Follicular fluid concentrations of adrenomedullin, vascular endothelial growth factor and nitric oxide in IVF cycles: relationship to ovarian response

Dolors Manau1, Juan Balasch1,3, Wladimiro Jiménez2, Francisco Fábregues1, Salvadora Civico1, Roser Casamitjana2, Montserrat Creus1 and Juan A. Vanrell1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Marked granulosa cell proliferation along with important changes in the vascular bed of the ovary characterize IVF cycles associated with multiple follicular growth and maturation. The present report investigated follicular fluid (FF) and circulating concentrations of adrenomedullin, vascular endothelial growth factor (VEGF) and nitric oxide (NO) in 70 IVF patients (14 of whom became pregnant); these three vasoactive substances may be implicated in extensive ovarian tissue remodelling. Serum and FF concentrations of oestradiol and progesterone were also measured in the 70 IVF cycles studied. Follicular fluid concentrations of VEGF and adrenomedullin but not nitrite/nitrate (the two stable oxidation products of NO metabolism) were significantly higher (P < 0.0001) than the corresponding circulating concentrations. Follicular fluid concentrations of oestradiol and progesterone were not correlated with those of adrenomedullin, VEGF or nitrite/nitrate. No relationship existed between circulating concentrations of adrenomedullin, VEGF or nitrite/nitrate on the day of oocyte aspiration and parameters of ovarian response to gonadotrophin stimulation. In contrast, FF adrenomedullin concentration showed a direct relationship with day 3 FSH serum concentration (r = 0.53, P < 0.01) and the number of ampoules of gonadotrophin administered (r = 0.36, P < 0.005), but an inverse correlation with the total number of oocytes retrieved (r = –0.29, P < 0.01) and the number of mature oocytes (r = –0.25, P < 0.05). A positive correlation was found for FF VEGF concentration and chronological age (r = 0.29, P < 0.05) and ampoules of gonadotrophins administered (r = 0.30, P < 0.05). There was no relationship between nitrite/nitrate FF concentrations and parameters of ovarian response. Neither serum concentrations nor FF concentrations of adrenomedullin, VEGF or nitrite/nitrate were correlated with IVF outcome. This study suggested for the first time that increased FF concentrations of adrenomedullin can be a marker of decreased ovarian response in IVF. Our results also provide further evidence favouring an association between FF VEGF and patient's age, while on the basis of our findings NO measurements are not a useful marker of ovarian response.

Key words: adrenomedullin/IVF/nitric oxide/ovarian response/VEGF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of the ovarian follicle from a small pre-antral follicle to the large pre-ovulatory follicle and the subsequent formation of a corpus luteum after ovulation, are key processes in mammalian reproduction. Marked changes in the vascular system accompany follicular development and thus, the pre-ovulatory follicle provides a unique physiological example of rapid growth associated with neovascularization. These processes are magnified in IVF cycles where ovarian stimulation with exogenous gonadotrophins is used in order to induce multiple follicular growth and maturation. According to recent investigations, both vascular endothelial growth factor (VEGF) and nitric oxide (NO) may play important roles in the process of folliculogenesis because they have marked angiogenic and/or vasodilator effects in addition to potential dual effects on cell growth and proliferation, displaying both promitogenic and antimitogenic actions (Koos, 1995Go; Powers et al., 1995Go; Roselli et al., 1998Go; Faller, 1999Go). In fact, both VEGF and NO are considered as markers of follicular hypoxia (Koos, 1995Go; Friedman et al., 1997Go, 1998Go; Roselli et al., 1998Go).

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., 1993Go). 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., 1996Go). In organs where expressed, adrenomedullin acts as a local vasodilatory hormone rather than as a circulatory hormone (He et al., 1995Go). 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., 1998Go). On the other hand, a recent report has shown for the first time that hypoxia induces adrenomedullin production (Nakayama et al., 1998Go).

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).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Seventy women with primary infertility undergoing IVF consented to the study, which was approved by the Investigation and Ethics Committee of our hospital. All patients had both ovaries and had regular menstrual cycles every 26 to 32 days and normal ovulatory function as shown by midluteal plasma progesterone and prolactin determinations, timed premenstrual endometrial biopsy and ultrasonographic scanning. The women were aged 26–40 years; mean (± SE) age was 35 ± 0.59 years. All had normal blood pressure and body mass index, were non-smokers and were not taking any medication or involved in intensive exercise. Twenty-nine of the women were aged >35 years, and 20 of these were aged >=37 years. These age populations were chosen arbitrarily in an attempt to exaggerate potential physiological differences in ovarian response among groups of women based on age. All women underwent cycle day 3 serum FSH measurement within 3 months of the IVF attempt. Mean basal FSH concentrations were slightly, albeit not significantly, higher in the older group (>=37 years) compared with younger women (7.6 ± 0.4 versus 6.9 ± 0.3 IU/l). Infertility aetiologies were similar in both groups of patients. For the older group, these included male factor (n = 8; 40%), tubal disease (n = 5; 25%), minimal/mild endometriosis (n = 4; 20%) and idiopathic infertility (n = 3; 15%). For the younger group, infertility diagnoses included male factor (n = 28; 56%), tubal disease (n = 10; 20%), minimal/mild endometriosis (n = 8; 16%) and idiopathic infertility (n = 4; 8%). No patient had any uterine anomaly.

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., 1996Go). Briefly, daily s.c. leuprolide acetate was started in the midluteal phase of the previous cycle, and gonadotrophin stimulation of the ovaries was commenced 12–14 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 35–36 h after HCG injection. The maturational status of the oocytes and the embryo grading were recorded according to published criteria (Veeck, 1986Go); 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., 1998Go). 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 oocyte–cumulus 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., 1992Go; Mendoza et al., 1999Go), 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., 1995Go; 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., 1996Go; Morales-Ruiz et al., 1997Go; Guevara et al., 1998Go; Jiménez et al., 1999Go; Pérez-Ruiz et al., 1999Go). 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., 1993Go), 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All patients had multiple follicular development and successful oocyte retrieval, the mean peak serum oestradiol concentration being 2180 ± 157 pg/ml and the mean number of oocytes obtained 10.1 ± 0.64. Fourteen women became pregnant, and no patient developed ovarian hyperstimulation syndrome. As expected, oestradiol and progesterone concentrations in FF were significantly higher than those found in serum. Follicular fluid concentrations of VEGF and adrenomedullin, but not nitrite/nitrate, were significantly higher than the corresponding circulating concentrations (Table IGo). Follicular fluid concentrations of oestradiol and progesterone were not correlated with those of adrenomedullin, VEGF or nitrite/nitrate (data not shown).


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Table I. Circulating and follicular fluid concentrations of adrenomedullin, vascular endothelial growth factor (VEGF), nitrite/nitrate, oestradiol and progesterone in the 70 patients investigated.
 
No relationship existed between circulating concentrations of adrenomedullin, VEGF or nitrite/nitrate on the day of oocyte aspiration and parameters of ovarian response to gonadotrophin stimulation (data not shown). Follicular fluid adrenomedullin concentration showed a direct relationship with day 3 FSH serum concentration and the number of ampoules of gonadotrophin administered, but an inverse correlation with the total number of oocytes retrieved and the number of mature oocytes (Figure 1Go). No correlation existed between FF adrenomedullin and the remaining parameters of ovarian response analysed. A positive correlation was found for FF VEGF concentration and chronological age and number of ampoules of gonadotrophins administered (Figure 2Go), but not with respect to the other parameters of ovarian response evaluated. There was no relationship between nitrite/nitrate FF concentrations and any of the parameters of ovarian response (data not shown).



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Figure 1. Relationship between (A) follicular fluid concentrations of adrenomedullin and basal FSH, (B) total ampoules of gonadotrophin administered, (C) total number of oocytes retrieved, and (D) number of mature oocytes obtained.

 


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Figure 2. Relationship between (A) follicular fluid concentrations of vascular endothelial growth factor (VEGF) and patient's age and (B) total ampoules of gonadotrophins administered.

 
Neither serum nor FF concentrations of adrenomedullin, VEGF or nitrite/nitrate were correlated with fertilization rate and embryo grading (data not shown). Similarly, no differences were detected between pregnant and non-pregnant women with respect to FF or circulating concentrations of adrenomedullin, VEGF, nitrite/nitrate, oestradiol and progesterone (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The data presented in this report provide some of the first evidence suggesting that adrenomedullin may be produced locally in the human ovary, and play a role in folliculogenesis. Several facts support this contention. First, there was a significantly higher amount of adrenomedullin present in FF relative to plasma. Second, although the precise role of adrenomedullin in the ovary remains to be determined, a recent study (Abe et al., 1998Go) showed that adrenomedullin was expressed in rat granulosa cells and enhanced the effects of FSH on these cells. An additive action of adrenomedullin and FSH existed on the production of cAMP, which is the intracellular messenger for FSH. From that study it was concluded that adrenomedullin may play a role in the process of granulosa cell differentiation as a local regulator through an autocrine/paracrine mechanism (Abe et al., 1998Go). Results in the present study would provide clinical evidence supporting this contention, as we were able to demonstrate a statistically significant correlation between FF adrenomedullin concentration and day 3 FSH serum concentration and the total amount of gonadotrophin administered, but an inverse correlation with the total number of oocytes retrieved and oocyte maturity. These findings are consistent with ovarian ageing or decreased ovarian reserve. Finally, a recent study (Nakayama et al., 1998Go) has demonstrated for the first time that hypoxia strongly stimulates production of adrenomedullin by cultured human cells. Moreover, treatment of cells with cobalt chloride, which mimics hypoxic states, significantly increased adrenomedullin production. Thus, it may be postulated that increased adrenomedullin FF concentrations are present in older women or low-responder patients as a result of relative follicular hypoxia. This would be in agreement with data from previous studies investigating other angiogenic substances such as VEGF, which are discussed below.

Follicular fluid VEGF concentrations in patients undergoing IVF have been investigated in recent reports. Similar to previous studies (Lee et al., 1997Go), 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., 1997Go). 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., 1997Go; Friedman et al., 1998Go) or the number of ampoules of gonadotrophin (Friedman et al., 1998Go) 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., 1997Go, 1998Go).

A previous observation (Lee et al., 1997Go) 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., 1998Go). 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., 1997Go) and negative (Friedman et al., 1998Go) 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., 1998Go) 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., 1998Go).

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.


    Acknowledgments
 
This work was supported by Grants from the Fondo de Investigación Sanitaria (FIS 96/0355) (J.B.), the Comissionat per a Universitat i Recerca-Generalitat de Catalunya (1997SGR 00213) (J.B.) and Dirección General de Investigación Científica y Técnica (SAF99-0016) (W.J.). Dr D.Manau received a grant from the IDIBAPS.


    Notes
 
3 To whom correspondence should be addressed at: Institut Clínic of Gynaecology and Obstetrics, Hospital Clínic; C/Casanova 143; 08036-Barcelona, Spain. E-mail: jbalasch{at}medicina.ub.es Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 22, 1999; accepted on February 15, 2000.