1 Department of Obstetrics and Gynaecology, University of Berne and 2 Central Laboratory, Inselspital, Berne, Switzerland
3 To whom correspondence should be addressed at: Universitäts-Frauenklinik Inselspital, Effingerstrasse 102, Berne, CH-3010, Switzerland. Email: dorothea.wunder{at}insel.ch
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Abstract |
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Key words: C-reactive protein/follicular fluid/IVF/leptin/serum
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Introduction |
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C-reactive protein (CRP) is an acute phase protein and is produced by the liver. Several studies have found that body mass index (BMI) is strongly associated not only with leptin but also with CRP levels (Mendall et al., 1996; Ford, 1999
; Visser et al., 1999
; Yudkin et al., 1999
). CRP was found to be increased after estrogen administration (Kluft et al., 2002
). An association between plasma CRP and leptin has been shown in humans (Shamsuzzaman et al., 2004
) and Bullo et al. even demonstrated that leptin could induce the production of CRP (Bullo et al., 2003
). Maruna et al. identified leptin as an acute phase reactant with potential haematopoietic, immunomodulatory and hepatocyte stimulating activity and also found a significant correlation between CRP and leptin (Maruna et al., 2001
). The available results on the usefulness of CRP concentrations as a marker of success in assisted reproductive technologies (ART) are controversial. In the study of Sacks et al., CRP levels have been measured before IVF stimulation and 14 days after egg collection. Women pregnant after IVF, especially pregnant women after ovarian hyperstimulation had significantly higher CRP levels compared to non-pregnant women 14 days after egg collection (Sacks et al., 2004
). Almagor et al. found serum CRP levels, measured on the day of embryo transfer to be correlated with the outcome of in vitro fertilization (IVF)-treatment (Almagor et al., 2004
), while Orvieto et al. (2004a)
did not.
Ovulation is an inflammatory process, and the interactions and correlations of leptin and CRP are thus of great interest in this context. To our knowledge only a few studies on CRP in assisted reproduction, and no data comparing CRP with leptin and steroids in this field, are available in the literature.
The aim of the present study was to examine whether leptin and CRP, which are both involved in inflammatory processes, are correlated with each other and whether they could be an outcome marker in IVF treatment.
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Materials and methods |
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For ovarian stimulation, the long protocol was used. With the agonist Triptorelin, 0.1 mg per day s.c. (Decapeptyl®, Ferring pharmaceuticals, Wallisellen, Switzerland), downregulation was performed either in the luteal phase of the previous or on the first day of the treatment cycle until complete pituitary desensitization was documented. After sonographic evidence indicated no ovarian follicular activity and serum levels of human chorionic gonadotrophin (hCG) and estradiol were below 2 mIU/ml and 130 pmol/l, respectively, stimulation was initiated in most cases with recombinant FSH (Gonal-F®, Serono Pharma, Geneva, Switzerland or Puregon®, Organon Pharmaceuticals, Pfäffikon, Switzerland) or with menopausal gonadotrophin (hMG, Pergonal®, Serono or Humegon®, Organon). The starting dose was adapted according to age and ovarian response; usually it was 150225 IU daily and for patients with an already known low response it was maximally 300450 IU daily. Ultrasound and serum estradiol measurements were performed to assess follicular maturation; hCG (5000 to 10 000 IU, Profasi®, Serono or Pregnyl®, Organon) was administered when at least three follicles exceeded 17 mm in diameter and the estradiol concentration per mature follicle exceeded 1000 pmol/l. 3536 h after the administration of hCG, the fluid containing the oocytes was retrieved from all follicles by needle aspiration, with transvaginal ultrasound guidance and under routine intravenous sedation.
Hyaluronidase denuded oocytes were assessed for maturity. Only metaphase II oocytes, identified by the presence of the first polar body, were chosen for fertilization. ICSI was performed 36 h after oocyte recovery by using previously described techniques and instrumentation (Tesarik and Sousa, 1995). Fertilization was assessed 17 h after ICSI. One to two normally fertilized oocytes (two pronuclei and two polar bodies) with the highest PN score and with the best morphological grade were considered for embryo transfer. Cryopreservation of the remaining PN was performed at this stage according to the Swiss law. The fertilized oocytes selected for transfer were cultured for another 2030 h at 37 °C in fresh CO2 equilibrated IVF medium (Vitrolife®, Göteborg, Sweden). Embryo development was evaluated 2 and 3 days after ICSI by determining the number of blastomeres and the relative proportion of anucleate cell fragments. Embryo scores were calculated by multiplying the fragmentation grade (<10% fragments, grade 4; 1020%, grade 3; 2030%, grade 2; >30% fragments, grade 1) with the number of blastomeres. In most cases two embryos (range 13) were transferred 2 or 3 days after oocyte retrieval. The Cumulated embryo score (CES) was calculated by adding up the individual scores of the transferred embryos for each patient.
Collection of serum and follicular fluid
Blood samples were drawn at three points: on the day of initiation of ovarian stimulation, on the day of or one day before hCG administration, and on the day of the oocyte pick-up, withdrawn immediately before the procedure. Only cycles with an available serum from all three time points were included in the study. Sera were prepared in the central routine laboratory and stored, after the immediate determination of estradiol, at 30 °C until assayed for the other analytes (see below) in batches.
Follicular fluid samples from individual follicles were pooled and centrifuged for 10 min at 500 g and the supernatants were stored at 30 °C until analysed further. Fractions of FF with massive blood contamination were excluded.
Measurement of steroid hormones, leptin and CRP in serum and follicular fluid
Serum 17-estradiol (E2) was assayed on the day of collection in the routine laboratory using an automated method (Elecsys, Roche, Basle, Switzerland). Progesterone was determined by competitive radio-immunoassay (RIA), using the coated tube (Coat-a-Count) kit manufactured by DPC, Los Angeles, CA, and obtained from Buhlmann Laboratories, Basle, Switzerland. Sera, obtained at the day of OPU, were used without dilution while the follicular fluids were diluted 1:500 in the diluent provided, and the 3 h incubation protocol was followed. Estradiol in the follicular fluids was similarly determined with the same RIA methodology, in an 1:250 dilution and following the protocol supplied by DPC.
Leptin was quantified with an ELISA developed in our laboratory using monoclonal matched-pair (capture and detection) antibodies obtained from R&D Systems, Abingdon, England. The recently described method (Malek et al., 2001) was followed with the exception that the samples (1:50 dilution for both sera and FF) and standards (100015.6 pg/ml in serial 1:2 steps) were incubated in RD5P medium (R&D Systems Europe, Abingdon, England). Subsequent incubations were in PBS containing 0.5% non-fat milk proteins (Blotto®, Pierce USA, obtained from Socochim, Lausanne, Switzerland) as described. The intra- and inter-assay coefficients of variance at midstandard level (125 pg/ml) were 2.7 and 8.9%, respectively.
CRP was determined by microplate ELISA in our laboratory as follows: rabbit polyclonal anti-human CRP (Sigma C3527) was added at 2 µg/ml in PBS (incubation volume = 100 µl/well) for overnight coating at 4 °C, then the contents were aspirated and the excess sites blocked with 250 µl BSA (0.5% w/v in PBS) during 1 h at 37 °C. After a washing step (two aspiration cycles using PBS containing Tween-20, Sigma, 0.1% v/v), standards (CRP Sigma C4064, 1000.39 ng/ml) or serum/FF samples (diluted in Blotto©) were added (100 µl/well) and the plates incubated for 2 h at 30 °C with a shaking speed of 500 r.p.m. Initial sample dilution was 1:250, but it was necessary to repeat some measurements at higher or lower dilution. After washing the wells three times with PBST, horseradish peroxidase conjugated rabbit anti-human CRP (Dako P227, Glostrup, Denmark, diluted 1:10 000 in Blotto) was added and the incubation continued for 1 h under the same conditions. Then the wells were washed four times with PBST, and ready-to-use TMB substrate solution (Zymed, obtained from Staehelin, Basle, Switzerland; 100 µl/well) was added in a timed sequence. After 1020 min of incubation at room temperature in the dark, the reaction was stopped by the addition of 100 µl 2M hydrochloric acid in the same sequence. The optical density was determined with a dual channel microplate reader (Bio-Rad, Hercules, USA, model 550) at 450 nm against a reference at 590 nm. The sensitivity of the assay was 0.30 ng/ml, but the lowest CRP concentration measured in this study was 18 ng/ml. The intra- and inter-assay coefficients of variance in the geometric mean of the standard curve were 5.54 and 10.65%, respectively (Malek et al., submitted).
Statistical methods
Concentrations of leptin, CRP and steroids between the subsequently pregnant (n=47) and non-pregnant (n=102) groups were compared non-parametrically with the MannWhitney U test. The same was done, over the whole study population (n=162, including the 13 cycles not leading to embryo transfer for reasons of fertilization or cleavage failure, i.e. CES = 0), when the concentrations of these markers were compared between the different time points. Correlations between the different markers were assessed by linear regression after logarithmic transformation and non-parametrically by Spearman rank correlation analysis. A P-value of 0.05 or less was considered to identify a significant difference.
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Results |
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Discussion |
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In agreement to several other studies (Mendall et al., 1996; Ford, 1999
; Visser et al., 1999
; Yudkin et al., 1999
), we have found a positive correlation between CRP and BMI. In the other study on CRP relating to IVF (Almagor et al., 2004
), data on BMI were not given. In contrast to Orvieto et al. (2004a)
, we also found a significant (P<0.001) difference in CRP levels between follicular fluid and serum taken on the day of OPU, with the FF concentration being lower. We believe that, for both observations comparing our study with the one cited (Orvieto et al., 2004a
), statistical significance was reached in ours because of the higher number of cases included, since the trend was the same and the range of measured values was wide.
Our results concur with the other studies (Almagor et al., 2004; Orvieto et al., 2004a
) showing no difference in serum CRP levels on the day of ovarian pick-up (OPU) between women subsequently becoming pregnant and those who did not. Leptin was also found not to be a prognostic marker in this context, which is however in contrast to others who did observe lower leptin levels to be associated with treatment success (Mantzoros et al., 2000
; Tsai et al., 2002
). In our study the estradiol concentration was the only prognostic parameter that was increased in the serum (as well as in the follicular fluid.) without a lot of women who became pregnant over those who did not on the day of hCG administration or hCG-1 and at OPU.
We could confirm the association of CRP and leptin levels found by others (Maruna et al., 2001; Shamsuzzaman et al., 2004
), and we concur with the findings of Orvieto et al. (2004a)
who did not find any correlations between steroid hormones and CRP or between steroid hormones and leptin. Our findings are also in full agreement with the recently published study by Chen et al. (2004)
, with an absence of correlation between serum or follicular fluid leptin levels and gonadal steroid levels or IVF outcome. Regarding CRP, our results as well as the findings by Orvieto et al. (2004a)
showed a significant increase in concentration on day of OPU when compared to the day hCG/-1 in spite of the decrease in E2 levels at this stage.
To our knowledge no data comparing CRP with leptin and steroids in this field are available in the literature. Our study is therefore unique since we have demonstrated that leptin and CRP, which are both involved in inflammatory processes, showed a significant difference in their serum concentration time course during ovarian stimulation. In contrast to leptin levels, which continually rose during the ovarian stimulation phase until the day of hCG/hCG-1, the concentrations of CRP did not change between the first day of stimulation and the day of hCG/hCG-1. This latter finding is in contrast to the one by Orvieto et al. (2004a) who reported a significant increase in serum CRP levels during stimulation and concluded that the controlled ovarian hyperstimulation potentiated a state of systemic inflammation. But our observations are in line with the recent report by Orvieto (2004b)
where he described a significant increase in CRP level on the day of ovum pick-up compared to the day of hCG adminstration despite the decrease in E2 level, and where he speculated that the inflammatory response was significantly stimulated by the exogeneous hCG. Orvieto et al. also reported on another inflammatory marker, leukocyte selectin (L-selectin), which was even found to be decreased during controlled ovarian hyperstimulation until peak estradiol was reached, and then significantly increased after hCG administration (Orvieto et al., 2001
). It seems that the situation is quite complex. An explanation for the discrepancies in the time course of the two inflammatory mediators leptin and CRP could be that they might be unrelated to each other or that they follow different pathways in the inflammatory process.
It has to be noted that blood sampling for measuring CRP and leptin was obtained before oocyte collection was started, and thus no ovulation or stress induction had taken place. Moreover, the lower CRP concentration in the FF than in the serum is indicating an absent or very low production of this protein in the ovary, and there are no data on in vitro CRP production by granulosa cells in the literature.
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Conclusions |
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References |
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Barash IA, Cheung CC, Weigle DS, Ren H, Kabigting EB, Kuijper JL, Clifton DK and Steiner RA (1996) Leptin is a metabolic signal to the reproductive system. Endocrinology 137, 31443147.[Abstract]
Barroso G, Barrionuevo M, Rao P, Graham L, Danforth D, Huey S, Abuhamad A and Oehninger S (1999) Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril 72, 10241026.[CrossRef][ISI][Medline]
Brzechffa PR, Jakimiuk AJ, Agarwal SK, Weitsman SR, Buyalos RP and Magoffin DA (1996) Serum immunoreactive leptin concentrations in women with polycystic ovary syndrome. J Clin Endocrinol Metab 81, 41664169.[Abstract]
Bullo M, Garcia-Lorda P, Megias I and Salas-Salvado J (2003) Systemic inflammation, adipose tissue tumor necrosis factor, and leptin expression. Obes Res 11, 525531.
Chen R, Fisch B, Ben-Haroush A, Kaplan B, Hod M and Orvieto R (2004) Serum and follicular fluid leptin levels in patients undergoing controlled ovarian hyperstimulation for in vitro fertilization cycle. Clin Exp Obstet Gynecol 31, 103106.[Medline]
Cioffi JA, Van Blerkom J, Antczak M, Shafer A, Wittmer S and Snodgrass HR (1997) The expression of leptin and its receptors in pre-ovulatory human follicles. Mol Hum Reprod 3, 467472.[Abstract]
Ford ES (1999) Body mass index, diabetes and C-reactive protein among U.S. adults. Diabetes Care 22, 19711977.[Abstract]
Janik JE, Curti BD, Considine RV, Rager HC, Powers GC, Alvord WG, Smith JW 2nd, Gause BL and Kopp WC (1997) Interleukin-1alpha increases serum leptin concentrations in humans. J Clin Endocrinol Metab 82, 30833086.
Karamouti M, Kollia P, Karligiotou E, Kallitsaris A, Prapas N, Kollios G, Seferiadis K, Vamvakopoulos N and Messinis IE (2003) Absence of leptin expression and secretion by human luteinized granulosa cells. J Mol Endocrinol 31, 233239.
Karlsson C, Lindell K, Svensson E, Bergh C, Lind P, Billig H, Carlsson LM and Carlsson B (1997) Expression of functional leptin receptors in the human ovary. J Clin Endocrinol Metab 82, 41444148.
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, 149154.[CrossRef][ISI][Medline]
Löeffler S, Aust G, Koehler U and Spanel-Borowski K (2001) Evidence of leptin expression in normal and polycystic human ovaries. Mol Hum Reprod 12, 11431149.[CrossRef]
Lord GM, Matarese G, Howard JK, Baker RJ, Bloom SR and Lechler RI (1998) Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature 394, 897901.[CrossRef][ISI][Medline]
Malek A, Willi A, Muller J, Sager R and Bersinger N (2001) Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early pregnancy. J Assist Reprod Genet 18, 299304.[CrossRef][ISI][Medline]
Malek A, Bersinger NA, Di Santo S, Mueller MD, Schneider H, Ghezzi F, Passi A and Raio L (2005) Production of Placental C-reactive protein: Conflicting findings with mRNA vs. protein expression. Placenta, submitted 2005.
Mantzoros CS, Cramer DW, Liberman RF and Barbieri RL (2000) Predictive value of serum and follicular fluid leptin concentrations during assisted reproductive cycles in normal women and in women with the polycystic ovarian syndrome. Hum Reprod 15, 539544.
Maruna P, Gürlich R, Frasko R and Haluzik M (2001) Serum leptin levels in septic men correlate well with c-reactive protein (CRP) and TNF-alpha but not with BMI. Physiol Res 50, 589594.[ISI][Medline]
Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nishimura H, Yoshimasa Y, Tanaka I, Mori T and Nakao K (1997) Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 3, 10291033.[CrossRef][ISI][Medline]
Matarese G, Alviggi C, Sanna V, Howard JK, Lord GM, Carravetta C, Fontana S, Lechler RI, Bloom SR and De Placido G (2000) Increased leptin levels in serum and peritoneal fluid of patients with pelvic endometriosis. J Clin Endocrinol Metab 85, 24832487.
Mendall MA, Patel P, Ballam L, Strachan D and Northfield TC (1996) C reactive protein and its relation to cardiovascular risk factors: A population based cross sectional study. Br Med J 312, 10611065.
Orvieto R, Ben-Rafael Z, Schwartz A, 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, 2933.[ISI]
Orvieto R, Chen R, Ashkenazi J, Ben-Harush A, Bar J and Fisch B (2004a) C-reactive protein levels in patients undergoing controlled ovarian hyperstimulation for IVF cycle. Hum Reprod 19, 357359.
Orvieto R (2004b) Controlled ovarian hyperstimulation-an inflammatory state. J Soc Gynecol Investig 11, 424426.[CrossRef][ISI][Medline]
Sacks GP, Seyani I, Lavery S and Trew G (2004) Maternal C-reactive protein levels are raised at 4 weeks gestation. Hum Reprod 19, 10251030.
Saraff P, Frederich RC, Turner EM, Ma G, Jaskowiac NT, Rivet DJ 3rd, Flier JS, Lowell BB, Fraker DL and Alexander HR (1997) Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia. J Exp Med 185, 171175.
Shamsuzzaman AS, Winnicki M, Wolk R, Svatikova A, Phillips BG, Davison DE, Berger PB and Somers VK (2004) Independent association between plasma leptin and C-reactive protein in healthy humans. Circulation 109, 21812185.
Sierra-Honigmann MR, Nath AK and Murakami C (1998) Biological action of leptin as an angiogenic factor. Science 281, 16831686.
Tesarik J and Sousa M (1995) Key elements of highly efficient intracytoplasmatic sperm injection technique: Ca+ fluxes and oocyte cytoplasmic dislocation. Fertil Steril 64, 770776.[ISI][Medline]
Tsai EM, Yang CH, Chen SC, Liu YH, Chen HS, Hsu SC and Lee JN (2002) Leptin affects pregnancy outcome of in vitro fertilisation and steroidogenesis of human granulosa cells. J Assist Reprod Gen 19, 169176.[CrossRef][ISI][Medline]
Visser M, Bouter LM, McQuillan GM, Wener MH and Harris TB (1999) Elevated C reactive protein levels in overweight and obese adults. JAMA 282, 21312135.
Welt KR, Schneyer AL, Heist K and Mantzoros CS (2003) Leptin and soluble leptin receptor in follicular fluid. J Assist Reprod Gen 20, 495501.[CrossRef][ISI][Medline]
Yudkin JS, Stehouwer CD, Emeis JJ and Coppack SW (1999) C-reactive protein in healthy subjects: associations with obesity, insulin resistance, and endothelial dysfunction: a potential role for cytokines originating from adipose tissue? Arterioscler Thromb Vasc Biol 19, 972978.
Submitted on November 17, 2004; resubmitted on December 31, 2004; accepted on January 10, 2005.
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