Povilas Sladkevicius, M.D.,†\ Lawrence Engmann, M.D.,† Nadia Payne, M.Sc.,* Jinan Bekir, M.D.,† Stuart Campbell, M.D.,†\ Gerard Conway, M.D.,* and Howard Jacobs, M.D.*† University College London Medical School, The Middlesex Hospital and London Women’s Clinic, London, United Kingdom
Received May 26, 1998; revised and accepted September 23, 1998. Reprint requests: Rina Agrawal, M.D., Department of Reproductive Endocrinology, Cobbold Laboratories, The Middlesex Hospital, Mortimer Street, London W1N 8AA United Kingdom (FAX: 0171-4875850; E-mail: ganapaty@dircon. co.uk). * University College London Medical School, Department of Reproductive Endocrinology, Cobbold Laboratories, The Middlesex Hospital, London. † London Women’s Clinic, London. ‡ Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada. § Epidemiology Unit, London School of Hygiene and Tropical Medicine, London. \ Department of Obstetrics and Gynecology, St. George’s Hospital Medical School, London. 0015-0282/99/$20.00 PII S0015-0282(98)00447-6
Objective: To explore the value of serum vascular endothelial growth factor (VEGF) concentrations during IVF cycles in predicting the risk of ovarian hyperstimulation syndrome (OHSS). Design: Prospective study. Setting: London Women’s Clinic. Patient(s): One hundred seven women undergoing IVF. Mild OHSS developed in 10 women, moderate OHSS in 7, and severe OHSS in 3. Intervention(s): Serum VEGF concentrations were measured before treatment, after pituitary desensitization, and on the days of hCG administration, oocyte collection, and ET. Main Outcome Measure(s): Serum VEGF concentrations. Result(s): Serum VEGF concentrations were higher in women in whom OHSS developed. The increase in the VEGF concentration that occurred between the day of hCG administration and the day of oocyte collection (the “VEGF rise”) was an important marker of OHSS. The VEGF rise was higher in women in whom OHSS developed. A higher VEGF rise predicted all cases of OHSS and moderate/severe cases of OHSS with a sensitivity of 100% and a specificity of 60%. A likelihood ratio test showed that adding the VEGF rise or the VEGF concentration on the day of oocyte collection to a regression model as a continuous variable to the number of follicles, the E2 concentration, and the presence of polycystic ovaries significantly contributed to predicting the risk of OHSS. Conclusion(s): The results support the role of VEGF as an important nonsteroidal index of ovarian response. The VEGF rise may have an advantage over the E2 concentration, the number of follicles, and the number of oocytes, which individually predict only 15%–25% of cases of OHSS. (Fertil Sterilt 1999;71:287–93. ©1999 by American Society for Reproductive Medicine.) Key Words: Risk prediction, likelihood ratio, OHSS, VEGF, E2, follicle numbers, oocytes, PCO
Predicting the development of ovarian hyperstimulation syndrome (OHSS) traditionally has involved indexing the endocrine response and ultrasound (US) findings after ovarian stimulation. Numerous reports attest to the value of measuring E2 concentrations in blood or urine; based on these concentrations, criteria have been developed for withholding hCG administration in protocols of ovarian stimulation (1). Although it is known that high E2 concentrations are an immediate precursor of OHSS,
E2 itself does not cause OHSS (2). Moreover, elevated serum E2 concentrations predict the development of OHSS in only one-fourth of all cases, and severe OHSS has developed in patients with partial 17, 20-desmolase deficiency despite low serum E2 concentrations (2). Other criteria for predicting the development of OHSS, such as the number of follicles on the day of hCG administration, the presence of polycystic ovaries (PCO) on US examination, and the number of oocytes retrieved, may 287
be subject to interobserver variations during US examination and interoperator differences during oocyte retrieval (3, 4). As individual markers of OHSS, both an increased number of follicles and an increased number of oocytes predict only one fourth of all cases of OHSS (6). Although the ovarian prorenin-renin-angiotensin system (7) and the ovarian cytokines (8) may be involved in the pathogenesis of OHSS, currently available measurements of these systems do not predict the risk of developing OHSS (5, 9). Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and multifunctional cytokine with potent angiogenic properties. Vascular endothelial growth factor is recognized as an important vasoactive substance and is the favored candidate as a mediator of OHSS (10 –12). Vascular endothelial growth factor messenger RNA expression becomes prominent in granulosa cells in the immediate preovulatory stage. After exposure to LH or hCG, the predominant site of VEGF production is the luteinized granulosa cell (13). Studies using bioassays (10) suggest that the release of VEGF mediates OHSS, and this relation has been confirmed with studies using immunologic assays performed in our own (14) and other laboratories (12). Therefore, VEGF provides a nonsteroidal index of the ovarian response to gonadotropin stimulation. The purpose of this study was to explore the value of the measurement of serum VEGF concentrations during IVF cycles compared with the value of the measurement of serum E2 concentrations and follicle numbers on the day of hCG administration, the number of oocytes retrieved on the day of oocyte collection, and the presence of PCO on US examination as a method of predicting ovarian overresponse and preventing the development of OHSS.
MATERIALS AND METHODS We recruited 107 consecutively seen women who underwent IVF at our institution between March 1996 and November 1996. Our institution’s ethics committee approved the study. According to established US and clinical criteria (15), 65 women had normal ovaries, 27 had PCO, and 15 had polycystic ovary syndrome (PCOS). The patients’ ages ranged between 27 and 47 years. Patients with concomitant pelvic pathology such as endometriosis, uterine fibroids, ovarian cysts, or pelvic inflammatory disease were excluded from the study. The causes of infertility were a male factor (56.8%), tubal disease (13.7%), unexplained causes (12.5%), mixed causes (16%), and other causes (2.6%). The serum and follicular fluid VEGF concentrations in the same group of patients in relation to OHSS and PCO have been described previously (16). According to the classification proposed by Golan et al. in 288
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1989 (17), 10 (9.3%) patients had mild OHSS, 7 (6.5%) had moderate OHSS, and 3 (2.8%) had severe OHSS. The women were divided into a “no OHSS” group (n 5 87) and an “OHSS” group (mild, moderate, or severe; n 5 20). In a further (post hoc) analysis, the women were divided into a “no/mild OHSS” group (n 5 97) and a “moderate/severe OHSS” group (n 5 10). This analysis was performed to facilitate prediction of the clinically important form of OHSS (i.e., moderate/severe) because it is this form of OHSS that is responsible for the considerable morbidity and occasional mortality associated with ovarian stimulation.
Treatment Protocol All patients underwent our standard “long” protocol of pretreatment with a GnRH analogue followed by ovarian stimulation with hMG (Pergonal; Serono Laboratories Ltd., Welwyn Garden City, Herts, United Kingdom, or Humegon; Organon Laboratories Ltd., Cambridgeshire, United Kingdom), FSH (Metrodin-HP; Serono Laboratories, or Orgafol, Organon Laboratories), or Normegon (Organon Laboratories) (18).
Pelvic US Examination and Doppler Blood Flow Velocity Measurements Ultrasound examination was performed with a 5-MHz transvaginal transducer with color and pulsed Doppler capabilities (128XP/10 OB; Acuson, Mountain View, CA). The peak systolic velocity and time-averaged maximal velocity (measurements of blood flow velocity), and the pulsatility index and the resistance index (measurements of the resistance to blood flow) were measured within both the ovarian blood vessels and the uterine arteries according to previously described methods (19). Observations were made in the early follicular phase of the menstrual cycle (day 2 or day 3) before the initiation of IVF treatment, after pituitary desensitization with buserelin acetate, and on the day of hCG administration for all patients. For 18 patients, Doppler blood flow measurements also were recorded on the day of oocyte retrieval (i.e., 36 hours after the administration of hCG and on the day of ET).
Vascular Endothelial Growth Factor Assay Blood samples for serum VEGF and hormone measurements were obtained between 8 AM and 12 PM immediately after the US examination for all patients. In addition, 20 women in whom OHSS developed and 10 women in whom it did not had blood samples taken 3, 5, and 7 days after ET. Serum was stored at 270°C for subsequent analysis. Vascular endothelial growth factor concentrations were measured with the use of an enzyme immunoassay (Cytokit Red EIA Kits; Peninsula Laboratories, Inc., College Park, MD). The sensitivity of the assay was 0.2 ng/mL (intra-assay coefficient of variation 5 7.8%, interassay coefficient of variation 5 12.2%). The cross-reactivities of the assay were ,0.5% against World Health Organization cytokine standards. The VEGF measured was the “total” circulating
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TABLE 1 Variables used for predicting the risk of ovarian hyperstimulation syndrome. Study group
Variable
Patients without OHSS (n 5 87)
Patients with OHSS (n 5 20)
Patients with moderate/severe OHSS (n 5 10)
P value
Serum No. of No. of No. of Serum Serum
5,759 6 3,285 15.9 6 9.4 10.6 6 5.2 26 (29.8) 0.87 6 0.30 4.42 6 0.80
9,762 6 5,844 29.2 6 8.9 21.6 6 7.8 17 (85) 1.56 6 0.50 5.37 6 0.90
12,525 6 7,141 33.1 6 6.7 23.6 6 7.5 10 (100) 1.58 6 0.60 5.68 6 1.10
,.001 ,.01 ,.005 ,.01 ,.0001 ,.01
E2 level (pmol/L) follicles oocytes obtained patients with PCO (%) VEGF rise (ng/mL) VEGF level on day of oocyte collection (ng/mL)
Note: All values are means 6 SD unless otherwise indicated. OHSS 5 ovarian hyperstimulation syndrome; PCO 5 polycystic ovaries. VEGF 5 vascular endothelial growth factor.
VEGF bound to plasma proteins (e.g., a2 macroglobulin) (19). The major circulating forms of VEGF are VEGF165 and VEGF121, whereas VEGF189 and VEGF206 are largely bound to cell membranes and do not represent measurable VEGF (20).
Hormone Assays Follicle-stimulating hormone and LH levels were measured with a microparticle enzyme immunoassay (Abbott AxSYM reagent Pack; Abbott Laboratories, Chicago, IL) and E2, progesterone, and testosterone levels were measured with chemiluminescent assays (Immulite Kits; Diagnostic Products Corporation, Los Angeles, CA). The intra-assay and interassay coefficients of variation were 2.9% and 3.7% for FSH, 1.8% and 4.1% for LH, 9.5% and 15% for E2, 8.6% and 12% for progesterone, and 6.6% and 9.2% for testosterone, respectively.
Statistical Analysis Data are expressed as means 6 SD. Hormone concentrations were logarithmically transformed to normalize the distribution of data. Mean values were compared across the categories of OHSS by analysis of variance. Post hoc analysis was performed with the use of Scheffe´’s test. Student’s t-test was used to compare continuous variables, and P values of ,.05 were considered statistically significant. A likelihood ratio test, based on logistic regression modeling using unit change in each predictive criterion of OHSS, was used to test whether VEGF concentrations made a significant contribution to predicting the development of OHSS. The sensitivity, specificity, and positive predictive value of various markers for OHSS in this study were investigated and compared. The optimum cutoff values for risk prediction for various variables were identified from receiver operating curves. FERTILITY & STERILITYt
RESULTS There were no statistically significant differences between women with and without OHSS with respect to age, duration of infertility, parity, previous IVF attempts, treatment received (IVF or intracytoplasmic sperm injection), cause of infertility, or basal serum FSH, LH, or E2 concentration. The mean (6SD) basal serum testosterone concentration was higher (1.54 6 1.1 nmol/L vs. 0.87 6 0.5 nmol/L, P ,.05) in the women in whom OHSS developed, probably because there was a higher proportion of women with PCOS in this group. Thus, women in whom OHSS developed were more likely to have PCO and PCOS (85%) than to have normal ovaries (29.8%; P ,.005) (Table 1). The mean (6SD) serum E2 concentration on the day of hCG administration in women who received FSH was lower (5,692 6 2,735 pmol/L) than in women who received hMG (8,006 6 5,860 pmol/L; P ,.01). This difference is not accounted for in predicting the risk of developing OHSS because there was no difference between these two groups in the proportion of patients in whom OHSS developed. The mean serum E2 concentration, mean number of follicles on the day of hCG administration, and mean number of oocytes retrieved in the various groups are shown in Table 1. The sensitivity, specificity, and positive predictive values of each of the variables as a marker of OHSS are shown in Table 2. The mean (6SD) serum VEGF concentration rose significantly in all the women during ovarian stimulation and was higher on the day of hCG administration (3.59 6 0.91 ng/mL) than in the early follicular phase (i.e., at the beginning of the IVF cycle) (2.93 6 0.77 ng/mL; P ,.0001). The mean (6SD) serum VEGF concentration rose further after hCG administration to 4.59 6 0.93 ng/mL on the day of oocyte retrieval and to 4.88 6 1.02 ng/mL on the day of ET. Women in whom OHSS developed had consistently 289
TABLE 2 Sensitivity, specificity, and positive predictive values of criteria used for estimating the risk of ovarian hyperstimulation syndrome. All cases of OHSS (n 5 20)
Variable Serum E2 level No. of follicles No. of oocytes Presence of PCO VEGF rise VEGF rise 1 follicles VEGF rise 1 follicles 1 PCO VEGF rise 1 follicles 1 PCO 1 E2 level VEGF rise 1 oocytes E2 1 follicles 1 VEGF rise E2 1 follicles 1 PCO
Moderate/severe cases of OHSS (n 5 10)
Sensitivity (%)
Specificity (%)
PPV (%)
Sensitivity (%)
Specificity (%)
PPV (%)
75 95 80 85 100 90 85 75 80 70 92
49.0 52.0 79.3 70.0 60.0 93.0 96.5 97.7 91.9 94.2 87.0
25.0 31.0 47.0 40.0 40.0 75.2 84.2 88.0 66.6 66.6 54.0
90 100 90 100 100 100 100 90 90 90 90
52.0 56.0 74.2 66.0 60.0 85.5 90.7 93.8 85.5 90.0 85.0
15.0 19.0 26.4 23.2 40.3 43.4 52.6 60.0 39.1 50.0 38.0
Note: OHSS 5 ovarian hyperstimulation syndrome; PCO 5 polycystic ovaries; PPV 5 positive predictive value; VEGF 5 vascular endothelial growth factor. Cut-off values for variables were established by receiver operating curves and were as follows: 6,500 pmol/L for serum E2 concentrations, 25 for number of follicles, 15 for number of oocytes retrieved, and 1 ng/mL for VEGF rise.
higher serum VEGF concentrations before and throughout their IVF cycles, except after pituitary desensitization (Fig. 1). The rise in the serum VEGF concentration that occurred between the day of hCG administration and the day of oocyte collection (subsequently referred to as the “VEGF rise”) proved to be a good marker of the development of OHSS (Table 2). The VEGF rise was higher in women in
whom OHSS developed than in those in whom it did not (Table 1). Although serum VEGF concentrations were higher in women in whom OHSS developed throughout the IVF cycle, including the early follicular phase (before the initiation of the IVF cycle), on the days of hCG administration, oocyte collection, and ET, serum VEGF concentrations as single markers did not predict the development of OHSS (statistical analysis not shown).
FIGURE 1 Differences in serum vascular endothelial growth factor (VEGF) concentrations in IVF cycles in women in whom mild ovarian hyperstimulation syndrome (OHSS) developed (n 5 10), moderate or severe OHSS developed (n 5 10), and OHSS did not develop (n 5 87). All differences were statistically significant (P ,.0001). GnRHa 5 GnRH agonist.
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follicles on the day of hCG administration also predicted the development of OHSS effectively (Table 2).
TABLE 3 Odds ratios and confidence intervals for variables that served as markers of ovarian response. Model number
Marker
Odds ratio
95% confidence interval
1
Log E2 level No. of follicles Presence of PCO VEGF rise Log E2 level No. of follicles Presence of PCO VEGF level on day of oocyte collection
5.32 1.08 5.75 11.30 4.80 1.10 2.57
1.10–24.60 1.00–1.10 0.67–49.00 1.40–90.40 1.10–20.20 1.02–1.19 0.35–18.40
3.01
1.01–8.90
2
Note: All values were determined by regression analysis and by the likelihood ratio test. PCO 5 polycystic ovaries; VEGF 5 vascular endothelial growth factor.
The sensitivity, specificity, and predictive values of various markers and a combination of markers showed that the VEGF rise, the number of follicles on the day of hCG administration, and a pretreatment diagnosis of PCO were the best markers of the development of OHSS (Table 2). The combination of these three factors gave a positive predictive value of 84.2% for all cases of OHSS (sensitivity 5 85%, specificity 5 96.5%) and of 52.6% for moderate and severe cases of OHSS (sensitivity 5 100%, specificity 5 90.7%). The combination of the VEGF rise and the number of
A likelihood ratio was calculated, which compared the log likelihood of two logistic regression models, one of which included all four continuous variables (i.e., number of follicles, log-transformed E2 concentration, presence of PCO, and VEGF rise) and the other of which excluded the VEGF rise. The result showed that adding the VEGF rise as a continuous variable significantly improved the model for calculating the odds of developing moderate and severe OHSS (P 5 .015) (Table 3). Similarly, adding the serum VEGF concentration on the day of oocyte collection as a continuous variable significantly improved the model (P 5 .02). The odds ratios and confidence intervals for the various markers are shown in Table 3. Within the ovarian stromal blood vessels, Doppler blood flow velocities (peak systolic velocity and time-averaged maximal velocity) were higher in women in whom OHSS developed than in those in whom it did not develop (Fig. 2). However, there were no differences in the pulsatility index and the resistance index within the ovarian stromal blood vessels and no differences in the blood flow velocities within the uterine arteries between the two groups of women. Although ovarian stromal blood flow velocities were higher in the women in whom OHSS developed than in those in whom it did not develop (probably because there were more patients with PCO in the group in whom OHSS developed), blood flow velocities on the day of hCG administration did not predict the risk of developing OHSS (statistical analysis not shown).
FIGURE 2 Changes in serum vascular endothelial growth factor (VEGF) concentrations and Doppler blood flow velocity (peak systolic velocity [PSV]) in IVF cycles in women in whom ovarian hyperstimulation syndrome (OHSS) did (n 5 20) and did not (n 5 87) develop. All differences were statistically significant (P ,.001). GnRHa 5 GnRH agonist.
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DISCUSSION Vascular endothelial growth factor is produced by the ovary after ovulation or after hCG administration (11). Therefore, the serum VEGF concentration before hCG administration does not predict the risk of OHSS. Although serum VEGF concentrations were higher in the women in whom OHSS developed than in those in whom it did not, the serum VEGF concentration on the day of hCG administration or earlier did not predict the risk of developing OHSS. The results of our study show that the rise in the serum VEGF concentrations that occurs between the day of hCG administration and the day of oocyte collection (the VEGF rise) may be an important nonsteroidal marker of OHSS. The VEGF rise predicted 40% of all cases of OHSS and 40.3% of cases of moderate and severe OHSS, with no false-negative results. However, the serum VEGF concentration as a single marker during the early follicular phase, on the day of hCG administration, on the day of oocyte collection, or on the day of ET did not predict the development of OHSS. These prediction rates for all cases of OHSS and for cases of moderate and severe OHSS were only marginally better than those of serum E2 concentrations on the day of hCG administration (Table 2). However, there were no falsenegative results with the use of the VEGF rise. It may be more important to have fewer false-negative results than to have higher prediction rates, because IVF is never a vital necessity but OHSS can be life-threatening (4). The biologic relation of OHSS with VEGF has been established in several studies (10 –12). However, serial measurement of the serum VEGF concentration before and after hCG administration has not been performed and its role in the prediction of the development of OHSS has not been studied. Ludwig et al. (21) did not find the serum VEGF concentration to be predictive of the clinical outcome of OHSS in IVF cycles. However, they did not evaluate its role in the prediction of risk. The diagnosis of PCO (synonymous with “necklace pattern of ovaries”) (22, 23) is a crucial prestimulation marker of OHSS, because OHSS occurs more commonly in women with PCO (22). With the use of immunohistochemical analyses, Kamat et al. (24) described increased expression of VEGF messenger RNA in the theca cells in PCO. Our recent work indicated that women with PCO have higher basal (prestimulation) serum concentrations of VEGF than women with normal ovaries on US examination (19). In the present study, we observed higher serum VEGF concentrations at all stages of ovarian stimulation in women with PCO compared with women with normal ovaries. Subjects in whom OHSS developed (85% of whom had PCO) had the highest VEGF concentrations compared with subjects who did not have PCO (29% of whom had OHSS). In the present study, as in our earlier report (18), the mean 292
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E2 concentration was significantly lower in patients who received FSH preparations than in those who received hMG; however, there was no difference in the risk of OHSS in relation to the type of gonadotropins used. Further, using an elevated serum E2 concentration on the day of hCG administration as a marker of OHSS, we could predict OHSS in only 25% of the cases. These results are similar to those published previously (25, 26). A clear cutoff value for the serum E2 concentration above which OHSS is likely to occur has not been established, in part because of differences in IVF techniques and variability in E2 assays. Thus, the serum E2 concentration on the day of hCG administration as a criteria for withholding hCG injection has varied from 800 pg/mL to 6,000 pg/mL (3, 4, 23, 25). Further, one would expect lower E2 concentrations as regimens of ovarian stimulation shift from the use of combinations of gonadotropins to preparations that contain FSH alone (18), making the use of E2 as a marker of OHSS increasingly unreliable. The number of follicles observed on the day of hCG administration and the number of oocytes retrieved are subject to variations among centers, US equipment, and operator techniques (3, 4). Previous reports have shown that an increased number of follicles on the day of hCG administration predicts the development of OHSS in only one-fourth of all cases (25). Using the same criteria, we were able to predict only 31% of all cases of OHSS and 19% of moderate and severe cases of OHSS. Again, there are no identified cutoff levels for the number of follicles (range, 10 –35) or the number of oocytes retrieved (range, 14 –30) above which the risk of developing OHSS is increased (3, 4, 25). Although several investigators have recommended the use of US examination and determination of the E2 concentration on the day of hCG administration for predicting the risk of OHSS (23), we found that the combination of a pretreatment diagnosis of PCO, the number of follicles on the day of hCG administration, and the VEGF rise gave the highest prediction rate for the development of OHSS (Table 2). The addition of the serum E2 concentration on the day of hCG administration improved the prediction rate marginally, but the rate of false-negative results increased from 15%– 25% for all cases of OHSS and from 0 –10% for moderate and severe cases of OHSS (Table 2). Further, a likelihood ratio test showed that adding the VEGF rise or the serum VEGF concentration on the day of oocyte collection as a continuous variable to the number of follicles, the serum E2 concentration on the day of hCG administration, and the presence of PCO during a regression analysis made a significant contribution to the model in predicting OHSS. No individual criteria predicted the risk of developing OHSS reliably. Delvigne et al. (22) also reported the lack of predictive power of individual criteria and suggested a step-
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wise discriminant analysis of several variables (log serum E2 rise, hMG dose, number of oocytes obtained, LH/FSH ratio, necklace sign of the ovaries) to provide a predictive mathematic function for evaluating the risk of OHSS before and after hCG administration. They obtained prediction rates of 76.1% and 78.5%, with a mean rate of false-negative results of 18.1% for all cases of OHSS. This result was superior to an evaluation based on the E2 concentration and/or the number of follicles or oocytes obtained.
9. 10.
11.
In clinical terms, it appears that VEGF may provide an important nonsteroidal index of ovarian response; this is biologically plausible because VEGF is also a mediator of OHSS. It is independent of the other markers of OHSS. We believe that the serum VEGF concentration on the day of hCG administration and on the day of oocyte collection, along with the total number of follicles on the day of hCG administration and a pretreatment diagnosis of PCO, may be more accurate than the serum E2 concentration in predicting the development of OHSS. The serum E2 concentration is becoming progressively less reliable as regimens of ovarian stimulation move from combined gonadotropin preparations to preparations containing FSH alone, because the serum E2 concentration may underrepresent the degree of follicular response to ovarian stimulation. Further prospective studies will be required to define the most reliable cutoff points for predicting and, therefore, ultimately preventing, OHSS.
12.
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