1 Williamson Laboratory, St Bartholomews and The London Hospitals Trust, London EC1A, 2 Centre for Reproductive Medicine, St Bartholomews and Royal London Hospital, Queen Mary School of Medicine and Dentistry, London E1 1BB, 3 Department of Biological and Applied Science, University of North London, Holloway Road, London and 4 The Bridge Centre, London Bridge, London SE19RY, UK
5 To whom correspondence should be addressed. e-mail: ggrudzinskas{at}thebridgecentre.co.uk
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Abstract |
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Key words: coasting/controlled ovarian stimulation/ovarian hyperstimulation syndrome/vascular endothelial growth factor
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Introduction |
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Withholding gonadotrophin and delaying hCG administration during COS in women at risk of developing OHSS has been shown to be an effective strategy in the prevention of OHSS without compromising the cycle outcome (Sher et al., 1995; Benadiva et al., 1997; Fluker et al., 1999
; Al-Shawaf et al., 2001
). Coasting is believed to be effective by diminishing the functional granulosa cell cohort with inhibition of granulosa cell proliferation and ultimately progressive granulosa cell apoptosis. A gradual decline in circulating levels of serum E2 is observed and, probably more importantly, there is a reduction in the chemical mediators that trigger OHSS such as VEGF. This study was designed to assess the effects of coasting on follicular fluid VEGF concentrations in individual follicles of different sizes in relation to the granulosa cell number, follicular fluid steroid levels, oocyte retrieval, fertilization and embryo quality.
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Materials and methods |
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All women underwent a long GnRH agonist stimulation protocol as previously described (Al-Shawaf et al., 2001). Women were identified as being at risk of developing OHSS if >20 follicles were noted on ultrasound with a serum E2 level >13 200 pmol/l. In these women, gonadotrophins were withheld, GnRH agonist continued and administration of hCG was delayed until the serum E2 level fell to <10 000 pmol/l.
Oocyte retrieval was performed 36 h later under ultra sound guidance and i.v. sedation. Oocytes were inseminated at 40 h post hCG administration and were checked the following day for the appearance of normal fertilization by the presence of two pronuclei.
Embryo grading
Embryos were graded according to their appearance with respect to how even the blastomeres appeared, degree of fragmentation and clarity of the cytoplasm. Grade 1 embryos had even-sized blastomeres, no fragmentation and good, clear cytoplasm.
Follicle aspiration and luteinized granulosa cell collection and isolation
Individual follicles of varying size (total of four to six follicles) were randomly selected prior to aspiration and measured in two dimensions by transvaginal ultrasound (model EUB-525; Hitachi, UK) in order to obtain a mean diameter. All measurements and aspirations were performed by the same operator (A.J.T.). Fluid was not collected from the first follicle aspirated as this had been previously found to contain large numbers of vaginal epithelium cells. However, each subsequent follicle aspirated was used for the study providing a range of follicle sizes were obtained. Following identification, the follicle was gently pierced using a double lumen needle and aspirated, allowing the follicle to collapse slowly around the needle. The follicle was then flushed with 4.5 ml of heparinized saline (3x1.5 ml automated flushes) and aspirated again to maximize the number of luteinized granulosa cells collected. Heavily blood-stained aspirates were discarded and a further follicle was measured and aspirated if appropriate. A record was also kept of the volume of fluid aspirated per individual follicle. Following examination of the follicular fluid from the individual follicle by the embryologist, the fluid was placed into a sterile tube and the oocyte number (if retrieved) recorded on the tube. The follicular fluids were taken to the laboratory immediately following all aspirations for isolation of the granulosa cells. Each tube was centrifuged at 1000 r.p.m. for 10 min and the supernatant frozen at 20°C for later analysis. The pellet was resuspended in 4 ml of medium [Roswell Park Memorial Institute 1640 with glutamine and NaHCO3 (Sigma, UK) supplemented with 10% fetal calf serum (Gibco BRL, UK) and 1% antibioticantimycotic containing 10 000 µg/ml penicillin G sodium, 25 µg/ml streptomycin and amphotericin B as fungizone (Gibco BRL)], and centrifuged at 1000 r.p.m. for 10 min. The pellet was again resuspended in 5 ml of medium and then incubated at 37°C for 30 min with 0.2% hyaluronidase (80 IU/ml; Medicult, UK) for cell dispersion.
Following further centrifugation at 1000 r.p.m. for 10 min, the pellet was resuspended with 2 ml of medium and layered over a 50% Percoll gradient [2 ml of Percoll (Pharmacia, UK) and 2 ml sterile PBS]. This was centrifuged at 1300 r.p.m. for 20 min to separate the luteinized granulosa cells from blood. The cells were removed using a pipette and washed with 8 ml of medium. After centrifugation at 1300 r.p.m. for 10 min the pellet was resuspended in 1 ml of medium for assessment of cell number using a haemocytometer (modified from Lee et al., 1997).
VEGF assay
VEGF concentration in follicular fluid was quantified using a commercial enzyme-linked immunosorbent assay (R&D Systems, UK). The intra-assay coefficient of variation (CV) of the assay was 6.2% and the inter-assay CV were 8.0, 5.7 and 9.5% at 100, 750 and 2400 pg/ml respectively. The assay recognizes VEGF165 as well as VEGF121 (major splice variants of VEGF-A) which are freely secreted isoforms.
Statistical analysis
Values are expressed as either mean ± SD where data were found to be normally distributed, or median (interquartile range; IQR) where data were found not to be normally distributed. Means were compared using the unpaired Students t-test and medians were compared using the MannWhitney test. Statistical significance was defined as P 0.05.
Written consent was obtained from each patient prior to commencing the study which was approved by the East London and The City Health Authority Research Ethics Committee on 18th February 1999 (Study number: P/98/222).
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Results |
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Discussion |
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VEGF expression by luteinized granulosa cells has been demonstrated in vitro with the extent of VEGF mRNA expression differing widely between individual patients (Yan et al., 1993). Certainly, we observed wide variations in follicular fluid levels of VEGF in follicles of the same size both in different patients and in the same patient, reflecting the unique and individual composition of each follicular environment. What was clear, despite these wide variations, was that the VEGF levels in the follicular fluid of follicles from the coasted group were consistently lower than in the control group. Whilst Agrawal et al. (1999
) and Artini et al. (1998
) found increased levels of VEGF in follicular fluid to be predictive of the development of OHSS, others have found that women at risk of developing OHSS have significantly lower levels of VEGF in follicular fluid and that lower levels are associated with good prognosis patients or hyper-responders (Friedman et al., 1998
; Pellicer et al., 1999
; Quintana et al., 2001
). It is hypothesized that withholding gonadotrophin reduces the functioning granulosa cell mass available for luteinization with the effect of decreasing any chemical mediators involved in the pathophysiology of OHSS. The lower levels of follicular fluid VEGF observed in the coasted group may be because gonadotrophin withdrawal has resulted in decreased granulosa cell production of VEGF or may reflect the level of follicular fluid VEGF before coasting since follicular fluid represents a composite of granulosa cell VEGF secretion over several days and may not entirely be representative of the functional capacity of the granulosa cells at the time of oocyte aspiration. The latter explanation seems more likely since the follicular fluid VEGF levels in the coasted group are well below those observed in the control group, and our findings therefore would be consistent with those of Pellicer et al. (1999
) and Quintana et al. (2001
). Coasting undoubtedly has an effect on follicular fluid VEGF levels with the loss of the correlation between follicle diameter and follicular fluid VEGF that is observed in the control group. There is also a much wider scatter of follicular fluid VEGF levels in the coasted group. Coasting probably affects all follicle sizes with differing effects on follicular fluid VEGF, reflecting the unique and individual nature of the follicles which show a variation in their susceptibility to gonadotrophin withdrawal. Interestingly, the type of gonadotrophin used had an impact on the follicular fluid VEGF concentration in the coasted group, with urinary products having significantly higher follicular fluid VEGF concentrations. This probably reflects the bioavailability of gonadotrophin used during the days of withdrawal of stimulation and may imply that urinary products would be better used in women suspected to be at high risk of OHSS. This aspect needs further evaluation.
VEGF concentration in follicular fluid is said to be dependent on the quality and number of granulosa cells responding (Van Blerkom et al., 1997). In both groups we observed a negative correlation between follicular fluid VEGF and granulosa cell number which was independent of follicle size. Greater granulosa cell numbers have been associated with more competent follicles (McNatty et al., 1979
) and lower follicular fluid VEGF levels with more oocytes (Friedman et al., 1998
) and better embryo quality (Barroso et al., 1999
). The fact that this correlation was more significant in the coasted group may be due to the differential effect of gonadotrophin withdrawal on individual follicles in favour of those follicles with greater numbers of granulosa cells/most competent.
It should be noted, however, that when granulosa cells are collected as part of the oocyte retrieval process, the cell numbers isolated from individual follicles are not the true numbers of cells within the follicle. It may be that in the smaller follicles greater shearing forces are present, thus mechanically increasing the number of cells retrieved. Interpretation of cell number has, therefore, inherent problems that are difficult to resolve. However, since each follicle has been aspirated in the same way by the same operator we hope we have allowed a comparative analysis to be made with data that is as reproducible and reliable as possible.
Like Lee et al. (1997), we found a positive correlation between follicular fluid VEGF levels and follicular fluid progesterone at the time of oocyte retrieval. This was observed in both groups. However, unlike Lee et al. (1997
), we also found a positive correlation between follicular fluid VEGF levels and follicular fluid estradiol levels in the control group. This was not seen in the coasted group. It is known that oocytes are lost during coasting, probably as a consequence of apoptosis of the granulosa cells followed by atresia of the oocyte. We might expect, therefore, to see higher VEGF levels in the follicular fluid of coasted patients in follicles from which an oocyte was not retrieved compared to follicles from which an oocyte was retrieved. We do see higher levels but the differences were not found to be statistically significantly different. Unlike Friedman et al. (1998
), we found no association between follicular fluid VEGF levels and fertilization in either group. Interestingly, we did find significantly lower levels of VEGF in follicular fluid from which oocytes were recovered which fertilized and cleaved to form grade 1 embryos compared to grade 2 and 3 embryos in the coasted group but not in the control group.
In conclusion, this study does not confirm or refute VEGF as being important in the pathophysiology of OHSS but does establish that follicular fluid VEGF concentrations, in highly responsive women who have undergone coasting, are significantly lower than the control group of women studied. Due to the study design, we cannot say whether the differences observed were secondary to the coasting process or typical of women at high risk of OHSS or a combination of both. Since none of the women developed OHSS despite lower levels of VEGF in the follicular fluid, withholding gonadotrophin could decrease the functional capacity of the granulosa cells to produce VEGF.
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Acknowledgements |
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References |
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Submitted on July 31, 2003; accepted on October 30, 2003.
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