Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of controlled ovarian hyperstimulation (COH). Its pathogenesis is not clarified yet. In the recent years a number of studies focused on the vascular endothelial growth factor (VEGF) and antihtrombin III (AT III) indicators. VEGF is homodimeric, heparin-binding glycoprotein, stimulating vascular permeability. Antithrombin III is protease inhibitor of activated clotting factors. This study aimed at examining the VEGF-A165 and AT III indicators with two OHSS patients. Two methods were used for the determination of the indicators of VEGF-A165 and AT III: ELISA for VEGF and chromogenic assay for ATT III. Kits of R/D Systems and American Diagnostica Inc. were used to estimate VEGF and AT III indicators in serum and plasma. There were higher indicators of VEGF-A165 (180pg/ml) and reduction of AT III indicators (48%) in the patient with a severe form of OHSS than in the control group while these indicators were normal in the patient with a moderate form of OHSS. Our results confirmed some published data concerning the importance of VEGF and AT III in the genesis of OHSS. This study should include a larger group of patients in order tofollow-up statistically and authentically the variations of the indicators of both factors and their importance for OHSS

Aleksandrov, K.

Cherneva, S.

Cvetkova, S.

Kovachev, E.

Kozovski, I.

Markova, V.

English

Varna Medical University Press: Journals

Scripta Scientifica Medica, vol. 35 (2003), pp 79-82 Copyright © Medical University, VarnaTHE ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND ANTITHROMBIN III IN THE PATHOGENESIS OF THE OVARIAN HYPERSTIMULATION SYNDROMEE. Kovachev, I. Kozovski, V. Markova, K. Aleksandrov, S. Cherneva, S. CvetkovaDepartment o f  Obstetrics and Gynaecology, Centre o f  Assisted Reproductive Technology, Prof. Paraskev Stoyanov Medical University o f  VarnaA B ST R A C TOvarian hyperstimulation syndrom e (OHSS) is an iatrogenic complication o f controlled ovarian hyperstim ulation (COH). Its pathogenesis is not clarified yet. In the recent years a number o f studies focused on the vascular endothelial growth factor (VEGF) and antihtrom bin III (AT III) indicators. VEGF is homodimeric, heparin-binding glycoprotein, stim ulating vascular permeability. Antithrom bin III is protease inhibitor o f activated clotting factors. This study aimed at exam ining the V EG F-A 165 and AT III indicators with two OHSS patients. Two methods were used for the determ ination o f the indicators o f VEGF-Aies and AT III: ELISA for VEG F and chrom ogenic assay for ATT III. Kits o f R/D Systems and AmericanDiagnostica  Inc. were used to estimate VEGF and AT III indicators in serum and plasma. There were higher indicators of VEGFA 165 (180pg/m l) and reduction o f AT III indicators (48% ) in the patient with a severe form o f  OHSS than in the control group while these indicators were normal in the patient with a moderate form o f OHSS. Our results confirmed some published data concerning the im portance o f  VEGF and AT III in the genesis o f  OHSS. This study should include a larger group o f patients in order to follow-up statistically and authentically  the variations o f the indicators o f both factors and their importance for OHSS.K ey w o rd s: O H SS, ART, V EG F, A T III, laboratory param eters, pathogenesisThe ovarian hyperstimulation syndrome (OHSS) is a seri­ous and potentially life-threatening physiologic complica­tion classically encountered in patients who undergo con­trolled ovarian hyperstimulation cycles (COH). It presents with a mild, moderate and severe form, according to Golan's classification. The following factors are of pathophysiological importance: polycystic ovarian syn­drome (PCOS) (4); high estradiol levels; elevation o f hu­man chorion gonadotropin (HCG); activation of the prorenin-renin-angiotensin chain (3); increased synthesis of prostaglandins (PG), histamine, serotonin, and cytokines such as IL; deficiency o f the enzyme 17-20 desmolase; role o f inhibins and activines in ovarian physiology and pathol­ogy as well as role o f the vascular endothelial growth factor (VEGF) and antithrombin III (AT III) (1,2). The increased capillary permeability and angiogenesis are significant fac­tors in OHSS pathogenesis (8) (Fig. 1).VEGF is a homodimeric, heparin-binding glycoprotein playing the key role o f mediator in angiogenesis and stimu­lating vascular permeability. There are two tyrosine kinase receptors for VEGF (9) which are produced in theca interna and human granulosa cells where two fractions -Address for correspondence:E. Kovachev, Dept, o f  Obstetr ics and Gynaecology,Prof. Paraskev Stoyanov Medical University o f  Varna, BG-9002 Varna, 55 Marin Drinov St., BulgariaVEGFA 165 and VEGFA 121 predominate (5). AT III is a pro­tease inhibitor o f clot-activating factors and its deficiency may cause thrombosis. One possible way of decreasing AT III in patients with severe OHSS is a shift o f albumin in extravascular space that may be accompanied by that o f AT III because o f its similar molecular weight. Thromboembolic incidents can occur in association with OHSS and can be observed in patients with hereditary AT III deficiency with plasma active levels o f 30-60% o f AT III. Acquired AT III deficiency can be suspected with liver disease, DIC, nephrotic syndrome, pulmonary embolism, stroke and thrombophlebitis. In addition, oral contraceptive use may reduce AT III levels (6,10).The objective o f the present study was to investigate the levels o f VEGFA165 and AT III with two patients with OHSS and to establish their role in OHSS pathogenesis.M A T E R IA L  A N D  M E T H O D SDuring the period from January, 2003 to January, 2004 in IVF Centre o f Varna, two patients with moderate and se­vere fomi of the OHSS were examined. 1 VF-procedure was performed with inhibition o f pituitary gland with GnRH-agonist, COH-long protocol - 'step up' depending on estradiol levels and follicular development. Exogenous gonadotropins were applied in a dose of5000E or 10000E79E. Kovachev, I. Kozovski, V. Markova .t  A D H  S N S  R A SST IM U L A T E SB A R O R E C E P T O R S1 B P /C VPA R T E R IO L A RVA SO D ILA TA TIO NR EN INH O R M O N E S A N D  V A SO A C T IV E  M ED IA TO R SI___B R A D Y K IN IN( OVARIAN V (STIMULATION/IA C U T E  IN FL A M M A T O R Y  R E S P O N S EiIN FL A M M A T O R Y  M ED IA TO R S1A C U T E  PH A SE  PR O T E IN Si— K A L L IK R IE N -K IN IN  SY ST E MR O L A C T INA N G IO G E N E S ISPREGNANCY ENDOGENOUS LHIL-8I t  M A C R O G L O B U L INVEGFî  V A SCU LA F PER M EA B ILITYFig. I. Pathogenesis o f  OH SSHCG (Pregnyl) and aspiration oocytes was done after 34-36h. On the third day after the punction, three embryos were transferred For lutheal support, Utrogestan was ap­plied. At the start o f the stimulation as well as before appli­cation o f  HCG, estradiol (E2) and the standard blood pa­rameters were measured. For establishing the levels of VEGF Ai65 in serum, ELISA was used and kits of R/D-Sysiems centrifuged at 2000x.g. and stored at -20°C were made use of. Plasma AT III levels were measured through chromogenic assay kit o f spectrophotometer - 405nm, centrifuged and stored at -20°C, 30min before use. Thrombin reagent, spectrozyme and dilution buffer of American Diagnostica Inc. were applied. For establishing the control levels o f VEGFA165 and calibration o f AT III standards, a control group o f 10 patients was formed who participated in IVF-programe - COH with no indication of OHSS, liver and hematologic diseases. VEGFAies and AT III were measured in serum and plasma. Blood samples for VEGF A ,65 and AT III were taken on the second day after HCG-application. Referent range for VEGF does not exist, therefore, the recommendation o f R/D-Systems is to estab­lish control levels in the group o f minimum 10 patients.R E SU L T S A N D  D ISC U SSIO NWith the patient with 'late' OHSS (severe) form, the diagno­sis was made on the 13th day after the injection o f Pregnyl5000E and the level o f  E2 before application was 2,800pg/ml, 10 dominant follicles in the 2 ovaries were present. After 36h a punction and aspiration o f 11 oocytes o f good quality was realized. ICSI-method was done be­cause the reason was azoospermia o f  the husband. On the 3rd day after the punction, embryo transfer o f 3 embryos took place. The patient was admitted on the 13th day in our hospital in Varna with some complaints such as malaise, vomiting, abdominal discomfort, and respiratory distress. It was established that ovaries were 4/11cm with theca-luttein cysts, large amounts o f ascites were revealed with ultra­sound examination. OHSS was classified as a severe form o f IV grade in reference to clinical and paraclinical parame­ters. The pregnancy test was positive, later on the ultra­sound examination showed multiple pregnancy (bigemini) which, additionally, exacerbated OHSS. The paracentesis was realized with a view to the increased quantity of ascites and all therapeutic procedures for treatment o f OHSS. Two live twins on the term were bom with Caesarean section in 38g. w. and their weights were 2,600g and 2,750g, respec­tively. With the second patient with OHSS the diagnosis was established on the 10th day after stimulation with gonadothropins. Preventive and therapeutic activities were done - 'step down' protocol, coasting HCG, early unilateral follicular aspiration, HCG o f 5000E, punction and aspira­tion o f 14 oocytes, infusion o f human albumin and antibi­otic treatment. On the 3rd day, embryo transfer o f  2 embryos80The role of vascular endothelial growth factor and antithrombin III in the .was conducted. The pregnancy was not proved, unfortu­nately. VEGFA 165 levels o f control group were between 30-105pg/ml. (67,5pg/ml). The normal laboratory levels ofwere normal, VEGFAies - 70 pg/ml and AT III - 95%, re­spectively. Table 1 shows the laboratory examinations of the patient with 'late' OHSS (severe) form.Table 1. Laboratory indices o f  the patient with severe fo rm  o f  OHSSLaboratory parameters normal rangeNumber o f daysafter the diagnosis of OHSS1 3 6 8 11hemoglobin 120-160 g/1 175 168 155 130 140hematocrit 0.36-0.48 0.58 0.52 0.40 0.42 0.42red blood cells 3.7-5.3*10l2/l 6.2 5.7 5.0 4.4 4.6leukocytes 3.5-10.5* 109/1 17.0 14.0 10.2 8.9 7.0platelets 130-360*109/1 380.0 358.0 305.0 309.0 260.0ESR up to 20mm/h 22mm/h - 17mm/h - 6mm/htime of bleeding 2 - 5  min 4.7 - 3.0 - -fibrinogen 2 - 4.5g/l 4.9 4.2 3.9 3.0 4.0fibrinogen degradation products < 10pg/ml 6.8 13.2 13.8 10.6 8.2prothrombin time 11.5-15sec 14.5 13.8 15.2 13.2 12.8activated partial thromboplastin time 28.0-38.0sec 36.0 - 31.0 - -total protein 60-83g/l 40.1 44.5 58.2 63.3 64.0ASAT 2-18U/1 20.2 - 14.0 - 6.4ALAT 2-18U/1 17.2 - 15.8 - 15.2urea 2.6-7.2mmol 8.8 8.0 7.3 6.3 -creatinine 58-9.6pmol/l 140 128 109 95 87K+ 3.5-5.6 mmol/1 5.5 5.1 3.9 4.0 3.6Na+ 136-151 mmol/1 148 - 140 - -pH 7.36-7.44 7.42 - 7.40 - -pOj 10.66-13.33kPa 11.2 10.72 - 11.6 12.0pC02 4.66-5,99kPa 6.1 5.82 - 4.92 5.2urine investigation for protein, sugar, ketons, bilirubin, urobilinogen24h diuresisE2 before application of HCG - 2,800ng/mlAT Ill-second day after HCG application - 48%75-125%VEGFA 165 - second day after HCGapplication - 180mg/ml - ■ ■ . ■ — "Control group 30-105ng/mlAT III in plasma were 75-125% and o f the control group - 80-109%. There was an increase o f VEGFAi65 (180 pg/ml) and a decrease o f AT III (48%) in the patient with 'late' OHSS. These parameters o f the other case with OHSSPathogenesis o f  OHSS is still unknown but the increased capillary permeability and angiogenesis are primary com­ponents in it. Some authors report o f OHSS complicated with thrombosis without hemoconcentration in a patientE. Kovachev, I. Kozovski, V. Markova .with AT HI deficiency, as well as of thromboembolic com­plications after hyperstimulation with hereditary AT III de­ficiency ( 10). The decrease o f AT III may be a result o f in­sufficient synthesis, high consumption, abnormal loss due to accompanying albumin in extravascular space (6). VEGF is the basic inductor o f  cappillary permeability in OHSS and the main cause o f ascites, pleural and pericardial effusion.It is suspected that HCG application in the stimulation pro­tocol causes VEGF synthesis (1). Other researchers con­sider VEGF a minor clinical marker o f OHSS. It is reported that VEGF participates in premature rupture of the mem­branes. They emphasize its role in the genesis of placental hypoxia and its examiantion as a potential early indicator of severe birth asphyxia OHSS is classified as 'early' that de­velops within 3-7 days after ovulatory dose o f HCG and 'late', after 12-17 days in women with multiple pregnancy. The parameters used during the stimulation for prediction of OHSS do not show any risk o f development o f  'late' OHSS.There are different opinions about the rate o f E2 for predic­tion o f OHSS. Some reports show a risk o f 38% for severe OHSS with peak o f E2 reaching 6000pg/ml on the day of HCG application (9). Their reports which use the same cri­teria predict only a risk o f 8,8% for development o f severe OHSS (7). Some investigators claim that 'late' OHSS does not correlate with the peak rate o f E2 like in our case.C O N C L U SIO NThe measurement o f VEGF rate between the days of HCG application and embryo transfer is a possible marker con­cerning the risk for 'late' OHSS. Our results support in part the data published in the literature available about the im­portance o f VEGF and AT III for OHSS pathogenesis. In our opinion, the study should include a larger group o f pa­tients to measure statistically the real fluctuation of the rates o f both factors and their importance for OHSS.R E FE R E N C E S1. Ковачев,  E.,  И. Козовски,  В. Маркова,  С. Чернева,  и др.  Някои аспекта наОХСС.- В: IV. Национален конгрес по стерилитет, контрацепция и хормонозаместителна терапия. Боровец, 2003 г.- Репрод. здраве, 2003, № 2.2. Ковачев,  E.,  И. Коз овски,  В.  Маркова,  С. Чернева,  и др.  Диагноза, клинична картина и проследяване на жени с ОХСС след ИВФ-ЕТ.- В: Юбилейна научна конференция. Стара Загора, 2002г.3. Козовски,  И.,  В. Маркова,  М. А нд он ов .  ОХСС при бременност след ИВФ-ЕТ.- В: Национална акушерска конференция. Китен,1997г.4. Козовски,  И.,  В. Маркова,  П. Шопова.  ОХСС не е усложнение само на методите за асистирана репродукция,- В: VII. Национален конгрес по акушерство и гинекология. Варна, 1998г.5. Geva,  E.,  J. В.  Jaffe .  Role o f VEGF in ovarian physiology and pathology.- Fertil. Steril., 74, 2000,No 3, 429-438.6. N eu le n ,  J., D. W enze l ,  C. Hornig,  E. Wunsch,  et al. Poor responder - high responder: the importanse o f soluble VEGF-receptor 1 in ovarian stimulation protocols.- Hum. Reprod., 16, 2001, p. 84.7. Levin,  E. R.,  G. F . Rosen ,  D. Cass ident i ,  B .Y ee ,  et al.  Role o f VEGF in ovarian hyperstimulation syndrome.- J. Clin. Invest., 102,1998, 1978-1985.8. Lee,  A . ,  L. Chr i s tenson,  L. Stouffer ,  A.  Burry,  et al.  VEGF levels in serum and follicular fluid o f patients undergoing in vitro fertilization.- Fertil. Steril., 68, 1997, 305-311.9. Ryo,  E.,  D. Hagino ,  N.  Yano ,  M. Sento ,  et a l . A case o f  ovarian hyperstimulation syndrome in which antithrombin III deficiency occurred because of  its loss into ascites.- Fertil. Steril., 71, 1999, 5.10. Kl igman,  I., N.  N o y e s ,  C. A.  Benadiva ,  Z. Ro senw aks ,  et al.  Massive deep vein thrombosis in a patient with antithrombin III deficiency undergo­ing ovarian stimulation for in vitro fertilization.- Fertil. Steril., 63, 1995, 673-676.82

The Role of Vascular Endothelial Growth Factor and Antithrombin III In The Pathogenesis of the Ovarian Hyperstimulation Syndrome

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The Role of Vascular Endothelial Growth Factor and Antithrombin III In The Pathogenesis of the Ovarian Hyperstimulation Syndrome

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