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Effect of GnRH agonist and hCG treatment on VEGF, angiopoietin-2, and VE-cadherin: trying to explain the link to ovarian hyperstimulation syndrome

2011; Elsevier BV; Volume: 95; Issue: 8 Linguagem: Inglês

10.1016/j.fertnstert.2010.12.054

1556-5653

Rosario Cerrillo Martín, Alberto Pacheco, Sara Rodríguez-Mora, Raúl Gómez, Francisco Delgado, António Pellicer, Juan A. García-Velasco,

Ovarian cancer diagnosis and treatment

This study evaluated the differentially modulated expression of vascular mediators in oocyte donors after hCG vs. GnRHa triggering, trying to understand ovarian hyperstimulation syndrome pathophysiology. Donors who received GnRH agonist triggering showed a statistically significant decrease in vascular endothelial growth factor in follicular fluid and in mRNA expression in granulosa cells, with no differences in angiopoietin 2 and vascular endothelial cadherin levels in serum or foicular fluid. This differential regulation of vascular endothelial growth factor by hCG might explain the higher likelihood of ovarian hyperstimulation syndrome following hCG administration compared with GnRH agonists. This study evaluated the differentially modulated expression of vascular mediators in oocyte donors after hCG vs. GnRHa triggering, trying to understand ovarian hyperstimulation syndrome pathophysiology. Donors who received GnRH agonist triggering showed a statistically significant decrease in vascular endothelial growth factor in follicular fluid and in mRNA expression in granulosa cells, with no differences in angiopoietin 2 and vascular endothelial cadherin levels in serum or foicular fluid. This differential regulation of vascular endothelial growth factor by hCG might explain the higher likelihood of ovarian hyperstimulation syndrome following hCG administration compared with GnRH agonists. After hCG is administered to induce final oocyte maturation in assisted reproductive treatments, ovarian hyperstimulation may occur as the ovaries release vasoactive substances that increase vascular permeability (1Balasch J. Fabregues F. Arroyo V. Peripheral arterial vasodilation hypothesis: a new insight into pathogenesis of ovarian hyperstimulation syndrome.Hum Reprod. 1998; 13: 2718-2730Crossref PubMed Google Scholar). Of the different vascular mediators released from the ovaries and other tissues in response to hCG, vascular endothelial growth factor (VEGF), angiopietin (Ang)-2, and vascular endothelial (VE)-cadherin appear to be key regulators (2McClure N. Healy D.L. Rogers P.A. Sullivan J. Beaton L. Haning R.V. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome.Lancet. 1994; 344: 235-236Abstract PubMed Scopus (342) Google Scholar, 3Abramov Y. Barak V. Nisman B. Schenker J.G. Vascular endothelial growth factor plasma levels correlate to the clinical picture in severe ovarian hyperstimulation syndrome.Fertil Steril. 1997; 68: 133-137Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 4Soares S.R. Gómez R. Simón C. Garcia-Velasco J.A. Pellicer A. Targeting the vascular endothelial growth factor system to prevent ovarian hyperstimulation syndrome.Hum Reprod Update. 2008; 14: 321-333Crossref PubMed Scopus (159) Google Scholar, 5Villasante A. Pacheco A. Ruiz A. Pellicer A. Garcia-Velasco J.A. Vascular endothelial cadherin regulates vascular permeability: implications for ovarian hyperstimulation syndrome.J Clin Endocrinol Metab. 2007; 92: 314-321Crossref PubMed Scopus (45) Google Scholar, 6Fraser H.M. Regulation of the ovarian follicular vasculature.Reprod Biol Endocrinol. 2006; 12: 4-18Google Scholar). Because hCG is the key mediator of OHSS, avoiding its use would be a good strategy for avoiding this syndrome (7Lanzone A. Fulghesu A.M. Apa R. Caruso A. Mancuso S. LH surge induction by GnRH agonist at the time of ovulation.Gynecol Endocrinol. 1989; 3: 213-224Crossref PubMed Scopus (45) Google Scholar, 8Gonen Y. Balakier H. Powell W. Casper R.F. Use of gonadotropin-releasing hormone agonist to trigger follicular maturation for in vitro fertilization.J Clin Endocrinol Metab. 1990; 71: 918-922Crossref PubMed Scopus (285) Google Scholar). Administration of GnRH agonist (GnRHa) causes an endogenous LH peak that is enough to induce final oocyte maturation, but has a much shorter duration than with hCG (9Griesinger G. Diedrich K. Devroey P. Kolibianakis E.M. GnRH Agonist for triggering final oocyte maturation in the GnRH antagonist ovarian hyperstimulation protocol: a systematic review and meta-analysis.Hum Reprod Update. 2006; 12: 159-168Crossref PubMed Scopus (215) Google Scholar, 10Kol S. Solt I. GnRH agonist for triggering final oocyte maturation in patients at risk of ovarian hyperstimulation syndrome: still a controversy?.J Assist Reprod Genet. 2008; 25: 63-66Crossref PubMed Scopus (27) Google Scholar). Our hypothesis would be that GnRHa would induce a milder and shorter stimulus of vascular mediators involved in vascular leakage compared with hCG. Therefore, we studied the expression of VEGF, Ang-2, and VE-cadherin in egg donors that received either hCG or GnRHa to induce final oocyte maturation. Sixty-four oocyte donors were recruited for this prospective cohort study that took place from June 2008 to January 2009 according to the Spanish Law on Human Reproduction and our own institutional criteria (4Soares S.R. Gómez R. Simón C. Garcia-Velasco J.A. Pellicer A. Targeting the vascular endothelial growth factor system to prevent ovarian hyperstimulation syndrome.Hum Reprod Update. 2008; 14: 321-333Crossref PubMed Scopus (159) Google Scholar). Informed consent was given by all the patients before recruitment, and the trial was approved by our institutional ethical committee. Donors were recruited and allocated at a 1:1 ratio to receive either hCG (n = 32) or GnRHa (n = 32) after controlled ovarian hyperstimulation. Four donors from the group receiving hCG were excluded because of follicular asynchrony (to achieve a mature oocyte cohort in the retrieval) and two more because of personal reasons (refused to continue with the stimulation). Both groups were stimulated with GnRH antagonist protocol, with 150 IU rFSH as a starting dose. When the leading follicle reached 13–14mm, GnRH antagonist was introduced. When at least two follicles reached 18 mm mean diameter, either 6,500 IU of hCG (Ovitrelle; Serono, Spain) or a single bolus of 0.2 mg of GnRHa triptorelin (Decapeptyl; Ipsen Pharma, Spain) was administered and egg retrieval was performed 36 h later. No luteal support was provided. Plasma samples were obtained from all donors the day of hCG-GnRHa administration and the day of oocyte retrieval. The samples were centrifuged at 900 × g for 10 min at room temperature, and the cell-free supernatants were stored immediately in aliquots at –80°C until assayed. Follicular fluid from the first two mature follicles was aspirated into 10-mL tubes. The needle was withdrawn and completely emptied before each puncture, and no culture medium was used in the collection tubes. After removal of oocytes, follicular aspirates were centrifuged at 200 × g for 5 min, and supernatant was stored at –80°C until assayed. Sample size was estimated based on the statistical assumption that 30 cases is a number large enough to draw significant conclusions in experimental settings (11Pocock S.J. Clinical trials: a practical approach. Wiley Science Series, Somerset, UK1984Google Scholar). Concentrations of soluble VEGF165, human Ang-2, and human VE-cadherin in serum and follicular fluid were evaluated with a commercially available ELISA kit (Quantikine; R&D Systems, Abingdon, United Kingdom; Bender MedSystems, Vienna, Austria) according to the manufacturer's instructions. The assay sensitivity was 5 pg/mL, 8.3 pg/mL, and 2.8 ng/mL, respectively. The mRNA expression of the three molecules was evaluated by real-time PCR, as described previously (12Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochemical. 1987; 162: 156-159Crossref PubMed Scopus (62899) Google Scholar). RNA extraction was performed according to the method described by Chomczynski and Sacchi (12Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochemical. 1987; 162: 156-159Crossref PubMed Scopus (62899) Google Scholar). Reverse transcription was performed using the Advantage RT-for-PCR Kit (Clontech Laboratories, Palo Alto, CA). Duplicate TaqMan PCR assays for the VEGF, Ang-2, and VE-cadherin gene targets were performed on cDNA samples in 96-well optical plates (Applied Biosystems, Foster City, CA). Each sample was amplified in duplicate for VEGF, Ang-2, VE-Cadherin, GAPDH, and β-actin. The n-fold differences in VEGF, Ang-2, and VE-cadherin gene expression relative to each of the housekeeping genes were generated using the 2−ΔΔCt method (13Livak K.J. Schmittgen T.D. Analysis of relative expression data using real-time quantitative PCR and the 2–ΔΔCt method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (116537) Google Scholar) using ABI PRISM 1.7 analysis software. Categorical data were expressed as numbers and percentages, and numerical data were expressed as mean ± SEM. Metric variables were analyzed by the independent t test, and nominal variables by the χ2 test; P<0.05 was considered statistically significant. All statistical analysis was performed with the SPSS version 10.0 (SPSS, Inc., Chicago, IL). The relevant clinical data from the two groups of oocyte donors were similar in terms of days of stimulation, total amount of rFSH administered, peak serum E2 or P, and number of oocytes retrieved. A shorter duration of the luteal phase was found in the group that received GnRHa (8.5 ± 0.7 vs. 5.2 ± 0.9; P<0.001). Similarly, referred symptoms (mild abdominal pain or discomfort or bloating) were lower during the luteal phase in donors who received GnRHa (42.3% [11/26 patients] vs. 15.6% [5/32 patients]; P<0.02). There were no cases of ovarian hyperstimulation syndrome (OHSS) in the GnRHa group, and two cases occurred in the hCG group, according to our OHSS classification (14Bellver J. Muñoz E.A. Ballesteros A. Soares S.R. Bosch E. Simón C. et al.Intravenous albumin does not prevent moderate-severe ovarian hyperstimulation syndrome in high-risk IVF patients: a randomized controlled study.Hum Reprod. 2003; 18: 2283-2288Crossref PubMed Scopus (65) Google Scholar). We did not find any differences in VEGF plasma levels between the groups on either the day of hCG-GnRHa administration or on the day of ovum pick-up. However, there was a significant reduction in the VEGF concentration of follicular fluid from the follicles of donors who received GnRHa instead of hCG (Table 1). Similarly, VEGF mRNA expression was significantly lower in granulosa cells obtained from donors receiving GnRHa. Plasma samples and follicular fluid levels of Ang-2 on the day of triggering and the day of ovum pick up, as well as mRNA expression in granulosa cells, were similar in the two groups (Table 1). The plasma and follicular fluid levels of sVE-cadherin were similar in the two groups as well, although there was a nonsignificant reduction in the group that received GnRHa (Table 1). VE-cadherin mRNA was not expressed in granulosa cell samples.Table 1VEGF, Ang-2, and VE-cadherin levels in plasma and follicular fluid.hCG(n = 26)GnRHa-triptorelin (n = 32)P valuePlasma VEGF (pg/mL) day hCG/GnRHa176 ± 84 (144–208)169 ± 75 (131–207)0.24Plasma VEGF (pg/mL) day ovum pick up144 ± 71 (117–171)163 ± 76 (137–189)0.33VEGF in FF (pg/mL)1395 ± 284 (1286–1504)1069 ± 122 (947–1191)<0.001Plasma Ang-2 (pg/mL) day hCG/GnRHa635 ± 426 (478–792)425 ± 297 (288–562)0.09Plasma Ang-2 (pg/mL) day ovum pick up435 ± 277 (333–537)472 ± 287 (340–562)0.66Ang-2 in FF (pg/mL)3504 ± 1167 (3072–3936)3110 ± 1195 (2558–3662)0.27Plasma sVE-cadherin (ng/mL) day hCG/GnRHa1.7 ± 0.9 (1.1–2.4)1.1 ± 0.8 (0.7–1.4)0.07Plasma sVE-cadherin (ng/mL) day ovum pick up1.7 ± 0.8 (1.0–2.3)1.1 ± 0.8 (0.7–1.4)0.06sVE-cadherin in FF (ng/mL)1.3 ± 0.3 (1.1–1.5)1.2 ± 0.2 (1.2–1.6)0.09Note: FF = follicular fluid. Open table in a new tab Note: FF = follicular fluid. Only a few studies have investigated the differential regulation of different vascular mediators when final oocyte maturation is induced by either hCG or a single bolus of GnRHa (15Parborell F. Irusta G. Rodríguez Celín A. Tesone M. Regulation of ovarian angiogenesis and apoptosis by GnRH-I analogs.Mol Reprod Dev. 2008; 75: 623-631Crossref PubMed Scopus (14) Google Scholar). Our study suggests that the differences observed in VEGF mRNA and protein expression in follicular fluid from donors receiving either hCG or GnRHa may have direct clinical consequences in the prevention of OHSS. The continuous stimulation of the corpora lutea and liberation of vascular mediators by hCG administration, because of its long half-life of approximately 7 days, increases the risk of OHSS in patients using assisted reproductive technology. More than 20 years ago, empirical observations confirmed that a bolus of GnRHa induced an endogenous LH surge (16Gonen Y. Balakier H. Powell W. Casper R.F. Use of gonadotropin-releasing hormone agonist to trigger follicular maturation for in vitro fertilization.J Clin Endocrinol Metab. 1990; 71: 918-922Crossref PubMed Google Scholar). This surge safely induces final oocyte maturation, and the risk of OHSS is decreased because of the short duration of this endogenous LH surge (17Bodri D. Guillén J.J. Polo A. Trullenque M. Esteve C. Coll O. Complications related to ovarian stimulation and oocyte retrieval in 4052 oocyte donor cycles.Reprod Biomed Online. 2008; 17: 237-243Abstract Full Text PDF PubMed Scopus (114) Google Scholar). Several studies have confirmed that VEGF and other proinflammatory cytokines play a critical role in the pathophysiology of OHSS (2McClure N. Healy D.L. Rogers P.A. Sullivan J. Beaton L. Haning R.V. Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome.Lancet. 1994; 344: 235-236Abstract PubMed Scopus (342) Google Scholar, 4Soares S.R. Gómez R. Simón C. Garcia-Velasco J.A. Pellicer A. Targeting the vascular endothelial growth factor system to prevent ovarian hyperstimulation syndrome.Hum Reprod Update. 2008; 14: 321-333Crossref PubMed Scopus (159) Google Scholar, 5Villasante A. Pacheco A. Ruiz A. Pellicer A. Garcia-Velasco J.A. Vascular endothelial cadherin regulates vascular permeability: implications for ovarian hyperstimulation syndrome.J Clin Endocrinol Metab. 2007; 92: 314-321Crossref PubMed Scopus (45) Google Scholar, 18Pellicer A. Albert C. Mercader A. Bonilla-Musoles F. Remohí J. Simon C. The pathogenesis of ovarian hyperstimulation syndrome: in vivo studies investigating the role of interleukin (IL)-1B, IL-6 and vascular endothelial growth factor (VEGF).Fertil Steril. 1999; 71: 482-489Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 19Gómez R. Simón C. Remohí J. Pellicer A. Vascular endothelial growth factor receptor-2 activation induces vascular permeability in hyperstimulated rats, and this effect is prevented by receptor blockade.Endocrinology. 2002; 143: 4339-4348Crossref PubMed Scopus (141) Google Scholar, 20Gómez R. Simón C. Remohí J. Pellicer A. Administration of moderate and high doses of gonadotropins to female rats increases ovarian vascular endothelial growth factor (VEGF) and VEGF receptor 1 expression that is associated to vascular hypermeability.Biol Reprod. 2003; 68: 2164-2171Crossref PubMed Scopus (89) Google Scholar). We found that VEGF was significantly lower in patients who received GnRHa, both at the mRNA and protein level. Thus, we suggest that the endogenous LH surge induced by GnRHa is not only of shorter duration, but is also a weaker activator of the LH-hCG receptor. Another vascular mediator we investigated was Ang-2, affecting vascular integrity and endothelial cell survival. Recently, Thomas et al. (21Thomas M. Augustin H.G. The role of the angiopoietins in vascular morphogenesis.Angiogenesis. 2009; 12: 125-137Crossref PubMed Scopus (304) Google Scholar) showed that in the presence of VEGF, Ang-2 mediated vascular leakage to the third space. We did not find evidence that hCG and GnRHa differentially regulated Ang-2 mRNA or protein expression. We may speculate that the reason for our negative results could be related to the timing of the events. VEGF showed an immediate response to either hCG or GnRHa, but Ang-2 expression and secretion could be modified at a later stage, when VEGF protein is already secreted. The increased vascular permeability that is characteristic of OHSS (22Vlahos N.F. Gregoriou O. Prevention and management of ovarian hyperstimulation syndrome.Ann N Y Acad Sci. 2006; 1092: 247-264Crossref PubMed Scopus (54) Google Scholar, 23Balasch J. Fabregues F. Arroyo V. Peripheral arterial vasodilation hypothesis: a new insight into pathogenesis of ovarian hyperstimulation syndrome.Hum Reprod. 1998; 13: 2718-2730Crossref PubMed Scopus (56) Google Scholar) led us to consider the role of cell adhesion molecules that mediate inflammatory and immune reactions. When we investigated whether VE-cadherin was differentially regulated, we observed a nonsignificant trend toward higher serum levels in the group that received hCG. As previously suggested, VE-cadherin concentration or gene expression may be a reflection of corpus luteum function after controlled ovarian hyperstimulation in response to hCG, rather than a direct indicator of OHSS risk (24Villasante A. Pacheco A. Pau E. Ruiz A. Pellicer A. Garcia-Velasco J.A. Soluble vascular endothelial-cadherin levels correlate with clinical and biological aspects of severe ovarian hyperstimulation syndrome.Hum Reprod. 2008; 23: 662-667Crossref PubMed Scopus (30) Google Scholar), which is in accordance with our negative findings. Our study has some limitations. First, we measured the protein concentration of the three vascular mediators by ELISA, but a significant proportion of these molecules are bound to tissue receptors. Thus, free-molecule concentrations are only an estimate of the total number of molecules present; however, this caveat can be addressed by analyzing the mRNA levels of these mediators. Second, our study was performed with healthy volunteers and not with symptomatic patients. It therefore remains unknown whether these molecules might behave differently in symptomatic patients or those in different age strata. We found that inducing final oocyte maturation with either hCG or a single bolus of GnRHa resulted in differential regulation of VEGF mRNA expression and protein concentration in the follicular fluid, which could have direct clinical consequences.