1 Laboratoire d'Histologie-Embryologie-Cytogénetique, Unité Inserm U327, 2 Service de Gynécologie-Obstétrique and 3 Unité de Biostatistique et d'Information médicale, Unité Inserm U444, Hopital Bichat-Claude-Bernard, Université Paris 7 Denis-Diderot, Service de Gynécologie-Obstétrique
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Abstract |
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Key words: endometriosis/follicular fluid/CD31/VCAM-1/VEGF
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Introduction |
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In women with normal non-stimulated cycles and those undergoing IVF, the local VEGF production in follicular fluid (follicular fluid) is correlated with the degree of follicular luteinization (Lee et al., 1997; Anasti et al., 1998
). Progesterone also appears to play a role in determining VEGF concentration in follicular fluid (Moncayo et al., 1998
). A positive correlation has also been observed between follicular fluid VEGF concentrations and patient age, especially in patients
38 years undergoing IVF (Friedman et al., 1997
; Manau et al., 2000
). Recently, it has been reported that elevated VEGF concentrations in follicular fluid might predict poor conception rates after IVF (Friedman et al., 1998
).
Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and vascular cell adhesion molecule-1 (VCAM-1) are transmembrane glycoproteins belonging to the immunoglobulin superfamily (Gearing et al., 1993; Almendro et al., 1996
). These molecules are known to be essential mediators of white blood cell adhesion and extravasation during inflammatory and immune reactions (Marik and Lo, 1996
; Cotran and Mayadas-Norton, 1998
). VCAM-1 and CD31 have also been used to assess angiogenesis; the expression of each is characterized by the release of an active soluble form (Banks et al., 1993
; Gearing et al., 1993
; Goldberger et al., 1994
). Recent investigation of the plasma and peritoneal fluid concentrations of soluble VCAM-1 (sVCAM-1) indicates that, like VEGF, VCAM-1 seems to play a role in the pathogenesis and progression of OHSS (Daniel et al., 1999
). To our knowledge, VCAM-1 and CD31 have never been studied in follicular fluid.
The aim of our study was to assess the concentrations of VEGF, sVCAM-1 and sCD31 in the follicular fluid of 75 women seeking to become pregnant with assisted reproductive technology and to assess their utility as markers or predictors of IVF outcome.
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Materials and methods |
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Population
The 75 patients were divided into three groups according to the cause of their infertility: for 50 women it was due only to laparoscopically confirmed tubal disease, without any male factors; 12 women had laparoscopy-proven severe endometriosis with no male factors and 13 women had partners diagnosed with severe oligoasthenoteratozoospermia that required intracytoplasmic sperm injection (ICSI). The records of all IVF and ICSI procedures were retrospectively reviewed to collect the following data: age, duration of stimulation, total ampoules of administered gonadotrophins, plasma oestradiol concentration on the day of HCG administration, number of retrieved oocytes, fertilization rate and pregnancy rate. None of them had more than three attempts at assisted reproduction and no patient was included twice considering sequential cycles. Fifty-six women did not become pregnant after assisted reproduction (group 1). Pregnancy (n = 19, group 2) was defined by significant HCG concentrations and the observation of embryonic cardiac activity during the transvaginal ultrasound examination performed 1 month after follicular aspiration.
IVF stimulation procedure
All patients underwent a standard stimulation protocol, described below. First, hormonal down-regulation was begun with a gonadotrophin-releasing hormone (GnRH) agonist (Triptorelin, Decapeptyl; Ipsen/Biotech, Paris, France). This treatment started on the third day of the menstrual cycle preceding IVF and was given i.m. at a daily dose of 0.1 mg for a minimum of 2 weeks. When the serum oestradiol concentration was <50 pg/ml, three ampoules of human menopausal gonadtotrophin (Menotropin, Humegon 75 IU; Organon, Saint-Denis, France) for 26 women or two ampoules of recombinant FSH (Follitropin alpha, Gonal-F 75 IU; Serono, Boulogne, France; or Follitropin beta, Puregon 100 IU; Organon, Saint-Denis, France) for 49 women were given i.m. each evening in addition to 0.05 mg of GnRH agonist. Follicular development was monitored by ultrasound with a 5 MHz transvaginal probe. The dose of gonadotrophin stimulation was then adjusted according to plasma oestradiol concentrations and the size of the ovarian follicles evaluated by ultrasound examination. Finally, when the plasma oestradiol concentration was >1800 pg/ml and when at least two follicles with a diameter of 20 mm were observed by ultrasound examination, 10 000 IU of HCG was given i.m. to induce follicular rupture. Transvaginal follicular aspiration was performed 3436 h after the HCG injection, under general anaesthesia using propofol (Diprivan; Zeneca, Cergy, France). After oocytes were identified and separated, the clear follicular fluid was collected and pooled for each woman. When blood contamination was observed, follicular fluid was discarded. This study considered follicular fluid from only one cycle for each patient. All samples were immediately centrifuged at 400 g for 10 min and the supernatant stored frozen at 40°C until they were assayed.
Determination of VEGF, sVCAM-1 and sCD31 concentrations in follicular fluid
These concentrations were assessed with the appropriate enzyme-linked immunosorbent assay test kits for human VEGF, human sVCAM-1, and human sCD31 (R&D Systems Minneapolis, USA). All samples were run in duplicate. For VEGF, VCAM-1 and CD31, all samples were diluted to 1/4, 1/20 and 1/5 respectively with the appropriate dilutant provided by the manufacturer. For VEGF, the sensitivity of the test was 9 pg/ml. The intra-assay and inter-assay coefficients of variation (CV) were 3.5 and 7% respectively. For sVCAM-1, the sensitivity of the assay was 2 ng/ml, and the intra-assay and inter-assay CV were 5 and 9% respectively. Finally, for sCD31, the sensitivity of the assay was 0.05 ng/ml, and the intra-assay and inter-assay CV were 7 and 6% respectively.
Statistical analysis
Student's t-test, the Kruskal-Wallis test and a Spearman correlation were all used for the statistical analysis. P < 0.05 was considered as significant. The sample size of our study provided a power of 97% for the t-test to detect the difference in VEGF concentrations previously observed (Friedman et al., 1998), with the variance estimates they reported.
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Results |
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Discussion |
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Thus, contrary to the results obtained by Friedman et al. (1998), our data suggest that VEGF concentrations in follicular fluid do not predict assisted reproduction outcome. However, some authors (Van Blerkom et al., 1997) have shown that the VEGF measurements of follicular fluid indicated a potential role for this factor in perifollicular angiogenesis and in the regulation of intrafollicular oxygen concentrations. Therefore, VEGF could be a marker for healthy individual follicle but not a clinical prognostic marker for the course of assisted reproduction, which would be consistent with our results. Moreover, the other angiogenesis factors we tested here, sCD31 and sVCAM-1, did not provide any additional information. It should nonetheless be borne in mind that the number of pregnancies in our series was limited and our sample collection design pooled follicular fluid of each woman (as did Friedman et al., 1998) and therefore did not allow identification of processes occurring in single follicles, which is a limitation of our study. However, our data showed that there was no statistically significant difference in the follicular fluid VEGF concentrations in patients conceiving and those failing to conceive. This suggests that VEGF production by granulosa cells is not directly correlated with assisted reproduction outcome or pregnancy potential.
VEGF has also been postulated to be a mediator of OHSS (Elchalal and Shenker, 1997; Rizk et al., 1997) but not an important clinical marker for the course of this condition (Ludwig et al., 1998
). In this context, a recent study reports that follicular fluid concentrations of VEGF are higher than serum concentrations (Artini et al., 1998
). No patient in our study had clinical OHSS, and only four patients had an oestradiol concentration that peaked at >3500 pg/ml on the day of HCG administration. The moderate increase in the follicular fluid concentration of VEGF in some patients, especially those
38 years old, might therefore simply reflect the variable ovarian response to gonadotrophin stimulation, rather than serve as a prognostic factor for assisted reproduction outcome.
Of the various endothelial markers known, CD31, which corresponds to the intercellular adhesion molecule PECAM-1, has been widely used in immunohistochemical analysis of different tumours (DeYoung et al., 1993). A soluble form of CD31 (sCD31), detected in the serum, has been proposed as a marker of angiogenesis (Goldberger et al., 1994
). CD31 is also expressed by the trophoblast (Baldwin et al., 1994
) and appears to be involved in the adhesion of trophoblast cells to arterial endothelium during implantation (Blankenship and Enders, 1997
). Some authors have also reported that sCD31 might be a predictive marker that could distinguish pre-eclamptic from normotensive pregnant women (Konijnenberg et al., 1997
; Krauss et al., 1997
). To our knowledge, our study was the first to analyse follicular fluid concentrations of sCD31 in patients treated with assisted reproduction, but we found no correlation between the sCD31 concentration and either the assisted reproduction outcome or patient age.
After cell activation, various cytokines induce VCAM-1 expression; it is released in an active soluble form (sVCAM-1) during inflammatory and immune reactions (Gearing et al., 1993; Marik and Lo, 1996
). Recent reports indicate that sVCAM-1 concentration in plasma and peritoneal fluid may be a predictive marker of OHSS (Daniel et al., 1999
). It is known to be expressed by the oocyte and early embryo (Campbell et al., 1995
). Our study confirmed for the first time the existence of sVCAM-1 in the follicular fluid. Like the other angiogenic factors tested in our study, follicular fluid sVCAM-1 does not appear to predict assisted reproduction outcome. Our data did, however, show a highly significant positive relation between follicular fluid sVCAM-1 concentrations and the fertilization rate. This concentration might thus be a marker of fertilization.
Besides the gonadotrophin-dependent production of VEGF by granulosa cells, other indications that cytokine interactions may be involved in the local regulation of VEGF production have appeared in recent studies (Cohen et al., 1996; Moncayo et al., 1998
). We observed a significant positive correlation between follicular fluid concentrations of sVCAM-1 and of VEGF. Thus, follicular fluid local production of these factors appears to be linked. Finally, our study suggests that both these angiogenic factors (VEGF and VCAM-1) are probably involved in fertilization, but their local regulation is currently unknown.
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Conclusion |
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Acknowledgements |
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Notes |
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References |
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Submitted on August 3, 2000; accepted on January 9, 2001.