Hum. Reprod. Advance Access originally published online on January 29, 2004
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Human Reproduction, Vol. 19, No. 3, 522-528,
March 2004
© 2004 European Society of Human Reproduction and Embryology
The effects of ‘coasting’ on follicular fluid concentrations of vascular endothelial growth factor in women at risk of developing ovarian hyperstimulation syndrome
1 Williamson Laboratory, St Bartholomew’s and The London Hospitals Trust, London EC1A, 2 Centre for Reproductive Medicine, St Bartholomew’s 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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BACKGROUND: The aim of this study was to assess the effect of withholding gonadotrophins (coasting) during controlled ovarian stimulation (COS) on individual follicle concentrations of follicular fluid vascular endothelial growth factor (VEGF) in women at high risk of developing ovarian hyperstimulation syndrome (OHSS). METHODS: Twenty-two women who had been coasted and 26 optimally responding women (control group) undergoing COS for IVF were studied. At the time of oocyte retrieval, the follicular fluid from four to six individual follicles of different sizes was collected for VEGF analysis. RESULTS: A total of 118 follicles was analysed in the coasted group and 137 in the control group. A negative correlation was observed between the follicle size and VEGF concentration (r = –0.18, P = 0.03) in the control group, which was not seen in the coasted group. Similarly, the correlation between oestradiol (E2) and VEGF (r = 0.4, P < 0.0001) observed in the control group was not apparent in the coasted group. Significantly lower concentrations of VEGF were seen in the follicular fluid of the coasted patients. CONCLUSIONS: It is clear that there are differences in follicular fluid VEGF concentrations between the two groups. It is possible that coasting alters the capacity of the granulosa cells to produce VEGF and/or their response to hCG and in this way acts to reduce the severity and incidence of severe OHSS.
Key words: coasting/controlled ovarian stimulation/ovarian hyperstimulation syndrome/vascular endothelial growth factor
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Introduction |
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Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication of controlled ovarian stimulation (COS) with severe OHSS occurring in 0.6–1.9% of gonadotrophin-stimulated ovarian cycles (Smitz et al., 1990
). Although the pathophysiology of this syndrome has not been fully elucidated, it seems likely that the release of vasoactive substances secreted by the ovaries, following exposure to hCG, plays a key role in triggering this syn drome (Tsirigotis et al., 1994; Goldsman et al., 1995). Vascular endothelial growth factor (VEGF), a potent vasoactive protein, has been implicated as a major factor in the pathophysiology of OHSS as high concentrations of VEGF have been found in ascites from patients with OHSS (McClure et al., 1994), elevated serum VEGF levels have been demonstrated in patients who developed OHSS (Krasnow et al., 1996) and VEGF expression in granulosa cells obtained from women undergoing oocyte retrieval for IVF has been found to be enhanced by hCG (Ravindranath et al., 1991; Neulen et al., 1995).
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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Patients
Patients consisted of two groups of women; 22 women who had been coasted for the prevention of OHSS and 26 women undergoing COS for IVF with an optimum ovarian response seen on ultrasound on the day of hCG administration (control group). An optimum response was defined as at least six follicles
16 mm in diameter but <20 follicles in total, with no evidence of endometriosis, ovulatory disorders or polycystic ovarian disease.
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% antibiotic–antimycotic 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 Student’s t-test and medians were compared using the Mann–Whitney 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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Clinical findings
Patient characteristics, ovarian stimulation and clinical outcome are shown in Tables I and II. No patient in either group developed moderate or severe OHSS. No significant differences were observed in the mean age, BMI, follicular phase FSH, LH or duration of infertility between the two groups. A significantly lower total dose of gonadotrophin was used for those women in the coasted group compared to the control group (1800 versus 2275 IU; P = 0.03) with significantly more follicles
14 and 15 mm developing (11.2 ± 7.3 and 22.8 ± 7.5 versus 6.5 ± 3.9 and 7.6 ± 2.8 respectively; P = 0.007 and P < 0.0001). No significant differences were observed between the serum levels of E2 and VEGF taken on the day of oocyte retrieval. Whilst a significantly lower fertilization rate was observed in the coasted group (47.1 versus 56.5%; P = 0.03), the number of embryos available for transfer was not significantly different and no differences were observed in the pregnancy rate per embryo transfer.
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Follicular fluid VEGF, follicle size and granulosa cell number
Follicular fluid was isolated and analysed for VEGF in 137 individual follicles in the control group and 118 follicles in the coasted group. Follicles ranged from 10 to 34 mm in diameter. In both groups a wide range of VEGF was seen for all follicle sizes both within the same woman and in different women. It is notable that for each follicle size, there was a trend for the median follicular fluid VEGF concentration to be lower in the coasted group. A negative correlation was seen between the follicle size and level of follicular fluid VEGF in the control group (r = –0.18; P = 0.03), which was not seen in those women who had been coasted (r = –0.003; P = 0.9). A much wider variation and hence scatter of VEGF levels was seen in the coasted group compared to the control group (Figure 1). A negative correlation was observed between follicular fluid VEGF levels and the granulosa cell number in both the coasted group and the control group (r = –0.3, P = 0.004; r = –0.2, P = 0.04 respectively) (Figure 2). There was no correlation seen between the granulosa cell number and follicle diameter in either group (coasted group: r = 0.1, P = 0.3; the control group: r = 0.06, P = 0.5).
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Correlation between follicular fluid VEGF, follicular fluid progesterone and follicular fluid E2
Both groups showed a significant correlation between follicular fluid VEGF and follicular fluid progesterone levels (coasted group: r = 0.3, P = 0.0008; control group: r = 0.3, P = 0.0004) with the scatter of VEGF levels once again being greater in the coasted group. A positive correlation was seen between follicular fluid VEGF and follicular fluid estradiol levels in the control group (r = 0.4, P < 0.0001), which was not apparent in the coasted group (r = 0.1, P = 0.2) (Figure 3).
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Follicular fluid VEGF, oocyte retrieval, fertilization and embryo quality
In the coasted group, 62 oocytes were retrieved from the 118 follicles analysed (52.5% retrieval rate), with the outcome of four oocytes being lost to follow-up. Of the remaining 58 oocytes, 34 fertilized to the two-pronuclear (2PN) stage (58.6%), three fertilized abnormally (5.2%) and 21 failed to fertilize (36.2%). In the control group, 101 oocytes were retrieved from the 137 follicles studied (retrieval rate of 73.7%), the outcome of four oocytes being lost to follow-up. Of the remaining 97 oocytes, 50 fertilized to the 2PN stage (51.5%), 13 fertilized abnormally (13.4%) and 34 oocytes failed to fertilize (35.1%). No significant differences were observed, in either group, in the follicular fluid VEGF levels of follicles from which an oocyte was retrieved or not (Table III) or if the oocyte fertilized normally, abnormally or failed to fertilize. The only significant finding was that in the control group, normal fertilization was associated with a greater follicle diameter than failed fertilization (20.6 ± 5.6 versus 17.3 ± 5.0 mm, P = 0.01) which was not observed in the coasted group. Significantly lower follicular fluid VEGF concentrations were observed in the coasted group compared with the control group whether an oocyte was retrieved or not (Table III) and whether the oocyte fertilized to 2PN or failed to fertilize [398.5 (205.7–1294.3) versus 1032.8 (776.7–1809.2) pg/ml; P = 0.0005 and 417.0 (173.1–2945.3) versus 1159.2 (745.8–1628.5) pg/ml; P = 0.005 respectively]. Significantly lower levels of follicular fluid VEGF were seen in the coasted group with grade 1 embryos compared to grade 2 and 3 embryos [335.6 (196.4–462.9) versus 1150.3 (308.4–2618.0) pg/ml, P = 0.05, Table IV].
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Follicular fluid VEGF and gonadotrophin used for COS
In the control group, no differences were observed in follicular fluid VEGF concentrations if pure FSH or FSH and LH were used for COS [1144.2 (693.6–2145.5) versus 1101.1 (761.5–1747.4) pg/ml, P = 0.9 respectively]. However, in the coasted group, significantly lower concentrations of follicular fluid VEGF were observed where pure FSH had been used compared to FSH and LH [340.2 (196.4–714.4) versus 935.1 (300.9–2267.3) pg/ml].
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Discussion |
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This is the first study, to our knowledge, that evaluates the effect of withholding gonadotrophins during COS in women at risk of developing OHSS on follicular fluid levels of VEGF in individual follicles of varying sizes in relation to the granulosa cell number, oocyte retrieval, fertilization and embryo quality. Whilst the ideal control group of women would have been those identified as at risk of developing OHSS but not coasted, this was not ethically possible since coasting has been successfully used to prevent severe OHSS in our unit for a number of years (Al-Shawaf et al., 2001
). We therefore selected a group of optimally responding women that excluded all poor and hyper-responders.
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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The authors would like to thank The Joint Research Board, St Bartholomew’s Hospital for funding this study.
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References |
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Submitted on July 31, 2003; accepted on October 30, 2003.
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