1 Department of Gynecology, 2 Sara Racine IVF Unit, Lis Maternity Hospital and 3 Internal Medicine D, all at Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel
4 To whom correspondence should be addressed at: Gynecology Department, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, 6 Weizman St, Tel-Aviv, 64239, Israel. e-mail: i-levin{at}barak-online.net
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: controlled ovarian stimulation/erythrocyte aggregation/ovarian hyperstimulation syndrome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many theories have evolved over the years regarding the aetiology of OHSS. These theories, at best, can only partly explain the full cascade of events leading to this clinical entity. Hyperoestrogenaemia as an example cannot be solely responsible for mediating OHSS, as patients with partial 17,20-desmolase deficiency with low estradiol levels were reported to have severe OHSS (Pellicer et al., 1991). Prostaglandins in high levels can only partly lead to OHSS, as indomethacin did not prevent the occurrence of OHSS (Pride et al., 1986
). Vascular endothelial growth factor (VEGF) has been assigned a significant role in mediating OHSS by its ability to induce vessel permeability (Levin et al., 1998
). VEGF receptor inhibitors demonstrated the capability of preventing the increase in vascular permeability brought about by the elevated levels of VEGF (Gomez et al., 2002
). Secretion of interleukin-6 by human lung microvascular endothelial cells was demonstrated to be increased following administration of hCG, causing an increase in vessel permeability (Albert et al., 2002
).
The common denominator of most theories has been endothelial activation and an increase in capillary permeability leading to fluid shifting to third spaces (Delvigne and Rozenberg, 2002).
An increase in erythrocyte aggregation is associated with unfavourable haemorrheological effects in terms of microcirculatory slow flow, tissue hypoxaemia and microcirculatory occlusions (Tateishi et al., 2001, 2002). These changes alter the peripheral resistance, reduce capillary perfusion and oxygen transfer to tissues and bring about a degeneration of the vascular wall leading to capillary leak (Rainer et al., 1987
; Soutani et al., 1995
; Cabel et al., 1997
; Pfafferott et al., 1999
; Bishop et al., 2001
).
Increased capillary leak brought about by the elevation of erythrocyte aggregation could be a newly discovered mediator in the pathophysiology of OHSS. In our present study we undertook to determine if women with COH and OHSS have increased red cell aggregation in their peripheral blood.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Twenty successive women from our IVF unit, undergoing COH from December 2001 to January 2002, were recruited for the study (group 2). The indications for IVF were male factor in five (25.0%) couples, mechanical factor in three (15.0%) women, unexplained in 11 (55.0%) women and lack of ovulation in one (5.0%) individual. COS in groups 1 and 2 was performed using down-regulation with GnRH analogues from day 1 of the cycle followed by gonadotrophin administration from cycle day 3 until the administration of hCG.
Twenty healthy control women (group 3) matched for age and body mass index were recruited for the study. Women from all groups were healthy and had no chronic disease. Coagulation disorders or past events of thrombosis were excluded.
Blood samples were drawn and prepared using the technique described below. Blood was collected from the hospitalized women (group 1) upon admission. Blood was drawn from the COH women (group 2) on the morning of day 1213 of the treatment cycle, after hCG administration.
Laboratory tests
Blood samples were analysed for cell count as well as the erythrocyte sedimentation rate (ESR) and fibrinogen concentration by the method of Clauss (1957).
Erythrocyte aggregation
The ErythrosenseTM biomarker is based on the previously described erythrocyte adhesiveness/aggregation test (Rogowski et al., 2000). Venous blood from the antecubital vein was obtained between 08:00 and 11:00. Blood was drawn into a syringe containing sodium citrate (one volume of 3.8% sodium citrate and three volumes of whole blood). One drop of the citrated whole blood was trickled onto a slide inclined at an angle of 30° and allowed to run down by gravity, leaving a fine film. The slides were left to dry in that position, at room temperature. A technician who was blinded to the clinical and laboratory results of the patients scanned the slides by using an image analysis system (InflametTM; Inflamet Ltd, Tel Aviv, Israel), as previously described by Fusman et al. (2002
).
The variable that was used to describe the state of erythrocyte aggregation was the erythrocyte percentage (EP). This is the slide area covered by the erythrocytes. When there is no aggregation, 100% of the slide area is covered with erythrocytes; during aggregation this percentage is reduced due to the appearance of clear areas between the groups of aggregated cells.
Statistical analysis
All the variables were analysed for the normality of their distribution by the one-sample KolmogorovSmirnov test procedure. Differences between parameters in different patient groups were evaluated using Fishers exact test, one-way analysis of variance, or the KruskalWallis test where appropriate. The non-paired t-test or post-hoc Bonferroni analyses were used to perform pair-wise comparisons between group means. A two-tailed Pearsons correlation was performed for the correlation coefficient and significance between EP and fibrinogen levels. According to power analysis, the minimum sample size required in each sub-group was 20 subjects (alpha of 5% and power of 80%). P < 0.05 was considered statistically significant. Calculations were performed using the SPSS software package (SPSS Inc., USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study clearly showed that enhanced erythrocyte aggregation as expressed by using the ErythrosenseTM can be detected in the peripheral blood of women following gonadotrophin administration. This phenomenon of erythrocyte aggregability can have a deleterious effect on the microcirculatory flow (Weng et al., 1998, 1999; Froom, 2000
; Bishop et al., 2001
). In fact, pathological red cell aggregation characterized by large aggregates with strong intercellular links is involved in microcirculatory sludging and stagnation, which alter the peripheral vascular resistance, reduce capillary perfusion and oxygen transfer to tissues and cause ischaemia, local metabolic acidosis as well as degeneration of the vascular wall and capillary leak (Rainer et al., 1987
; Soutani et al., 1995
; Cabel et al., 1997
; Pfafferott et al., 1999
; Bishop et al., 2001
; Tateishi et al., 2001
, 2002; Suckfull, 2002
). Moreover, in a recent study by Baskurt et al. (2004
) it was shown that enhanced red blood cell aggregation results in suppressed expression of NO synthesizing mechanisms. In an animal model of enhanced erythrocyte aggregation, they showed that this suppression causes an alteration in vasomotor tone. The mechanisms involved most likely relate to decreased wall shear stresses due to decreased blood flow and/or increased axial accumulation of red blood cells.
We believe that increased erythrocyte aggregation in patients with COH and OHSS after the administration of gonadotrophins is brought about by hyperfibrinogenaemia. It has been shown that fibrinogen has a major role in the induction and/or maintenance of increased erythrocyte aggregation in the peripheral blood (Weng et al., 1996). The enhanced synthesis of this macromolecule is probably a result of the presence of enhanced IL-6 concentrations in the peripheral blood (Gabay et al., 1999
). In fact, enhanced concentrations of IL-6 have been shown in OHSS (Aboulghar et al., 1999
). In the present study, the fibrinogen concentration was significantly correlated with erythrocyte aggregation (Figure 3). The findings of the present study are therefore in accordance with previous observations related to the role of this macromolecule in the tendency of red blood cells to aggregate during hyperfibrinogenaemic conditions (Schechner et al., 2003
).
Patients with OHSS demonstrated erythrocyte aggregation to a larger extent than patients undergoing COH. This further increase in aggregation may be the necessary step in the evolution from a silent state to a clinical entity.
Finally, we would like to comment on the potential relationship, if any, between the degree of erythrocyte aggregation and the day of the menstrual cycle. In an ongoing study, we have evaluated 418 apparently healthy women and found no clear evidence that the degree of erythrocyte aggregation changes significantly according to the day of the menstrual cycle.
We conclude that following gonadotrophin administration for ovarian stimulation, enhanced red blood cell aggregation can be detected in the peripheral blood. These findings may have pathophysiological relevance in terms of capillary slow flow, tissue hypoxia, changes in vasomotor tone, reduced NO synthase expression and capillary leak. These observations are important in order to define therapeutic strategies that might be applied to those women who develop life-threatening side effects during ovarian hyperstimulation.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albert C, Garrido N, Mercader A, Rao CV, Remohi J, Simon C and Pellicer A (2002) The role of endothelial cells in the pathogenesis of ovarian hyperstimulation syndrome. Mol Hum Reprod 8,409418.
Baskurt OK, Yalcin O, Ozdem S, Armstrong JK and Meiselman HJ (2004) Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation. Am J Physiol Heart Circ Physiol 286,H222H229.
Bishop JJ, Nance PR, Popel AS, Intaglietta M and Johson PC (2001) Effect of erythrocyte aggregation on velocity profiles in venules. Am J Physiol 280,H222H236.
Cabel M, Meiselman HJ, Popel AS and Johnson PC (1997) Contribution of red blood cell aggregation to venous vascular resistance in skeletal muscle. Am J Physiol 272,H1020H1032.[Medline]
Clauss A (1957) Gerinnungsphysiologische Shnellmethode zur Bestimmung des Fibrinogens. Acta Haematol Basel 17,237246.
Delvigne A and Rozenberg S (2002) Epidemiology and prevention of ovarian hyperstimulation syndrome (OHSS): a review. Hum Reprod Update 8,559577.
Delvigne A and Rozenberg S (2003) Review of clinical course and treatment of ovarian hyperstimulation syndrome (OHSS). Hum Reprod Update 9,7796.
Froom P (2000) Blood viscosity and the risk of death from coronary heart disease. Eur Heart J 21,513514.
Fusman G, Mardi T, Justo D, Rozenblat M, Rotstein R, Zeltser D, Rubinstein A, Koffler M, Shabtai E, Berliner S et al (2002) Red blood cell adhesiveness/aggregation, C-reactive protein, fibrinogen and erythrocyte sedimentation rate in healthy adults and in those with atherosclerotic risk factors. Am J Cardiol 90,561563.[CrossRef][Medline]
Gabay C and Kushner I (1999) Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340,448454.
Golan A, Ron-el R, Herman A, Soffer Y, Weinraub Z and Caspi E (1989) Ovarian hyperstimulation syndrome: an update review. Obstet Gynecol Surv 44,430440.[Medline]
Gomez R, Simon C, Remohi J and Pellicer A (2002) Vascular endothelial growth factor receptor-2 activation induces vascular permeability in hyperstimulated rats, and this effect is prevented by receptor blockade. Endocrinology 143,43394348.
Levin ER, Rosen GF, Cassidenti DL, Yee B, Meldrum D, Wisot A, Pedram A (1998) Role of vascular endothelial cell growth factor in ovarian hyperstimulation syndrome. J Clin Invest 102,19781985.
Myrianthefs P, Ladakis C, Lappas V, Pactitis S, Carouzou A, Fildisis G and Baltopoulos G (2000) Ovarian hyperstimulation syndrome (OHSS): diagnosis and management. Intens Care Med 26,631634.[CrossRef][ISI][Medline]
Pellicer A, Miro F, Sampaia M, Gomez E and Bonilla-Musoles FM (1991) In vitro fertilization as a diagnostic and therapeutic tool in patients with partial 17,20 desmolase deficiency. Fertil Steril 55,970975.[ISI][Medline]
Pfafferott C, Moessmer G, Ehrly AM and Bauersachs RM (1999) Involvement of erythrocyte aggregation and erythrocyte resistance to flow in acute coronary syndromes. Clin Hemorheol Microcirc 21,3543.[Medline]
Pride SM, Ho Yuen B, Moon YS and Leung PC (1986) Relationship of gonadotropin-releasing hormone, danazol and prostaglandin blockage to ovarian enlargement and ascites formation of the ovarian hyperstimulation syndrome. Am J Obstet Gynecol 154,11551160.[Medline]
Rainer C, Kawanishi DT, Chandraratna PA, Bauersachs RM, Reid CL, Rahimtoola SH and Meiselman HJ (1987) Changes in blood rheology in patients with stable angina pectoris as a result of coronary artery disease. Circulation 76,1520.[Abstract]
Rogowski O, Zeltser D, Rotstein R, Shapira I, Avitzour D, Fusman R, Mardi T, Prochorov V, Arber N, Berliner S (2000) Correlated expression of adhesive properties for both white and red cells during inflammation. Biorheology 37,361370.[Medline]
Schechner V, Shapira I, Berliner S, Comaneshter D, Hershcovici T, Orlin J, Zeltser D, Rozenblat M, Lachmi K, Hirsh M and Beigel Y (2003) Significant dominance of fibrinogen over immunoglobulins, C-reactive protein, cholesterol and triglycerides in maintaining increased red blood cell adhesiveness/aggregation: a model in hypercholesterolemic patients. Eur J Clin Invest 33,955961.[CrossRef][Medline]
Soutani M, Suzuki Y, Tateishi N and Maeda N (1995) Quantitative evaluation of flow dynamics of erythrocytes in microvessels: influence of erythrocyte aggregation. Am J Physiol 268,H1959H1965.[Medline]
Suckfull M (2002) Fibrinogen and LDL apheresis in treatment of sudden hearing loss: a randomised multicentre trial. Lancet 360,18111817.[CrossRef][Medline]
Tateishi N, Suzuki Y, Cicha I and Maeda N (2001) O2 release from erythrocytes flowing in a narrow O2-permeable tube: effects of erythrocyte aggregation. Am J Physiol Heart Circ Physiol 281,H448H456.
Tateishi N, Suzuki Y, Shirai M, Cicha I, Maeda N (2002) Reduced oxygen release from erythrocytes by the acceleration-induced flow shift, observed in an oxygen-permeable narrow tube. J Biomech 35,12411251.[CrossRef][ISI][Medline]
Weng X, Cloutier G, Beaulieu R and Roederer GO (1996) Influence of acute-phase proteins on erythrocyte aggregation. Am J Physiol 271,H2346H2352.[Medline]
Weng X, Roederer GO, Beaulieu R and Cloutier G (1998) Contribution of acute-phase proteins and cardiovascular risk factors to erythrocyte aggregation in normolipidemic and hyperlipidemic individuals. Thromb Haemost 80,903908.[ISI][Medline]
Weng X, Cloutier G and Genest J Jr (1999) Contribution of the 455G/A polymorphism at the beta-fibrinogen gene to erythrocyte aggregation in patients with coronary artery disease. Thromb Haemost 82,14061411.[Medline]
Whelan JG III and Vlahos NF (2000) The ovarian hyperstimulation syndrome. Fertil Steril 73,883896.[CrossRef][ISI][Medline]
Submitted on December 5, 2003; accepted on February 11, 2004.
|