1 Division of Clinical Laboratories and 2 Department of Obstetrics and Gynecology, Bikur Cholim Hospital, Jerusalem, Israel
3 To whom correspondence should be addressed at: Division of Clinical Laboratories, Bikur Cholim Hospital, 5 Strauss Street, Jerusalem 91004, Israel. e-mail: malmagor{at}bikurholim.org.il
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Abstract |
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Key words: C-reactive protein/cytokine/IVF/uterine receptivity
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Introduction |
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The active role of these cytokines in the process of implantation, on the one hand, and their effect on acute phase proteins and CRP generation, on the other, encouraged us to explore the hypothesis that the concentrations of CRP in blood of women undergoing IVF may reflect the outcome of treatment.
CRP is a pentameric protein (mol. wt 1.15 kDa) synthesized by liver hepatocytes in response to acute stimuli (Pepys et al., 2003). Its half-life in the circulation is
19 h, and it has been reported to reflect ongoing inflammation and/or tissue damage (Pepys et al., 2003
).
Since CRP is easily monitored in clinical laboratories and is considerably less expensive to assay in comparison with LIF and IL-1, we undertook a prospective study of women who underwent IVF treatment. The levels of serum CRP were evaluated from the day of oocyte retrieval, on embryo transfer day, during the implantation period and until the 12th day after embryo transfer.
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Materials and methods |
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Luteal phase support was provided by administration of progesterone (Gestone; Ferring, UK) 50100 mg/day (i.m.) starting on the day of oocyte retrieval.
Venous blood samples were obtained on admission for oocyte retrieval, on the day of embryo transfer (before procedures were performed), on day 5, 6 or 7 afterwards, as well as on day 12.
Serum was separated by centrifugation (3000 g, 10 min) and stored at 40°C. The serum samples were analyzed for CRP by immunoturbidometry using the Integra-700 chemistry analyzer (Roche Diagnostics, Switzerland). The normal values of CRP in our laboratory are <5 mg/l.
The study protocol was reviewed and approved by Bikur Cholim Hospital review board. Informed consent was given by all participants.
Statistical methods
CRP concentrations and ratios relative to baseline values at oocyte pick-up were compared between the pregnant and non-pregnant groups using the t-test because the skewness of the distribution of the smaller group was moderate. The use of pooled variances was determined following Levenes test for equality of variances. A discriminating cut-off value for the transfer/pick-up ratio was determined by the Classification and Regression Trees (CART) methodology (Steinberg et al., 1995). Statistical analysis was performed using SPSS for Windows (version 11) and CART (version 3.6.3). Data were considered statistically significant if P
0.05.
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Results |
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The characteristics of the study participants are presented in Table I. The mean age of the women who achieved pregnancy was not significantly different from that of the non-pregnant group (Table II). The mean concentrations of CRP in the sera of all participants were 6.8 ± 9.5 mg/l on the day of oocyte pick-up, 7.1 ± 7.3 mg/l on the day of embryo transfer and 14.6 ± 12.5 mg/l 57 days post-transfer. The 2-fold increase in CRP on days 57 was highly significant as compared to the first days of treatment (P < 0.0001). Parallel levels of CRP in conception and non-conception groups are summarized in Table III. Although CRP values did not change significantly between the two groups, they seemed higher on oocyte retrieval day in the pregnancy group (Table III). Therefore, we tested the assumption that the ratio of CRP rather than the actual daily concentration may significantly differ between successful and unsuccessful cycles. As shown in Table III, the mean of transfer/pick-up ratios was 1.2 in women who conceived versus 2.5 in the non-pregnant group (P = 0.01).
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Discussion |
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Although the hypothesis that successful implantation may be associated with a significant change in circulating levels of CRP has been negated, our findings indicate that the CRP ratio (embryo transfer/oocyte pick-up, Table III) was significantly lower in conception cycles as compared to unsuccessful cycles. These results may reflect the low-grade inflammation or proinflammatory effect of CRP in women who achieve pregnancy. It is plausible that the extent of the inflammatory response of the endometrium may play a role in establishing optimal receptivity. As CRP can interact with both humoral and cellular effector systems of inflammation, it seems that some degree of immunosuppression may be essential for proper maturation of the endometrium and embryo apposition.
Because CRP is rapidly synthesized (Pepys et al., 2003), it may be possible that its increase on transfer day reflects response to the actual procedure of oocyte retrieval. However, the observation that its relative increment was significantly higher in unsuccessful cycles (Table III) implies that intrinsic factors are involved in eliciting the acute reaction.
Previous studies evaluated the timing of implantation in natural conceptions (Wilcox et al., 1999) and in women treated by IVF (Liu et al., 1991, 1993). Implantation was detected 612 days after ovulation (Wilcox et al., 1999
) and 614 days after egg retrieval (Liu et al., 1991, 1993). Since we evaluated CRP only on days 79 after egg retrieval, the possibility that CRP levels further elevate on the following 3 days cannot be ruled out.
Brumsted et al. (1990) demonstrated that the tumor marker CA-125 is produced by the endometrium in naturally ovulating women. Because endometrial receptivity is an important factor in IVF pregnancy success, the concentrations of CA-125 as predictors of pregnancy before embryo transfer in IVF (Miller et al., 1996
; Chryssikopoulos et al., 1996
; Brandenberger et al., 1998
; Noci et al., 1999
) and ICSI (Tavmergen et al., 2001
) were determined. Some reports showed that increased CA-125 levels were associated with pregnancy (Miller et al., 1996
; Chryssikopoulos et al., 1996
; Tavmergen et al., 2001
), whereas others reported no prognostic significance (Brandenberger et al., 1998
; Noci et al., 1999
). Although the source of high serum CA-125 concentrations was not fully elucidated, Tavmergen et al. (2001
) have shown that women with CA-125 >10 IU/ml on the day of oocyte retrieval had very high pregnancy rates. Our attempt to determine a cut-off value for CRP ratio, which may serve as a predictive marker for the early phase of embryo implantation, resulted in acceptable sensitivity, but unsatisfactory specificity. It would be of interest to evaluate simultaneously both CRP and CA-125 in IVF practice. It seems reasonable to assume that in combination, CRP and CA-125 may provide a more sensitive indication of uterine receptivity.
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Acknowledgements |
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References |
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Submitted on March 10, 2003; resubmitted on August 21, 2003; accepted on September 17, 2003.