Department of Obstetrics and Gynecology, National Taiwan University College of Medicine and Hospital, 7 Chung-Shan South Road, Taipei, Taiwan
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Abstract |
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Key words: cytokine/follicular fluid/ovarian hyperstimulation syndrome/prediction/vascular endothelial growth factor
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Introduction |
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Accurate prediction of the likelihood of development of OHSS in an individual treatment cycle remains a difficult task (Mathur et al., 1996). It is increasingly recognized that traditional determinants [e.g. serum oestradiol concentration on the day of human chorionic gonadotrophin (HCG) administration and the number of oocytes retrieved] do not adequately define the risks for this syndrome and do not accurately predict its occurrence (Morris et al., 1995
; Chen et al., 1997
). The potential value of cytokine measurements (local and systemic) as complements to hormone tests in the prediction of OHSS has not been fully investigated. Ideally, this prediction is best made prior to the administration of the ovulatory HCG trigger, allowing cancellation of truly high-risk treatment cycles. Previous work (Loret de Mola et al., 1996
; Lee et al., 1997
) has shown that pre-ovulatory serum concentrations of IL-6, IL-1 receptor antagonist, TNF-
, and VEGF cannot predict the occurrence of OHSS.
Because the onset of early-form OHSS (moderate or severe) always presents several days after HCG administration (Lyons et al., 1994), if changes of these mediators occurred between the days of HCG administration (pre-ovulatory) and embryo transfer, these may be used to predict more specifically which patients will develop this complication.
The present study was undertaken to examine the possible role of serum and follicular fluid concentrations of pro-inflammatory cytokines (IL-6, IL-8, TNF-) and VEGF as predictors of OHSS. Simultaneous assessment made it possible to determine whether interactions occurred between these mediators. This study was performed to facilitate prediction of the clinically important form of OHSS (i.e. moderate/severe) because it is this form of OHSS that is responsible for the considerable morbidity associated with ovarian stimulation (Golan et al., 1989
).
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Materials and methods |
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The study group was comprised of 12 patients who developed early-form moderate (n = 7) or severe (n = 5) OHSS requiring hospitalization. The two control groups were comprised of a randomized selection of 12 high-risk women and 12 low-risk women in whom OHSS did not develop.
To define patients at high risk for the development of OHSS, the records of the women in the study group who subsequently developed moderate/severe OHSS were reviewed. Because it was revealed that the lowest serum oestradiol concentration on the day of HCG administration in these cases was 1665 pg/ml (conversion factor to SI units, 3.671), this concentration was chosen as the threshold value for selection of control patients. Serum oestradiol concentration was chosen to define high-risk patients because this is the most commonly accepted risk factor for the development of OHSS (Delvigne et al., 1993).
Ovarian stimulation protocols
The ovarian stimulation protocols were performed as previously described (Chen et al., 1999). Briefly, pituitary desensitization was initiated using buserelin (Supremon®; Hoechst, Frankfurt, Germany) by a long protocol, which was administered by intranasal spray from the mid-luteal phase of the previous cycle at a dose of 200 µg, four times a day, until the day of menses. In the short protocol, buserelin was started on the second day of the treatment cycle and continued at a dose of 200 µg, given four times a day until the day before transvaginal aspiration of oocytes. Follicle stimulating hormone (FSH, Metrodin®; Serono, Rome, Italy) (150 IU/day) and human menopausal gonadotrophin (HMG, Pergonal®; Serono) (150 IU/day) were injected from cycle days 36 in the long protocol, while FSH and HMG were administered on cycle days 5 and 6 in the short protocol. Individualized injections of HMG were then continued until the administration of HCG (10 000 IU, Profasi®; Serono) when two or more leading follicles had reached a diameter of 18 mm. Transvaginal oocyte retrieval was performed 3436 h later.
In all patients, peripheral venous blood was drawn in the morning on the days of HCG administration, oocyte retrieval, and embryo transfer. Transvaginal oocyte retrieval was performed 3436 h after HCG administration. All embryo transfers were performed 2 days after oocyte retrieval. During oocyte retrieval, clear follicular fluid from individual follicles of 18 mm was collected and pooled in each patient. The follicular fluid was centrifuged immediately (1000 g), and the supernatant was stored, together with the serum samples, at 70°C before assay for cytokines and steroids.
Cytokine and hormone assays
Concentrations of IL-6, IL-8 and TNF- in serum and follicular fluid were measured with a solid-phase chemiluminescent enzyme immunoassay system (Immulite®; Diagnostic Products Corporation, Los Angeles, CA, USA). The sensitivity of these cytokine assays was 1 pg/ml for IL-6, 2 pg/ml for IL-8, and 1.7 pg/ml for TNF-
. Serum and follicular VEGF concentrations were quantified using an enzyme-linked immunosorbent assay (ELISA) (Quantikine®; R & D System, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. The sensitivity of the assay was 5 pg/ml. All samples were assayed at the same time to avoid interassay variations.
Serum and follicular fluid oestradiol and progesterone concentrations were assayed using a chemiluminescent immunoassay (Immulite®; Diagnostic Products Corporation). The intra-assay and interassay coefficients of variation for oestradiol at a concentration of 480 pg/ml were 6.3 and 6.4% respectively, and 6.3 and 5.8% for progesterone at a concentration of 7.2 ng/ml.
Statistical analysis
Statistical calculations were done using the Statistical Package for the Social Sciences (version 9.0, SPSS Inc., Chicago, IL, USA). For data that were not normally distributed, the MannWhitney U test was used if only two groups were being compared; the KruskalWallis one-way analysis of variance was used if more than two groups were being compared. For normally distributed data, analysis of variance was used. Spearman rank correlation was used to determine if there were correlations between variables. Results are expressed as means ± SD and means with 95% confidence intervals as appropriate. Statistical significance was defined as a value of P < 0.05.
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Results |
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Discussion |
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A previous study has shown that IL-6 ribonucleic acid is produced during follicular neovascularization (Motro et al., 1990). The potential role of IL-6 in ovulation could be to enhance vascular permeability. Elevated concentrations of IL-6 in ascites and serum of women with OHSS were found (Friedlander et al.,1993), suggesting its role as a marker of OHSS. In addition, it was found that elevated concentrations of IL-6 in the follicular fluid at the time of oocyte retrieval may predict the development of early-form OHSS in high responders (oestradiol concentrations >3000 pg/ml and/or 20 oocytes recovered) (Geva et al., 1997
). However, no comparison was made with normal responders in whom OHSS did develop. The results of the current study confirm the observation that increased follicular fluid IL-6 concentrations at the time of oocyte retrieval may indicate the early stages of OHSS in IVF patients. The study further expanded on the assay of circulating IL-6 concentration by showing that serum IL-6 concentrations differed significantly on the days of HCG administration in the OHSS group compared to the low risk group. There was no difference between the OHSS and high-risk group without OHSS. Serum concentrations of IL-6 were also correlated to oestradiol concentrations. It is of note that the statistical results for serum IL-6 concentrations on the days of HCG administration were on the lower detection concentrations for the assay and probably not clinically significant. This suggests the ovary may be the main resource of mediators that work as the initiators of the changes and lead to the full appearance of OHSS.
The findings of this study agree with previous studies (Krasnow et al., 1996; Geva et al., 1999
), where no difference in follicular fluid VEGF concentrations was detected between OHSS and controls. However, lower concentrations of VEGF were found in the follicular fluid of patients who were at risk for OHSS (Pellicer et al., 1999
). This finding is difficult to understand because excessive ovarian angiogenesis is a key factor involved in the syndrome. The authors speculated that the ovary might not be involved initially in the pathogenesis of OHSS. Rather, a systemic response to HCG seems plausible. Whether these opposite patterns of VEGF found in follicular fluid are representative of regulation of the cytokine by other cytokines or steroid hormones requires further study.
Although some studies (Abramov et al., 1997; Artini et al., 1998
; Pellicer et al., 1999
; Agrawal et al., 1999
) have established correlations between the development of OHSS and detectable circulating VEGF concentrations, the correlation is somewhat inconsistent. Pellicer et al. (1999) showed that there was a significant increase in serum VEGF concentrations after HCG administration in patients who were at risk for OHSS compared with those who were not at risk. Agrawal et al. (1999) reported that the increase in the VEGF concentration that occurred between the day of HCG administration and the day of oocyte retrieval (the `VEGF rise') was an important non-steroidal marker of OHSS. The VEGF rise predicted 40.3% of cases of moderate and severe OHSS, with no false-negative results. However, the serum VEGF concentrations as a single marker during the early follicular phase, on the days of HCG administration, oocyte retrieval and embryo transfer did not predict the development of OHSS. Ludwig et al. (1999) described a correlation between the values of free VEGF on the day of HCG and the occurrence of OHSS, but could not show this effect for the total VEGF. The divergent results may be explained by different patient groups with inclusion of even lower degree of OHSS and the small sample sizes in these studies. Another factor may be the kit used to measure VEGF concentration in serum or plasma (Ludwig et al., 1999
).
Moreover, Lee et al. (1997) recently reported that serum VEGF concentrations at the time of oocyte retrieval are not useful in predicting which patients undergoing IVF are at increased risk for the development of OHSS. Furthermore, Krasnow et al. (1996) did not find any significant difference in pre-ovulatory and on day 7 after the ovulatory dose of HCG plasma VEGF concentrations between women who subsequently developed OHSS and matched controls who did not. Thus, based on the work presented here and previous reports (Krasnow et al., 1996; Lee et al., 1997
; Ludwig et al., 1998
), circulating VEGF concentrations from the day of HCG administration up to the days of oocyte retrieval or embryo transfer do not appear to be a good marker or predictor of OHSS. The presence of other factors that interact with VEGF may be required for the development of severe OHSS (Geva et al., 1999
). Another possible explanation is that the VEGF may be involved, but it is not necessarily implicated in the early events of the syndrome when its action would be clinically relevant.
Interleukin-8, a cytokine with neutrophil chemotatic activity, is a potent angiogenic factor (Koch et al., 1992). Recently, it was found that IL-8 concentrations were significantly higher in peritoneal fluid in 12 patients with severe OHSS compared with 20 controls (Revel et al., 1996
), but no statistically significant difference was observed in the serum concentrations of patients and controls. The potential value of IL-8 measurements for predicting OHSS has not been fully investigated. The present results showed that a significantly higher serum concentration of IL-8 on the day of embryo transfer were observed in the OHSS group compared to the two control groups. Serum IL-8 concentrations may be a good parameter to predict an OHSS, even if it is only possible on the day of embryo transfer. However, it is of note that serum concentrations of IL-8 were also correlated strongly to serum oestradiol concentrations in this study.
Of interest are the relationships between the individual cytokines and steroid hormones in serum and follicular fluid. It is important to state that in many of its functions IL-6 probably acts in concert with other cytokines (Jones, 1994). Interleukin-6 also has been shown to induce the expression of VEGF in several cell lines (Cohen et al., 1996
). The data of the current study showed that serum IL-6 and IL-8 concentrations tend to increase with an increase in serum oestradiol concentration, and there is no correlation between serum VEGF and oestradiol concentrations. This indicates that a complex pattern of cytokines exists in serum and follicular fluid from patients with OHSS, suggesting the importance of `profiling' cytokines rather than monitoring the concentration of only a single type. It is speculated that the presence of other factors that interact with IL-6 and IL-8 may be required for the development of severe OHSS. The interrelations between the individual cytokines remain to be established in further studies, as does the feasibility of performing measurements in serum and follicular fluid in clinical practice.
This study had limitations. The present study design did not answer the question of whether serum IL-6 and IL-8 are independent risk factors, or just an epiphenomenona of high oestradiol values. A more reasonable study design would have been to obtain another control group without OHSS including patients who are matched to the group of patients with OHSS according to both age at oocyte retrieval and oestradiol values on day of HCG administration. Furthermore, we have no prospective experience with measurements of serum and follicular fluid, pro-inflammatory cytokines and VEGF in a different series of patients. Therefore, a prospective study is necessary to evaluate the proposed predictors of OHSS.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 16, 1999; accepted on February 4, 2000.