1 Institut Clínic of Gynaecology, Obstetrics and Neonatology and 2 Hormonal Laboratory, Faculty of Medicine-University of Barcelona, Hospital Clínic-Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
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Abstract |
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Key words: adrenomedullin/IVF/nitric oxide/ovarian response/VEGF
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Introduction |
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Adrenomedullin is a novel vasorelaxant peptide recently isolated from the acid extract of human phaeochromocytoma that was discovered as its stimulating action on platelet cAMP production was monitored (Kitamura et al., 1993). Subsequent studies have shown that adrenomedullin is synthesized in several organs in normal conditions, including vascular tissue, heart, adrenal medulla, lungs, brain, gastrointestinal tissue and kidney (Massart et al., 1996
). In organs where expressed, adrenomedullin acts as a local vasodilatory hormone rather than as a circulatory hormone (He et al., 1995
). Remarkably, a recent study showed for the first time that adrenomedullin is expressed in rat granulosa cells and enhances the effects of FSH treatment, acting additionally to produce cAMP in the cells (Abe et al., 1998
). On the other hand, a recent report has shown for the first time that hypoxia induces adrenomedullin production (Nakayama et al., 1998
).
On the above evidence, the specific aims of this study were to: (i) quantify serum and follicular fluid (FF) concentrations of VEGF, nitrite/nitrate (the two stable oxidation products of NO metabolism) and adrenomedullin in IVF cycles on the day of egg retrieval; (ii) define the relationship between FF concentrations of VEGF, nitrite/nitrate and adrenomedullin and those of ovarian steroids (oestradiol, progesterone); and (iii) assess the relationship between both circulating and FF concentrations of VEGF, adrenomedullin and nitrite/nitrate and parameters of ovarian response (age, basal FSH levels, ampoules of gonadotrophins used, peak oestradiol serum concentration, oocytes retrieved and oocyte maturity) to stimulation with gonadotrophins as well as the IVF outcome (fertilization rate, embryo quality and clinical pregnancy).
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Materials and methods |
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Stimulation protocol and retrieval
IVF was carried out according to a protocol previously reported, including gonadotrophin ovarian stimulation under pituitary suppression with gonadotrophin-releasing hormone (GnRH) agonist (Balasch et al., 1996). Briefly, daily s.c. leuprolide acetate was started in the midluteal phase of the previous cycle, and gonadotrophin stimulation of the ovaries was commenced 1214 days later when the plasma oestradiol concentration declined to <50 pg/ml and a vaginal ultrasonographic scan showed an absence of follicles >10 mm diameter. On days 1 and 2 of ovarian stimulation, six ampoules/day of highly purified FSH (Neo-Fertinorm; Serono S.A., Madrid, Spain) were administered s.c. On days 3, 4 and 5 of ovarian stimulation, two ampoules/day of FSH were administered to each patient. From day 6 onward, FSH was administered on an individual basis according to the ovarian response.
Sequential transvaginal ultrasonography and serum oestradiol measurements were carried out to assess follicular development. Ultrasonic scans were performed with a 5 mHz vaginal transducer attached to an Aloka sector scanner (model SSD-620, Aloka, Tokyo, Japan). Finally, human chorionic gonadotrophin (HCG; 5000 IU) (Profasi; Serono S.A.) was administered i.m. when a consistent rise in serum oestradiol concentration was associated with the presence of two or more follicles of 18 mm diameter. Oocyte aspiration was performed by vaginal ultrasonography 3536 h after HCG injection. The maturational status of the oocytes and the embryo grading were recorded according to published criteria (Veeck, 1986
); embryos of Veeck grades 1 or 2 were considered high quality. Blood samples were obtained from each subject on the day of oocyte retrieval for comparative purposes with FF.
Pregnancy was diagnosed by increasing serum concentrations of ß-HCG after embryo transfer, and the subsequent demonstration of an intrauterine gestational sac by ultrasonography.
Sample collection and processing
Venous blood samples were collected after overnight fasting and after 1 h of bed rest (i.e. ~13 h after the last meal) for measurement of serum concentrations of oestradiol, progesterone, VEGF and nitrite/nitrate, and plasma concentrations of adrenomedullin. Serum samples were allowed to clot and then centrifuged at 3000 g for 10 min. Centrifugation was always performed within 30 min after blood withdrawal to minimize the potential contribution of VEGF released from platelets during blood clotting (Webb et al., 1998). Plasma samples were identically processed. For FF measurement of VEGF, adrenomedullin and NO, as well as oestradiol and progesterone, the content of all mature follicles (
14 mm diameter) containing the oocytecumulus complex was collected. After identification and removal of the oocytes, the clear FF from each patient was pooled, its volume determined, and the samples centrifuged at 3000 g for 10 min to separate out cellular contents and debris. Follicular fluid supernatant was transferred to sterile polypropylene tubes and frozen at 70°C for further analysis. Since a wide interfollicular variation in intrafollicular steroid and cytokine concentrations has been reported as a reflection of interfollicular asynchrony during ovarian stimulation for IVF (Barak et al., 1992
; Mendoza et al., 1999
), it was elected to use pooled aspirated FF from each patient in an attempt to assess whole ovarian production as previously recommended (Orvieto et al., 1995
; Friedman, 1997, 1998), rather than to evaluate each follicle separately.
Laboratory methods
The laboratory methods for FSH, oestradiol, VEGF, adrenomedullin and NO measurement used have been reported previously (Balasch et al., 1996; Morales-Ruiz et al., 1997
; Guevara et al., 1998
; Jiménez et al., 1999
; Pérez-Ruiz et al., 1999
). FSH serum concentrations were measured using an immunoradiometric assay (Immunotech International, Marseilles, France) and data were expressed in terms of International reference preparation (IRP) 78/549. Oestradiol and progesterone concentrations in serum and FF were measured by direct radioimmunoassay (bioMérieux, Marcy l'Etoile, France for oestradiol; Immunotech International, Marseilles, France for progesterone).
Serum and FF VEGF concentrations were measured using an enzyme-linked immunosorbent assay (Quantikine Human VEGF Immunoassay, R&D Systems Inc., Minneapolis, MN, USA) that recognizes the soluble isoforms VEGF121 and VEGF165. Intra- and inter-assay coefficients of variation (CV) in serum samples were 7.1 and 9.2% respectively. In FF, these values were 4.7 and 7.5% respectively. The portion of inhibition produced by serial dilution of FF samples (n = 5) paralleled the standard curve (data not shown). The recovery of 400 pg of recombinant human VEGF165 added to follicular samples was 108%, and the recovery of 100 pg was 106%.
Plasma and FF adrenomedullin concentrations were measured by radioimmunoassay (Phoenix Pharmaceuticals, Mountain View, CA, USA) after extraction of adrenomedullin on Sep-Pack C18 cartridges (Waters Associates, Mildford, MA, USA). Briefly, plasma or follicular samples (2 ml) were acidified with 4% acetic acid (3 ml) and applied twice to cartridges pre-activated with methanol, distilled water and 4% acetic acid. Cartridges were then washed with distilled water and 25% ethanol, and adrenomedullin was eluted with 4 ml glacial acetic acid in 86% ethanol. The eluted adrenomedullin was then dried and reconstituted for radioimmunoassay. The recovery rate for the extraction procedure was 79%, as determined by the addition of 125I-labelled adrenomedullin to plasma or FF. Maximum binding of the anti-adrenomedullin antibody in the radioimmunoassay was 40.6%. Intra- and inter-assay CV were 12.4 and 13.6% respectively. Dilution curves obtained from plasma or follicular extracts paralleled the standard curve.
Serum and FF concentrations of nitrite/nitrate were determined by a fluorometric method (Misko et al., 1993), the fluorescent signal being measured (Perkin Elmer, Foster City, CA, USA) at excitation and emission wavelengths of 365 and 425 nm respectively. Intra- and inter-assay CV were 8.4 and 14.8% respectively.
Statistical analysis
Data were analysed using SPSS statistical software using paired and unpaired Student's t-tests, and Pearson's correlation coefficient when appropriate. Results are expressed as mean ± SE and were considered significant at a P-value < 0.05.
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Results |
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Discussion |
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Follicular fluid VEGF concentrations in patients undergoing IVF have been investigated in recent reports. Similar to previous studies (Lee et al., 1997), we found that FF VEGF concentrations were more than 10-fold greater than serum concentrations at the time of egg retrieval, thus implying that the peri-ovulatory, luteinizing follicle produces significant amounts of VEGF (Lee et al., 1997
). In addition, mean VEGF concentrations detected in both studies were very similar. Previous studies have also reported a similar degree of correlation between FF VEGF concentrations and patient age (Lee et al., 1997
; Friedman et al., 1998
) or the number of ampoules of gonadotrophin (Friedman et al., 1998
) as found in our study. This has been explained on the basis of an enhanced VEGF production because of relative hypoxia in these ovarian follicles, a fact supported by studies in vitro investigating the effects of hypoxic conditions and treatment with cobalt chloride on granulosa cell cultures (Friedman et al., 1997
, 1998
).
A previous observation (Lee et al., 1997) that FF VEGF was positively correlated with both FF and serum progesterone concentration has been considered as an additional argument favouring the hypoxia hypothesis because premature luteinization may be an early manifestation of limited ovarian reserve or ovarian ageing (Friedman et al., 1998
). However, such a correlation was lacking in the present study where pituitary suppression with GnRH agonist was used and thus, premature luteinization as the stimulant for elevated FF VEGF concentrations is hardly supportable. On the other hand, both positive (Lee et al., 1997
) and negative (Friedman et al., 1998
) correlations between FF VEGF concentrations and the number of oocytes obtained have been reported. Our results are in agreement with those of the previous negative study in this regard. Finally, as for adrenomedullin, no differences were found with respect to circulating and FF concentrations of VEGF between pregnant and non-pregnant women. This is in contrast to the published study (Friedman et al., 1998
) which reported elevated FF VEGF concentration in non-pregnant versus pregnant women. In that study, however, women conceiving were slightly younger than those failing to conceive, and marked overlapping existed between individual values in pregnant and non-pregnant groups (Friedman et al., 1998
).
Neither serum nor FF nitrite/nitrate measurements were found to be useful markers of ovarian response or pregnancy in IVF cycles in the present study. A possible explanation of this might be that NO is a highly diffusible, labile gas with a half-life of a few seconds; moreover, the high instability of NO makes measurement of changes in its concentration or production very difficult to perform.
In summary, this study suggested for the first time that increased FF concentrations of adrenomedullin may serve as a marker of decreased ovarian response in IVF cycles, as indicated by day 3 FSH concentrations, the number of ampoules of gonadotrophins used, the number of eggs retrieved, and oocyte maturity. In addition, our results provide further evidence favouring an association between FF VEGF concentration and patient age, as well as the total dose of gonadotrophins used. Finally, on the basis of our findings, nitrite/nitrate measurements in either serum or FF are not useful markers of ovarian response.
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Acknowledgments |
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Notes |
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References |
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Balasch, J., Creus, M., Fábregues, F. et al. (1996) Inhibin, follicle-stimulating hormone, and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin-releasing hormone agonist-treatment. Am. J. Obstet. Gynecol., 175, 12261230.[ISI][Medline]
Barak, V., Mordel, N., Zajicek, G. et al. (1992) The correlation between interleukin 2 and soluble interleukin 2 receptors to oestradiol, progesterone and testosterone levels in periovulatory follicles of in-vitro fertilization patients. Hum. Reprod., 7, 926929.[Abstract]
Faller, D.V. (1999) Endothelial cell responses to hypoxic stress. Clin. Exp. Pharmacol. Physiol., 26, 7484.[ISI][Medline]
Friedman, C.I., Danforth, D.R., Herbosa-Encarnación, C. et al. (1997) Follicular fluid vascular endothelial growth factor concentrations are elevated in women of advanced reproductive age undergoing ovulation induction. Fertil. Steril., 68, 607612.[ISI][Medline]
Friedman, C.I., Seifer, D.B., Kennard, E.A. et al. (1998) Elevated level of follicular fluid vascular endothelial growth factor is a marker of diminished pregnancy potential. Fertil. Steril., 70, 836839.[ISI][Medline]
Guevara, M., Ginés, P., Jiménez, W. et al. (1998) Increased circulating levels of adrenomedullin in patients with cirrhosis. Relationship with hemodynamic abnormalities and vasoconstrictor systems. Gastroenterology, 114, 336343.[ISI][Medline]
He, H., Bessho, H., Fujisawa, Y. et al. (1995) Effects of a synthetic rat adrenomedullin on regional hemodynamics in rats. Eur. J. Pharmacol., 273, 209214.[ISI][Medline]
Jiménez, W., Ros, J., Morales-Ruiz, M. et al. (1999) Inducible nitric oxide synthase expression in peritoneal macrophages of cirrhotic patients. Hepatology, 30, 670676.[ISI][Medline]
Kitamura, K., Kangawa, K., Kawamoto, Y. et al. (1993) Adrenomedullin: a novel hypotensive peptide isolated from human pheocromocytoma. Biochem. Biophys. Res. Commun., 192, 553560.[ISI][Medline]
Koos, R.D. (1995) Increased expression of vascular endothelial growth/permeability factor in the rat ovary following an ovulatory gonadotropin stimulus: potential roles in follicular rupture. Biol. Reprod., 52, 14261435.[Abstract]
Lee, A., Christenson, L.K., Stouffer, R.L. et al. (1997) Vascular endothelial growth factor levels in serum and follicular fluid of patients undergoing in vitro fertilization. Fertil. Steril., 68, 305311.[ISI][Medline]
Massart, P.E., Hodeige, D. and Donckier, J. (1996) Adrenomedullin: view on a novel vasodilatory peptide with natriuretic properties. Acta Cardiol., LI, 259269.
Mendoza, C., Cremades, N., Ruiz-Requena, E. et al. (1999) Relationship between fertilization results after intracytoplasmic sperm injection, and intrafollicular steroid, pituitary hormone and cytokine concentrations. Hum. Reprod., 14, 628635.
Misko, P.T., Schilling, R.J., Salvemini, D. et al. (1993) A fluorometric assay for the measurement of nitrite in biological samples. Ann. Biochem., 214, 1116.
Morales-Ruiz, M., Jiménez, W., Ros, J. et al. (1997) Nitric oxide production by peritoneal macrophages of cirrhotic rats: a host response against bacterial peritonitis. Gastroenterology, 112, 20562064.[ISI][Medline]
Nakayama, M., Takahashi, K., Murakami, O. et al. (1998) Induction of adrenomedullin by hypoxia and cobalt chloride in human colorectal carcinoma cells. Biochem. Biophys. Res. Commun., 243, 514517.[ISI][Medline]
Orvieto, R., Voliovitch, I., Fishman, P. and Ben-Rafael, Z. (1995) Interleukin-2 and ovarian hyperstimulation syndrome: a pilot study. Hum. Reprod., 10, 2427.[Abstract]
Pérez-Ruiz, M., Ros, J., Morales-Ruiz, M. et al. (1999) Vascular endothelial growth factor production in peritoneal macrophages of cirrhotic patients: regulation by cytokines and bacterial lipopolysaccharide. Hepatology, 29, 10571063.[ISI][Medline]
Powers, R.W., Chen, L., Russel, P.T. and Larse, W.J. (1995) Gonadotropin-stimulated regulation of blood-follicle barrier is mediated by nitric oxide. Am. J. Physiol., 269, E290E298.
Roselli, M., Keller, P.J. and Dubey, R.K. (1998) Role of nitric oxide in the biology, physiology and pathophysiology of reproduction. Hum. Reprod. Update, 4, 324.
Veeck, L.L. (1986) Atlas of the Human Oocyte and Early Conceptus. Williams & Wilkins, Baltimore.
Webb, N.J.A., Bottomley, M.J., Watson, C.J. and Brenchley, P.E.C. (1998) Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease. Clin. Sci., 94, 395404.[ISI][Medline]
Submitted on November 22, 1999; accepted on February 15, 2000.