C-reactive protein levels in patients undergoing controlled ovarian hyperstimulation for IVF cycle

Raoul Orvieto1, Roni Chen, Jacob Ashkenazi, Avi Ben-Harush, Jacob Bar and Benjamin Fisch

Department of Obstetrics and Gynecology, Rabin Medical Center, Petah Tiqva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Rabin Medical Center, Petah Tiqva 49 100, Israel. e-mail: orvieto{at}clalit.org.il


    Abstract
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 Abstract
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 Patients and methods
 Results
 Discussion
 References
 
BACKGROUND: The aim of the present study was to determine serum and follicular fluid C-reactive protein (CRP) levels in patients undergoing controlled ovarian hyperstimulation (COH) for IVF-embryo transfer cycle, and their possible correlation to COH variables. PATIENTS AND METHODS: The subjects were 16 consecutive patients undergoing our routine IVF long GnRH agonist protocol. Blood was drawn three times during the COH cycle: (i) the day on which adequate suppression was obtained (Day-S); (ii) the day of, or prior to HCG administration (Day-HCG); and (iii) the day of (and before) oocyte pick-up (Day-OPU). Levels of sex steroids and serum and follicular fluid CRP were compared among the three time points. Serum and follicular fluid CRP were measured with a commercial immunoturbidimetric assay. RESULTS: Serum levels of CRP were significantly higher on Day-OPU and Day-HCG than on Day-S, and significantly higher on Day-OPU than on Day-HCG. No difference was observed between follicular and serum CRP levels on Day-OPU. No significant correlations were found between serum and follicular fluid CRP, or between serum CRP-to-BMI ratio and serum sex steroid levels or IVF treatment variables. CONCLUSIONS: The significant increase in serum CRP levels during COH, especially after HCG administration, suggests that COH potentiates a state of systemic inflammation.

Key words: BMI/C-reactive protein/follicular fluid/ovulation induction/sex steroids


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
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Controlled ovarian hyperstimulation (COH) is apparently a key factor in the success of IVF-embryo transfer. One of the major complications of COH is severe ovarian hyperstimulation syndrome (OHSS), which is characterized by marked ovarian enlargement and acute third-space fluid sequestration that almost always develops after HCG administration or in early pregnancy. OHSS is similar to vascular leak syndrome (Orvieto et al., 1995Go), which may be attributable to the massive increase in systemic inflammatory cytokines observed during the course of severe OHSS, or to neutrophil activation (Carey et al., 1997Go). In earlier studies, our group found that neutrophil and endothelial activations are significantly stimulated by HCG administration (Orvieto et al., 2000Go; 2001Go).

C-reactive protein (CRP) is a biological marker of systemic inflammation, produced by the liver. It was recently demonstrated to have strong prognostic value for cardiovascular events (Ridker et al., 2002Go). CRP levels have no diurnal variation, are stable over long periods (Meier Ewert et al., 2001Go) and increase after estrogen administration (Kluft et al., 2002Go). The aim of the present prospective study was to longitudinally investigate serum and follicular fluid CRP levels during different time points of COH, and to determine whether they correlate with serum sex steroid levels or other COH variables.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The study population consisted of 16 consecutive patients attending the IVF unit of our department for treatment of infertility [anovulatory (n = 4), male factor (n = 5), unexplained (n = 5), mechanical (n = 2)]. The study required no modification of our routine human IVF protocols. Briefly, patients were pretreated with GnRH agonist in the mid-luteal phase using a long protocol. Fifteen days later, when adequate suppression was obtained, the patients underwent ovarian stimulation with HMG. The gonadotropin dosage was adjusted individually according to serum estradiol (E2) levels and vaginal ultrasound measurements of follicular diameter, obtained every 1 or 2 days. HCG 10 000 IU was administered when the leading follicle reached a minimum of 18 mm diameter, with a peak serum E2 level of >2000 pmol/l. Oocytes were aspirated by the transvaginal ultrasonographic route ~34 h after HCG injection. We use pooled clear aspirated follicluar fluid from each patient in an attempt to assess whole ovarian CRP production, rather than to evaluate each follicle separately (Orvieto et al., 1995Go). The follicular fluid was collected, centrifuged for 10 min at 1000 g, and then stored in aliquots at –20°C until assayed.

For the purposes of the study, in addition to the routine monitoring during the COH cycle, blood samples were drawn to determine the hormonal profile (E2, progesterone and HCG) and serum CRP levels at three time points: (i) the day on which adequate suppression was obtained (Day-S); (ii) the day of or prior to HCG administration (Day-HCG); and (iii) the day of (and before) oocyte pick-up (Day-OPU). For serum CRP determination, blood samples were centrifuged for 10 min at 1000 g, and the plasma was stored in aliqouts at –20°C until assayed.

Serum and follicular fluid concentrations of CRP were determined with a high-sensitivity immunoturbidimetric assay (Roche Diagnostics Corporation, Indianapolis, IN, USA) using an automated clinical chemistry analyser. The intra-assay and interassay coefficients of variation were 1.0% and 2.9%, respectively. Blanks and controls were included in all experiments.

Informed consent was obtained from all patients before participation in the study, and the study was approved by the Clinical Research Committee.

The results are expressed as means ± SD. The statistical analysis was performed with paired Student’s t-test and correlation analysis. P-values of ≤0.05 were considered significant.


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 Patients and methods
 Results
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The mean age of the study group was 35 ± 5.2 years, and mean BMI was 25.4 ± 4.1 kg/m2. The mean number of gonadotropin ampoules used during the COH cycle was 37.2 ± 14.4, mean number of oocytes retrieved was 13.9 ± 8.9, and mean fertilization rate was 59 ± 24%. The pregnancy rate was 31.25%.

Mean serum E2, progesterone and CRP levels on Day-S, Day-HCG and Day-OPU, and follicular fluid CRP level on Day-OPU, are presented in Table I. As expected, serum E2 and progesterone levels were significantly higher on Day-OPU than Day-S (P < 0.01 for both). The serum E2 level was significantly higher on Day-HCG than Day-OPU (P < 0.01), whereas serum progesterone was significantly lower (P < 0.01).


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Table I. Hormone profile and CRP levels during COH (n = 16)
 
Serum CRP levels were significantly higher on Day-OPU and Day-HCG than on Day-S (P < 0.0006 and 0.008, respectively), and significantly higher on Day-OPU than on Day-HCG (P < 0.004) (Figure 1). Follicular fluid CRP level did not differ from serum level on Day-OPU (P < 0.07). No significant correlations were observed between serum and follicular fluid CRP, or between the serum CRP-to-BMI ratio and E2 or progesterone level. The CRP-to-BMI ratio was chosen for comparison because of the known significant correlation between CRP levels and body weight (Tchernof et al., 2002Go). There were no significant correlations between serum CRP level and patient age, cause of infertility, amount of gonadotropins, number of oocytes retrieved, or fertilization or pregnancy rates.



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Figure 1. Individual serum CRP levels of patients during COH. Day-S = day on which adequate suppression was achieved; Day-HCG = day of or prior to HCG administration; Day-OPU = day of (and before) oocyte pick-up.

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The present study shows that serum CRP is significantly increased during COH until the peak E2 level, with a further significant increase after HCG administration. No significant difference was observed between serum and follicular fluid CRP levels, and no correlation was found between serum CRP and serum sex steroid levels, IVF treatment variables or pregnancy rate.

To the best of our knowledge, the present study is the first to provide information on CRP levels during COH. We observed a 60% increase in CRP levels from Day-S to Day-HCG, which is in accordance with the positive effect of E2 level on CRP level (Kluft et al., 2002Go; Tchernof et al., 2002Go). The increase in CRP is known to be coincident with an increase in other acute-phase proteins (fibrinogen, ceruloplasmin, von Willebrand factor originating from the liver and vessel walls) (Kluft et al., 2002Go). This observation is also in agreement with our previous in vitro study (Orvieto et al., 2003Go) on cytokine production by peripheral lymphocytes of patients undergoing COH, wherein whole-blood culture interleukin-2 was found to be significantly increased during COH until peak E2. Moreover, the significant (30%) increase in CRP level on Day-OPU compared with Day-HCG, despite the decrease in E2 level, substantiates our previous studies, which showed that neutrophil and endothelial activations, as reflected by the observed increase in L-selectin and E-selectin levels, respectively, are significantly stimulated by HCG administration (Orvieto et al., 2000Go; 2001Go). Furthermore, CRP levels on Day-OPU rise significantly above the cutoff value of 0.5 mg/dl, which indicates the presence of an inflammatory process (Thomas, 1992Go).

The present finding of an observed increase in serum CRP, which reflects a state of systemic inflammatory response, may further substantiate the role of systemic inflammation in the pathophysiology of OHSS. It is in line with the suggestion of our group in an earlier work that hyperstimulated human ovaries may contain an as yet undetermined factor that might cause OHSS (Orvieto et al., 1995Go). Moreover, the absence of a correlation between CRP and E2 levels supports our previous conclusion that the occurrence of different, unrelated ovarian responses to COH could account for both the ovarian enlargement and excessive steroidogenesis, and the release of the intermediate factor, causing an increase in capillary permeability (Orvieto, 2003Go).

Additional studies are required to elucidate the role of COH in systemic inflammatory response and its relation to the pathophysiology of OHSS. These may ultimately lead to new strategies in the prevention and treatment of complications of COH.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Carey PD, Wakefield CH and Gulillou PJ (1997) Neutrophil activation, vascular leak toxicity, and cytolysis during interleukin-2 infusion in human cancer. Surgery 122,918–926.[ISI][Medline]

Kluft C, Leuven JA, Helmerhorst FM and Krans HM (2002) Pro-inflammatory effects of oestrogens during use of oral contraceptives and hormone replacement treatment. Vascul Pharmacol 39,149–154.[CrossRef][ISI][Medline]

Meier Ewert HK, Ridker PM, Rifai N, Price N, Dinges DF and Mullington JM (2001) Absence of diurnal variation of C-reactive protein levels in healthy human subjects. Clin Chem 47,426–430.[Abstract/Free Full Text]

Orvieto R (2003) Prediction of ovarian hyperstimulation syndrome: challenging the estradiol mythos. Hum Reprod 18,665–667.[Abstract/Free Full Text]

Orvieto R, Voliovitch I, Fishman P and Ben-Rafael Z (1995) Interleukin-2 and ovarian hyperstimulation syndrome – a pilot study. Hum Reprod 10,24–27.[Abstract]

Orvieto R, Schwartz A, Bar Hava I, Abir R, Ashkenazi J, La Marca A and Ben Rafael Z (2000) Controlled ovarian hyperstimulation – a state of endothelial activation. Am J Reprod Immunol 44,257–260.[CrossRef][ISI][Medline]

Orvieto R, Ben-Rafael Z, Schwartz A, Abir R, Fisch B, La Marca A and Bar Hava I (2001) Soluble L-selectin levels during controlled ovarian hyperstimulation. Gynecol Endocrinol 15,29–33.[ISI]

Orvieto R, Elites T, Abir R, Bar J, Yoeli R, Feldberg D and Fisch B (2003) Interleukin-2 production in whole blood cell cultures of women undergoing controlled ovarian hyperstimulation for assisted reproductive technology cycles. Am J Reprod Immunol 50,220–223.[CrossRef][ISI][Medline]

Ridker PM, Rifai N, Rose L, Buring JE and Cook NR (2002) Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med 347,1557–1565.[Abstract/Free Full Text]

Tchernof A, Nolan A, Sites CK, Ades PA and Poehlman ET (2002) Weight loss reduces C-reactive protein levels in obese postmenopausal women. Circulation 105,564–569.[Abstract/Free Full Text]

Thomas L (1992) Labor und Diagnose, 4th edn. Die Medizinische Verlagsgesellschaft, Marburg, Germany, p. 781.

Submitted on August 7, 2003; resubmitted on September 30, 2003; accepted on October 27, 2003.