Abstract. In this prospective study the concentration of circulating vascular endothelial growth factor (VEGF) was followed in 10 patients with severe ovarian ...
Human Reproduction vol.14 no.10 pp.2437–2441, 1999
Prediction of severe ovarian hyperstimulation syndrome by free serum vascular endothelial growth factor concentration on the day of human chorionic gonadotrophin administration M.Ludwig1,3, W.Jelkmann2, O.Bauer1 and K.Diedrich1 1Department
of Gynecology and Obstetrics and 2Institute of Physiology, Medical University of Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany 3To
whom correspondence should be addressed
In this prospective study the concentration of circulating vascular endothelial growth factor (VEGF) was followed in 10 patients with severe ovarian hyperstimulation syndrome (OHSS) after ovarian stimulation and in 15 patients without OHSS. VEGF was assayed by means of two different commercially available kits as either free or total VEGF in serum. The concentration of free VEGF was significantly higher on the days of human chorionic gonadotrophin (HCG) administration (309.4 K 165.0 versus 190.3 K 127.8 pg/ml, P < 0.05) and embryo transfer (315.0 K 125.2 versus 209.3 K 137.2 pg/ml, P < 0.05) in the OHSS compared to the control group. No such difference existed with respect to total circulating VEGF. In addition, there was no significant rise in the free or in the total serum VEGF concentration in the OHSS patients or the controls from the day of HCG administration up to the days of oocyte retrieval or embryo transfer. A cut-off concentration of 200 pg/ml free serum VEGF concentration on the day of HCG treatment resulted in a sensitivity of 90% and a specificity of 80% for the prediction of OHSS development. This is the first report on the parallel measurement of free and total VEGF in serum following ovarian stimulation. The value of the proposed cut-off concentration should be confirmed in a study of a larger group of women. Key words: OHSS/ovarian stimulation/pregnancy/VEGF
Introduction The pathogenesis of ovarian hyperstimulation syndrome (OHSS), one of the most severe complications of ovarian stimulation with human chorionic gonadotrophin (HCG), is still not finally described. Vascular endothelial growth factor (VEGF) is proposed to have an impact on the development of this clinical entity (Rizk et al., 1997). This has been shown in vitro (Neulen et al., 1995; Lee et al., 1997a) as well as in vivo (Krasnow et al., 1996; Lee et al., 1997b; Abramov et al., 1997; Agarwal et al., 1997, 1998; Artini et al., 1998; Ludwig et al., 1998a). Indeed, VEGF is not only an important mediator of angiogenesis, but also a potent stimulator of vascular permeability and inflammation. However, some authors have proposed that the increase in serum or ascitic © European Society of Human Reproduction and Embryology
VEGF concentrations is just an epiphenomenon of an activated kallikrein system (Kobayashi et al., 1998). Recently, we have provided evidence that the course of severe OHSS cannot be predicted by the overall pattern of circulating free VEGF (Ludwig et al., 1998a), and that other factors must also be involved in the pathogenesis of spontaneous or iatrogenic OHSS (Ludwig et al., 1998b). Nevertheless, other authors have considered the possibility of predicting the onset of an OHSS from the concentration of circulating VEGF on the day of HCG administration for ovulation induction (Artini et al., 1998; Agarwal et al., 1998, 1999). We performed a prospective study to evaluate the possibility of OHSS prediction based on serum VEGF concentration measurements on the days of HCG administration, oocyte retrieval and embryo transfer. This approach is similar to the one chosen by Agarwal et al. (1999). However, to improve the understanding of the role of VEGF in the pathogenesis of OHSS we used two different commercially available kits to measure side by side the concentrations of free and of total VEGF in serum. Materials and methods Patients From February 1997 to July 1998, serum from 782 consecutive patients undergoing an in-vitro fertilization (IVF) or IVF/intracytoplasmic sperm injection (ICSI) treatment was obtained from routine blood samples taken on the day of HCG administration, oocyte retrieval and embryo transfer. Complete sets of serum samples from all three days were present from 423 patients. The patients were stimulated according to the long luteal protocol, using a gonadotrophin releasing hormone (GnRH) agonist depot preparation (triptorelin, Decapeptyl®; Ferring Arzneimittel GmbH, Kiel, Germany; goserelin, Zoladex®; ICI Pharma, Plankstadt, Germany) and either urinary gonadotrophins (HMG, Menogon®; Ferring Arzneimittel GmbH) or recombinant follicle stimulating hormone (FSH) (recFSH; Gonal F, Serono Pharma GmbH, Mu¨nchen, Germany). Ovulation was induced if at least three follicles were ù18 mm in diameter by administration of 10 000 IU HCG (Predalon®; Organon GmbH, Mu¨nchen, Germany; Choragon, Ferring Arzneimittel GmbH). OHSS was defined according to the WHO criteria (1973). Only women with severe (type III) OHSS were included in this study (n 5 10). There were no cases of mild or moderate OHSS. From the remaining patients with available serum samples 15 were randomly chosen as controls. Blood sampling Blood was sampled in the morning and immediately centrifuged, divided into aliquots and stored at –20°C until measurement of VEGF. This measurement was done within 1 month after blood sampling was completed.
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Measurement of free VEGF in serum The concentration of free VEGF (isoform 165) in serum was measured using the Quantikine® human VEGF enzyme-linked immunosorbent assay (ELISA) from R&D Systems Europe (Abingdon, Oxon, UK). The immobilized antibody was monoclonal, while the second horseradish peroxidase-conjugated antibody was polyclonal. We have previously determined the intra- and interassay coefficients of variation of this system to be 5 and 7.5% respectively. The lower detection limit was 9 pg/ml (Heits et al., 1997). Measurement of total VEGF in serum The concentration of the total (bound 1 unbound) amount of VEGF in serum was measured using the Accucyte® VEGF ELISA from CytImmune (College Park, ML, USA). In this system, assay plates pre-coated with goat anti-rabbit IgG antibodies were used to capture the VEGF complexes in each sample consisting of rabbit anti-VEGF antibody, biotinylated VEGF, and sample or standard. The biotinylated VEGF conjugate and the VEGF in the samples and standards competed for the VEGF-specific antibody binding sites. Thus, as the concentration of VEGF in the sample increased, the amount of biotinylated VEGF captured by the antibody decreased. The reaction was visualized by the addition of streptavidin-conjugated alkaline phosphatase and colour reagent. The assay range was 0.195–50.0 ng/ ml and the intra- and interassay coefficients of variation were 8.0 and 11.8% according to the manufacturer. Statistics All statistical analyses were done using a one-sided heteroscedastic Student’s t-test.
Results Patients’ characteristics as well as treatment outcome and the results of serum VEGF measurements are summarized in Table I. There was no significant difference with respect to the number of oocytes and number of embryos transferred when the control group and the group of patients developing OHSS were compared. However, the concentration of free VEGF in serum was significantly higher on the days of HCG administration and embryo transfer in the OHSS group compared to the controls. Also, the sum of the free VEGF values on all 3 days when it was measured was significantly higher in the OHSS group. The concentration of total VEGF in serum tended to be higher in the OHSS group, but this difference was not statistically significant. Sensitivity, specificity, positive and negative predictive values were calculated for different cut-off values of free VEGF as a prospective marker of the development of OHSS (Table II). The optimal combination of positive (75%) and negative predictive values (92%) was obtained with a cut-off value of 200 pg/ml on the day of HCG administration. If the cut-off value was lowered to 180 pg/ml the negative predictive value was still 92%; however, the positive predictive value was reduced to 69%. Similar values were obtained with a cutoff value of 620 pg/ml for the sum of free VEGF serum concentrations of all three days (positive predictive value: 69%; negative predictive value: 92%). The highest positive predictive value (80%) was obtained with a cut-off value of 650 pg/ml. 2438
Discussion In the present study data are given on the free and total serum VEGF concentration during ovulation induction for IVF treatment. Both parameters remained constant from the day of HCG administration up to the day of embryo transfer. However, significantly higher values of free serum VEGF were obtained on the days of HCG administration and embryo transfer in the OHSS group compared to the controls. The total serum VEGF concentration was comparably high in both groups on all days. The observation that the administration of HCG does not result in a rise of the concentration of free VEGF in serum confirms data by Licht et al. (1997). They measured serum VEGF (ELISA kit; DPC, Minneapolis, MN, USA) at 15 min intervals from 5 h before up to 20 h after ovulation induction with 10 000 IU HCG in six volunteers in an IVF programme. The authors did not identify any apparent response of VEGF serum concentration to the induction of ovulation (Licht et al., 1997). It may be, however, that the time period in that study was too short to include the rise of VEGF, which may start later than the time point of ovulation. Others have described a substantial rise of VEGF in vivo and in vitro (Neulen et al., 1995; Krasnow et al., 1996; Lee et al., 1997a, b; Abramov et al., 1997; Artini et al., 1998; Ferrara et al., 1998; Agarwal et al., 1998, 1999). Therefore, our data are not in accordance with observations made in two other studies. In a prospective study in 15 patients at risk to develop OHSS, VEGF was measured in plasma on the days of HCG and oocyte retrieval (Artini et al., 1998). The authors describe a significantly higher VEGF plasma concentration on the day of oocyte retrieval in the patients who developed OHSS (n 5 4), compared to those who were at risk but did not show OHSS (n 5 11), or in the control group (n 5 15). The VEGF serum concentration was in the range of 26.26–41.50 pg/ml on the day of HCG administration, and 109.50 pg/ml and between 25.00–38.00 pg/ml on the day of oocyte retrieval, in the patients developing OHSS and in the remaining ones respectively. In that study an enzyme immunoassay (CYTElisa VEGF; Peninsula Laboratories Inc., Belmont, CA, USA) was used. Others also observed a higher rise in VEGF in patients developing OHSS compared with those who did not (Agarwal et al., 1998). Out of a cohort of 107 patients receiving ovarian stimulation for IVF, 20 developed a mild (n 5 10), a moderate (n 5 7) or a severe form (n 5 3) of OHSS. The authors described consistently rising VEGF serum concentrations during the phase of ovarian stimulation. The VEGF serum concentrations were significantly higher on the day of HCG administration compared to the beginning of ovarian stimulation (P , 0.0001). They rose thereafter on the days of oocyte retrieval and embryo transfer. In women who developed OHSS, VEGF concentrations continued to rise thereafter, while in women who did not develop OHSS VEGF concentrations did not rise. VEGF concentrations, although significantly higher on the day of HCG administration, did not predict the development of OHSS with adequate sensitivity and specificity (Agarwal et al., 1998), but the rise of VEGF thereafter did predict the development of OHSS in another evaluation of the same
OHSS and VEGF
Table I. Patients’ characteristics, laboratory results and treatment outcome of OHSS and control patient groups. Values are either absolute values or mean 6 SD OHSS Age (years) Duration of infertility (years) No. of polycystic ovaries on baseline scan (%) Basal serum FSH concentrations (U/l) Tubal infertility (%)a Body mass index (kg/m2) Dose of gonadotrophins (U) No. of stimulation days Oestradiol on day of HCG (pg/ml) Number of oocytes Number of embryos transferred Clinical pregnancies (n) Abortions (n) Free serum VEGF (pg/ml) HCG oocyte retrieval embryo transfer sumb Total serum VEGF (ng/ml) HCG oocyte retrieval embryo transfer sumb aAll other patients were treated because bSum of VEGF concentration on day of
NS 5 not significant.
Control
P value
31.3 6 2.8 4.25 6 2.25 3 (30)
30.3 6 3.2 5.18 6 4.22 1 (6.7)
NS NS ,0.05
5.41 6 1.57
5.78 6 3.45
NS
20 25.64 40.38 14.00 1694.6 15.1 2.8 6 2
6 6 6 6 6 6
3.13 12.11 2.20 1291.6 5.8 0.4
309.4 297.7 315.0 922.1
6 6 6 6
165.0 110.3 125.2 293.4
28.2 32.2 29.4 80.78
6 6 6 6
18.8 16.7 13.9 49.9
13.3 23.71 50.00 16.36 1743.8 13.5 2.8 4 1
NS NS NS NS NS NS NS – –
6 6 6 6 6 6
3.35 16.98 3.98 986.8 6.9 0.6
190.3 219.3 209.3 618.9
6 6 6 6
127.8 124.5 137.2 378.5
,0.05 50.06 ,0.05 ,0.05
24.8 24.3 27.3 76.3
6 6 6 6
9.3 11.7 13.4 24.8
NS NS NS NS
of male factor infertility. HCG, on day of oocyte retrieval and on day of embryo transfer.
Table II. Sensitivity, specificity and predictive values of free VEGF serum measurement in OHSS prediction Parameter VEGF on HCG day (ovulation induction) VEGF on oocyte retrieval day
Cut-off value to predict OHSS (pg/ml)
180 200 220 240 200 220 240 260 VEGF on 200 embryo transfer 220 day 240 VEGF sum 620 650
Sensitivity Specificity Positive% Negative predictive value % predictive value % % % 90 90 80 60 80 70 70 50 80 70 60 90 80
data (Agarwal et al., 1999). This group used an enzyme immunoassay, that was apparently sensitive for measurement of total VEGF in serum (Cytokit Red EIA kits; Peninsula Labs Inc., Minneapolis, MN, USA). The divergent results may be explained by different patient groups with inclusion of even lower degrees of OHSS and the small dataset sizes of patients with severe OHSS in these studies. Another factor may be the kit used to measure VEGF concentration in serum or plasma. One has to be aware that the concentration of total circulating VEGF was about 100-fold higher than the concentration of free VEGF in serum in our own study. This observation provides an explanation for the large variation in published absolute values of the serum VEGF concentration in OHSS
73 80 80 80 60 73 80 80 73 87 87 73 87
69 75 73 67 57 64 70 63 67 78 75 69 80
92 92 86 75 82 79 80 71 85 81 76 92 87
patients as well as in normal pregnancies and those complicated by pre-eclampsia (Sharkey et al., 1996; Evans et al., 1997; Lyall et al., 1997). For example, Sharkey et al. (1996) measured VEGF concentrations, which were 10 000-fold higher in preeclampsia patients compared to a similar population published by Lyall et al. (1997). Measurements of cytokines and growth factors in serum or other body fluids should therefore be specified with respect to the fraction that is detected by the assay system used for study. Sandwich ELISA do usually detect free cytokines, while the total pool of cytokines cannot be identified because carrier proteins and soluble receptors may obstruct antibody binding sites (Radoux and De Groote, 1994). This phenomenon has also been discussed for VEGF previously (Anthony et al., 1997; Evans et al., 1997). Clearly, 2439
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however, the concentration of free hormone determines its acute effects on the target cells. The problem of an uncritical use of commercially available kits has been published before for other cytokines (Bienvenu et al., 1993). Furthermore, the different fluids used for the measurements (serum versus plasma) may also contribute to the differences observed in the study by Artini et al. (1998) and our own data. A 10-fold higher concentration can be expected when serum instead of EDTA or heparin plasma is used for measurement. Finally, the storage temperature of –20°C in our study may have influenced the total VEGF serum concentration. Until now, however, there have been no data published regarding the effect of temperature on serum VEGF concentration. Previous studies have not fully clarified whether VEGF is really involved in the pathogenesis of OHSS, or if its increase is only an epiphenomenon of other regulatory mechanisms. It has been shown by several authors that the source of VEGF really seems to be the ovary, since in follicular fluid the VEGF concentration is up to 100-fold higher compared to serum samples (Krasnow et al., 1996; Lee et al., 1997; Abramov et al., 1997). Others demonstrated that the rise in permeability induced by ascites in OHSS patients could not be blocked by administration of anti-VEGF antibodies (Kobayashi et al., 1998). These authors proposed that the kallikrein–kinin system is one of the main factors in the pathogenesis of OHSS, since the permeability, raised by ascitic fluid from OHSS patients, could be blocked by trasylol, which is known to inhibit bradykinin synthesis, and captopril, known as a kininase II inhibitor. While some authors share this concept (Kodama et al., 1997), others believe that the renin–angiotensin–aldosterone system is involved as the main pathogenic mechanism (Delbaere et al., 1997; Elchalal and Schenker, 1997; ItskovitzEldor et al., 1997; Aboulghar et al., 1998). However, even if the raised VEGF serum concentration is only an epiphenomenon of OHSS, it is apparently a good marker to predict the onset of OHSS, even if the course of OHSS seems to be independent of the VEGF serum concentration (Ludwig et al., 1998a, b). As shown in Table II, a cut-off value of 200 pg/ml free serum VEGF on the day of HCG administration was a good indicator to estimate the individual risk in patients undergoing ovarian stimulation for IVF. Despite a low positive predictive value of only 75%, the negative predictive value was 92%. That means that in only 8% of the women undergoing ovarian stimulation, OHSS occurred despite a VEGF serum concentration below 200 pg/ml on the day of HCG. Similar predictive values were obtained when the sum of free VEGF measured on HCG day, oocyte retrieval day, and the day of embryo transfer was calculated. However, this procedure may not enable one to prevent the onset of OHSS – if a real prevention is possible at all. Possible techniques could be to lower the HCG dose to 5000 IU, to withhold HCG for a couple of days (coasting) or to start an i.v. albumin administration. Coasting, however, should start earlier than at a stage at which follicles had reached a size that would allow HCG administration (Benadiva et al., 1997; Lee et al., 1998) Since the total VEGF serum concentration did not differ significantly between OHSS patients and controls, this may 2440
be the mechanism by which the i.v. administration of albumin can help to prevent the onset of OHSS, or the onset of less severe forms of OHSS, although this topic is one of controversy (Ben Rafael et al., 1995; Ng et al., 1995; Shalev et al., 1995; Shaker et al., 1996; Orvieto and Ben-Rafael, 1996, 1998; Sabatini et al., 1997). Albumin may act as a non-specific VEGF binding protein, which helps to withdraw active VEGF from the circulation. Recently, we showed in a case of spontaneous OHSS in a triploid pregnancy that free VEGF concentrations were low before the onset of OHSS but increased to nearly 1000 pg/ml when OHSS started to develop (Ludwig et al., 1998b). VEGF values remained high, even when the clinical picture disappeared. This case confirmed the results of our observations of a lack of correlation between VEGF serum concentrations and the clinical course of OHSS (Ludwig et al., 1998a). Therefore, albumin administration must start on the day of oocyte retrieval because at that time OHSS seems to be triggered by VEGF. A later administration of albumin may nevertheless lower the VEGF serum concentration but cannot help to change the clinical course of OHSS, which is then maintained by other mechanisms. Recently, Abramov et al. (1999) have published their results concerning the albumin concentration in severe OHSS cases. The authors found a significant reduction in blood, and an increased albumin concentration in ascitic fluid of OHSS patients. This observation supports our hypothesis, since total VEGF remained unchanged, while free VEGF serum concentration increased – possibly due to a reduced albumin concentration. Others, however, have recently shown that the VEGF mRNA expression of luteinized granulosa cells in vitro is enhanced in the presence of albumin (Doldi et al., 1999). These authors could not explain their findings in the background of the apparent protective effect of an albumin administration on the day of oocyte retrieval. The possibility of predicting an OHSS by measuring free serum VEGF concentration on the day of HCG may become more important in the near future, when more recombinant FSH is used in ovarian stimulation. It is known that recombinant FSH results in lower oestradiol concentrations because of a total lack of LH activity in its preparation. In our study population, there was no difference between the oestradiol concentrations of the OHSS and the control patients. This may be mainly due to the fact that 7/10 patients in the OHSS group and 2/15 patients in the control group received recombinant FSH for ovarian stimulation. The others received HMG. A prediction of OHSS by oestradiol concentrations alone was therefore not possible. To conclude, we propose a novel tool to predict the onset of OHSS, namely the free VEGF serum concentration on the day of HCG. Clearly, prior to its use in clinical routine the proposed threshold of 200 pg/ml should be ascertained in a larger, prospectively designed study, in which only patients at high risk for OHSS are included. The further clinical course of these patients must be evaluated and related to the measured VEGF serum concentrations. References Aboulghar, M.A., Mansour, R.T., Serour, G.I. et al. (1998) Elevated concentrations of angiogenin in serum and ascitic fluid from patients with severe ovarian hyperstimulation syndrome. Hum. Reprod., 13, 2068–2071.
OHSS and VEGF Abramov, Y., Barak, V., Nisman, B. and Schenker, J.G. (1997) Vascular endothelial growth factor plasma concentrations correlate to the clinical picture in severe ovarian hyperstimulation syndrome. Fertil. Steril., 67, 261–265. Ambramov,Y., Naparstek,Y., Elchalal,U. et al. (1999) Plasma immunoglobulins in patients with severe ovarian hyperstimulation syndrome. Fertil. Steril., 70, 102–105. Agarwal, R., Conway, G., Sladkevicius, P. et al. (1998) Serum vascular endothelial growth factor and Doppler blood flow velocities in in vitro fertilization: relevance to ovarian hyperstimulation syndrome and polycystic ovaries. Fertil. Steril., 70, 651–658. Agarwal, R., Conway, G.S., Payne, N. and Jacobs, H.S. (1997) Vascular endothelial growth factor secretion in women undergoing ovarian stimulation: relevance to ovarian hyperstimulation syndrome. Joint meeting of the British Endocrine Societies, Harrogate, UK, 07.10.04, abstract 16. Agarwal, R., Tan, S-L., Wild, S. et al. (1999) Serum vascular endothelial growth factor concentrations in in vitro fertilization cycles predict the risk of ovarian hyperstimulation syndrome. Fertil. Steril., 71, 287–293. Anthony, F.W., Evans, P.W., Wheeler, T. and Wood, P.J. (1997) Variation in detection of VEGF in maternal serum by immunoassay and the possible influence of binding proteins. Ann. Clin. Biochem., 34, 276–280. Artini, P.G., Fasciani, A., Monti, M. et al. (1998) Changes in vascular endothelial growth factor concentrations and the risk of ovarian hyperstimulation syndrome in women enrolled in an in vitro fertilization program. Fertil. Steril., 70, 560–564. Ben Rafael, Z., Orvieto, R., Dekel, A. et al. (1995) Intravenous albumin and the prevention of severe ovarian hyperstimulation syndrome [letter; comment]. Hum. Reprod., 10, 2750–2752. Benadiva, C.A., Davis, O., Kligman, I. et al. (1997) Withholding gonadotropin administration is an effective alternative for the prevention of ovarian hyperstimulation syndrome. Fertil. Steril., 67, 724–727. Bienvenu, J., Coulon, L., Doche, C. et al. (1993) Analytical performances of commercial ELISA-kits for IL-2, IL6 and TNF-α. A WHO study. Eur. Cytokine Netw., 4, 447–451. Delbaere, A., Bergmann, P.J., Gervy-Decoster, C. et al. (1997) Prorenin and active renin concentrations in plasma and ascites during severe ovarian hyperstimulation syndrome. Hum. Reprod., 12, 236–240. Doldi, N., Destefani, A., Gessi, A. et al. (1999) Human albumin enhances expression of vascular endothelial growth factor in cultured human luteinizing granulosa cells: importance in ovarian hyperstimulation syndrome. Hum. Reprod., 14, 1157–1159. Elchalal, U. and Schenker, J.G. (1997) The pathophysiology of ovarian hyperstimulation syndrome – views and ideas. Hum. Reprod., 12, 1129–1137. Evans, P.W., Wheeler, T., Anthony, F. and Osmond, C. (1997) Maternal serum vascular endothelial growth factor during early pregnancy. Clin. Sci., 92, 567–571. Ferrara, N., Chen, H., Davis-Symth, T. et al. (1998) Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nature Med., 4, 336–340. Heits, F., Katschinski, D.M., Wiedemann, G.J. et al. (1997) Serum vascular endothelial growth factor (VEGF), a prognostic indicator in sarcoma and carcinoma patients. Int. J. Oncol., 10, 333–337. Itskovitz-Eldor, J., Kol, S., Lewit, N. and Sealey, J.E. (1997) Ovarian origin of plasma and peritoneal fluid prorenin in early pregnancy and in patients with ovarian hyperstimulation syndrome. J. Clin. Endocrinol. Metab., 82, 461–464. Kobayashi, H., Okada, Y., Asahina, T. et al. (1998) The kallikrein–kinin system, but not vascular endothelial growth factor, plays a role in the increased vascular permeability associated with ovarian hyperstimulation syndrome. J. Mol. Endocrinol., 20, 363–374. Kodama, H., Takeda, S., Fukuda, J. et al. (1997) Activation of plasma kinin system correlates with severe coagulation disorders in patients with ovarian hyperstimulation syndrome. Hum. Reprod., 12, 891–895. Krasnow, J.S., Berga, S.L., Guzick, D.S. et al. (1996) Vascular permeability factor and vascular endothelial growth factor in ovarian hyperstimulation syndrome: a preliminary report. Fertil. Steril., 65, 552–555. Lee, A., Christenson, L.K., Patton, P.E. et al. (1997a) Vascular endothelial growth factor production by human luteinized granulosa cells in vitro. Hum. Reprod., 12, 2756–2761. Lee, A., Christenson, L.K., Stouffer, R.L. et al. (1997b) Vascular endothelial growth factor concentrations in serum and follicular fluid of patients undergoing in vitro fertilization. Fertil. Steril., 68, 305–311. Lee, C., Tummon, I., Martin, J. et al. (1998) Does withholding gonadotrophin administration prevent severe ovarian hyperstimulation syndrome? Hum. Reprod., 13, 1157–1158.
Licht, P., Engel, N., Fischer, O. et al. (1997) HCG does not directly regulate circulating concentrations of vascular endothelial growth factor in IVF and embryo transfer cycles. Hum. Reprod. (abstract book), 12, 6. Ludwig, M., Bauer, O., Lopens, A. et al. (1998a) Serum concentration of vascular endothelial growth factor cannot predict the course of severe ovarian hyperstimulation syndrome. Hum. Reprod., 13, 30–32. Ludwig, M., Gembruch, U., Bauer, O. and Diedrich, K. (1998b) Ovarian hyperstimulation syndrome (OHSS) in a spontaneous pregnancy with fetal and placental triploidy: information about the general pathophysiology of OHSS. Hum. Reprod., 13, 2082–2087. Lyall, F., Greer, I.A., Boswell, F. and Fleming, R. (1997) Suppression of serum vascular endothelial growth factor immunoreactivity in normal pregnancy and in pre-eclampsia. Br. J. Obstet. Gynaecol., 104, 223–228. Neulen J., Yan Z., Raczek S. et al. (1995) Human chorionic gonadotropindependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome. J. Clin. Endocrinol. Metab., 80, 1967–1971. Ng, E., Leader, A., Claman, P. et al. (1995) Intravenous albumin does not prevent the development of severe ovarian hyperstimulation syndrome in an in-vitro fertilization programme. Hum. Reprod., 10, 807–810. Orvieto, R. and Ben-Rafael, Z. (1998) Role of intravenous albumin in the prevention of severe ovarian hyperstimulation syndrome. Hum. Reprod., 13, 3306–3309. Orvieto, R. and Ben Rafael, Z. (1996) Prophylactic intravenous albumin for the prevention of severe ovarian hyperstimulation syndrome [letter]. Hum. Reprod., 11, 460–461. Radoux,D. and DeGroote,D. (1994) The total cytokine concept: the influence of soluble receptors in the cytokine measurement Contrib. Onco., 46, 251–259. Rizk, B., Aboulghar, M., Smitz, J. and Ron-El, R. (1997) The role of vascular endothelial growth factor and interleukins in the pathogenesis of severe ovarian hyperstimulation syndrome. Hum. Reprod. Update, 3, 255–266. Sabatini, L., Wilson, C., al Shawaf, T. et al. (1997) Efficacy of serum albumin to prevent ovarian hyperstimulation syndrome? [letter; comment]. Fertil. Steril., 67, 587–589. Shaker, A.G., Zosmer, A., Dean, N. et al. (1996) Comparison of intravenous albumin and transfer of fresh embryos with cryopreservation of all embryos for subsequent transfer in prevention of ovarian hyperstimulation syndrome. Fertil. Steril., 65, 992–996. Shalev, E., Giladi, Y., Matilsky, M. and Ben Ami, M. (1995) Decreased incidence of severe ovarian hyperstimulation syndrome in high risk in-vitro fertilization patients receiving intravenous albumin, a prospective study. Hum. Reprod., 10, 1373–1376. Sharkey, A.M., Cooper, J.C., Balmforth, J.R. et al. (1996) Maternal plasma concentrations of vascular endothelial growth factor in normotensive pregnancies and in pregnancies complicated by pre-eclampsia. Europ. J. Clin. Invest., 26, 1182–1185. WHO (1973) Agents stimulatins gonadal function in human. World Health organ. Tech. Rep. Ser., 514–520. Received on February 22, 1999; accepted on June 18, 1999
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