Thrombosis Research 130 (2012) 919–924

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Fibrinolysis alterations in infertile women during controlled ovarian stimulation:Influence of BMI and genetic components

Elena Sticchi a, Ilaria Romagnuolo a,b, Anna Paola Cellai a, Donatella Lami a, Sandra Fedi a, Domenico Prisco a,Ivo Noci c, Rosanna Abbate a, Cinzia Fatini a,⁎a Department of Medical and Surgical Critical Care, Thrombosis Center, University of Florence, Azienda Ospedaliero-Universitaria Careggi, Florence, Italyb Fiorgen Foundation, Florence, Italyc Department of Gynecology, Perinatology and Reproductive Medicine, University of Florence, Florence, Italy

Abbreviations: IVF, in vitro fertilization; ICSI, intraGnRH, gonadotropin-releasing hormone; CLT, clot lysis timbrinolysis inhibitor; t-PA, tissue plasminogen activator; PAtor; FGA, fibrinogen α-chain; FGB, fibrinogen β-chain; FGGgene; CBP2, TAFI gene; F13, factor XIII; PLG, plasminogen.⁎ Corresponding author at: Department ofMedical and

of Florence, Largo Brambilla 3, 50134 Florence, Italy. Tel0557949929.

E-mail address: cinzia.fatini@unifi.it (C. Fatini).

0049-3848/$ – see front matter © 2012 Elsevier Ltd. Alldoi:10.1016/j.thromres.2012.07.005

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 30 March 2012Received in revised form 21 June 2012Accepted 7 July 2012Available online 25 July 2012

Keywords:CLTCandidate genes polymorphismsFibrinolysisOvarian stimulationPAI-1TAFI

Introduction: Ovarian stimulation protocols have been described to induce prothrombotic phenotype throughalterations of both coagulation and fibrinolysis pathways. We investigated fibrinolytic changes during ovar-ian stimulation through a global test (CLT) and PAI-1 and TAFI concentrations at different times of ovarianstimulation procedure, and the influence of polymorphisms in genes encoding for fibrinogen chains (FGA,FGB, FGG), t-PA (PLAT), TAFI (CBP2), FXIII (FXIIA1, FXIIIB), plasminogen (PLG) and PAI-1 (PAI1) on their inter-mediate phenotype.Materials and methods: We evaluated fibrinolytic and genetic parameters in 110 infertile women undergoingovarian stimulation procedure (in vitro fertilization, IVF or intracytoplasmic sperm injection, ICSI). Allwomen were observed during the mid-luteal phase of cycle (T0) and on day 5 (T1), 7 (T2) and 9 (T3) of theovarian stimulation.Results: Significant changes in fibrinolytic parameters from T0 to T3 of ovarian stimulation were found (CLTp=0.003; TAFI p=0.009 and PAI-1 p=0.003). CLT values, TAFI and PAI-1 concentrations significantly in-

creased from baseline to T1 (pb0.0001, p=0.01, p=0.005, respectively), and decreased at T2, but remainedhigher than those at T0. Moreover, at baseline overweight women showed longer CLT, higher TAFI and PAI-1concentrations than normal weight women, as well as at T1 two-fold longer CLT and higher PAI-1 concentra-tions were observed (p=0.001 and p=0.05, respectively). Significant differences of TAFI and PAI-1 concen-trations during ovarian stimulation according to TAFI and PAI1 polymorphisms were observed.Conclusions: This study shows alterations of fibrinolysis and suggests the contribution of TAFI and PAI1 genesin modulating fibrinolysis changes during the ovarian stimulation cycle.

© 2012 Elsevier Ltd. All rights reserved.

Introduction

Ovarian stimulation procedures, such as in vitro fertilization (IVF)or intracytoplasmic sperm injection (ICSI), have been demonstratedto induce a prothrombotic phenotype through alterations of both co-agulation and fibrinolysis pathways [1].

An emerging role for fibrinolytic system in modulating miscarriage[2,3] and implantation failure [4] has been described. Nevertheless,

cytoplasmic sperm injection;e; TAFI, thrombin-activatable fi-I, plasminogen activator inhibi-, fibrinogen γ-chain; PLAT, tPA

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few information and conflicting reports on the influence of ovarianstimulation in fibrinolysis are available [5–7].

Beyond traditional laboratory assays of fibrinolytic parameters,such as fibrinogen, PAI-1 and t-PA, the measurement of clot lysis time(CLT), a global test able to evaluate the whole fibrinolytic process, andof thrombin-activatable fibrinolysis inhibitor (TAFI) antigen levels, maycontribute to better investigate the fibrinolytic response to hormonaltreatment in infertilewomenundergoing ovarian stimulation procedures.

Fibrinolytic process may be genetically influenced, so we could hy-pothesize a genetic contribution inmodulating the fibrinolytic responseto ovarian stimulation procedure.

Polymorphisms in fibrinolysis candidate genes have been relatedto alterations of fibrinolytic process. In particular, data from literaturereported a functional role for polymorphisms in genes encoding for fi-brinogen Aα-, Bβ- andγ-chain genes (FGA, FGB, FGG), which contributedto modulate fibrinogen levels or changes in fibrin structure and function[8–10], and for polymorphisms in genes encoding for TAFI and PAI-1,known to be associated with their circulating levels [11,12]. Moreover,

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polymorphisms in other candidate genes have also been related to fibri-nolysis alterations [13–15].

No data concerning the role of polymorphisms in fibrinolysis genesable tomodulate the fibrinolytic phenotype in women undergoing ovar-ian stimulation procedures are available.

Aims of this study were to evaluate in a group of healthy infertilewomen undergoing controlled ovarian stimulation, previously inves-tigated for the hemorheological profile [16]:

1. the fibrinolytic response to ovarian stimulation by a global test (CLT)and by PAI-1 and TAFI plasma concentrations

2. the relationship between functional polymorphisms in fibrinolysiscandidate genes and fibrinolysis parameters.

Materials and methods

Study population and experimental procedure

We performed this study in 110 consecutive infertile women[median age 37 yrs, range (20–42)] who underwent in vitro fertiliza-tion (IVF) (n=59, 53.6%) or intracytoplasmic sperm injection (ICSI)(n=51, 46.4%) procedure, enrolled at Fertility Center of the Depart-ment of Sciences for Woman and Child's Health, University of Florencefrom October 2007 to March 2008, and previously investigated for thehaemorheological profile [16]. Eighty-four women (76.4%) showed pri-mary and 26 (23.6%) secondary infertility, 59 (53.6%) underwent previ-ous IVF/ICSI procedure; 18 (16.4%) were overweight and 14 (12.7%)smokers.

Women were treated by a long gonadotropin-releasing hormone(GnRH) agonist down regulation protocol, as described elsewhere [16].

All women were observed during the mid-luteal phase of cycle(T0) and on day 5 (T1), 7 (T2) and 9 (T3) of ovarian stimulation [16].

Informed written consent was obtained from all women, and thestudy was approved by the Institutional Review Board of the AziendaOspedaliero-Universitaria , Careggi, Florence.

Fibrinolysis parameters

Blood samples were collected from the antecubital vein into evacuat-ed plastic tubes (Vacutainer) in the morning, after an overnight fastingon days from T0 to T3. Plasma sample was obtained by centrifugingblood at 2000 g for 10 min at 4 °C.

Complete blood cell count was performed by using the SysmexXE-2100 hematology analyzer (Sysmex, Kobe, Japan). Fibrinogen wasassessed by coagulation system, PT-base method (RecombiPlas Tin 2G,Instrumentation laboratory, Lexington, USA).

The CLT analysis was performed as previously described [17]. Plas-ma TAFI antigen level and PAI-1 antigen levels were measured withcommercially available ELISA kits (Asserachrom TAFI, and AsserachromPAI-1, Diagnostica Stago, Asnieres, France).

Genetic polymorphisms

Polymorphisms in fibrinolysis candidate genes are reported inSupplemental Table 1.

FGA (rs6050, rs2070011), FGB (rs1800790, rs4220), FGG (rs1049636),PLAT (rs2020918), CBP2 (rs3742264, rs35814191, rs1926447) were ana-lyzed through GenomeLab SNPStream technology (Beckman Coulter inc.Fullerton, CA), a DNA microarray technology based on primer extensionreaction able to perform the simultaneous detection of several singlenucleotide polymorphisms (SNPs) [18]. PCR and extension primer se-quences are reported in Supplemental Table 2.

F13A1 rs5985, F13B rs6003, PLG rs4252125 and PAI1 rs1799889were detected through TaqMan technology using the 9700 GeneAmpPCR system (Applied Biosystems). F13A1 rs5985 and PAI1 rs1799889polymorphisms were genotyped through TaqMan designed assays

according to Tjarnlund A et al. [19]. F13B rs6003 and PLG rs4252125polymorphisms were analyzed by TaqMan SNP genotyping assays(C_ 2532034_10 and C_25614474_20).

PLAT Alu I/D polymorphism was analyzed through PCR reaction,performed in a final volume of 20 μl using primers 5′-AGTTAAGGGTCCTGGCCTGT-3′ (forward) and 5′-TCATCTTGACCTTGCAGCAC-3′ (re-verse) at an annealing temperature of 62 °C.

Statistical analysis

Statistical analysis was performed by using the SPSS (StatisticalPackage for Social Sciences, Chicago, USA) software for Windows(Version 11.5). Theχ2 testwas used to testHardy-Weinberg equilibrium.

The statistical analysis was performed according to the differentphases of ovarian stimulation procedure (T0–T3).

Continuous variables are expressed as median and range. The non-parametric Wilcoxon and Friedman tests for paired data were usedfor comparisons between and among groups, as appropriate. Thenon-parametric Mann-Whitney test was used for analysis of unpaireddata.

Correlation analysis was measured by using the Spearman's corre-lation test.

A p-valueb0.05 was considered to indicate statistical significance.

Results

In 110 consecutive infertile women [16] who underwent IVF/ICSIprocedure significant changes of fibrinolysis parameters during ovar-ian stimulation were found (CLT p=0.003; TAFI p=0.009 and PAI-1p=0.003). A significant increase of CLT (6%), TAFI concentrations(10%) and PAI-1 levels (28%) from baseline to T1 was observed(Fig. 1); values decreased, even if not significantly, from T1 to T2, pos-sibly due to modifications of the hormonal burden during the ovarianstimulation phases [16], although remaining higher than those at base-line (T0). At T1, total cholesterol levels significantly increased (29%),whereas fibrinogen concentrations significantly decreased (15%), in linewith our previous results [16].

CLT significantly correlated with PAI-1, TAFI and cholesterol levels(R=0.35 pb0.0001, R=0.28 pb0.0001, R=0.19 p=0.02, respec-tively). The correlations between CLT and biohumoral parameters atall times of the ovarian stimulation cycle are shown in Table 1a. Nocorrelations between estradiol levels and fibrinolysis parameters atall phases of ovarian stimulation were observed (Table 1b).

Fibrinolytic parameters were evaluated according to BMI, age andsmoking habit (Table 2). At baseline women with BMI >25 Kg/m2

showed longer CLT (4%) and higher TAFI and PAI-1 concentrations(15% and 25%) in comparison to that observed in women with BMIb25 Kg/m2 (Table 2). At T1 fibrinolytic parameters increased inboth groups; overweight women showed a two-fold longer CLT andhigher PAI-1 concentrations than women with BMI b25 Kg/m2 (p=0.001 and p=0.05, respectively) (Table 2).

No difference in CLT, TAFI and PAI-1 values according to age (higher/lower than 35 yrs) and smoking habit at baseline and during the ovar-ian stimulation was observed (Table 2).

In order to investigate the influence of polymorphisms in genesencoding for components of the fibrinolytic system [fibrinogen α, β,and γ-chain (FGA,FGB and FGG), t-PA (PLAT), TAFI (CBP2), factor XIIIA and B-subunit (F13A1 and F13B), plasminogen (PLG) and PAI-1(PAI1)] in modulating changes in CLT, we analyzed CLT values at allphases of ovarian stimulation in women carrying functional rare allelescomparedwithwomen homozygous for wild-type alleles. No deviationfrom Hardy-Weinberg equilibrium for all polymorphisms investigatedwas found.

CLT values were not significantly different between women carry-ing and not carrying rare alleles at baseline and during ovarian stim-ulation (data not shown).

Fig. 1. Histograms showing CLT, and TAFI and PAI-1 values [median (range)] according to all phases of ovarian stimulation (T0–T3) are reported. Statistical comparisons (Wilcoxontest) between biohumoral parameters values at T1, T2 and T3 phase vs baseline (T0) are indicated.

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The combined effect of the number of the unfavourable alleles on CLTlengthening was investigated: no significant difference in CLT changesfrom T0 to T1 betweenwomen carryingmore than 5 unfavourable allelesandwomen carrying 5 or less rare variants [Δ (CLT)To–T1: 7.0±13.8 minvs Δ (CLT)To–T1: 4.2±16.4 min, p=0.09] was found.

In addition, the relationship between polymorphisms in genesencoding for fibrinogen α, β, and γ-chain (FGA,FGB and FGG), TAFI(CBP2) and PAI-1 (PAI1) and their biohumoral intermediate phenotypewas evaluated. At baseline, no difference in fibrinogen concentrationsbetweenwomen carrying and not carrying FGA,FGB and FGG rare alleleswas reported, and no influence on changes in fibrinogen levels duringovarian stimulation phases was observed (data not shown).

TAFI concentrations were significantly different at baseline betweenwomen carrying and not carrying CBP2 −2345 1G and 325Ile rare al-leles (p=0.02 and p=0.04, respectively), whereas no difference inTAFI levels between women carrying and not carrying CBP2 147Thrrare allele was found (p=0.08) (Fig. 2). Significant differences in TAFI

Table 1aCorrelations between CLT and biohumoral parameters according to ovarian stimulationphases.

CLT

T0 T1 T2 T3

R p⁎ R p⁎ R p⁎ R p⁎

PAI-1 (ng/ml) 0.24 0.01 0.47 b0.0001 0.26 0.04 0.43 0.005TAFI (μg/ml) 0.14 0.1 0.36 b0.0001 0.32 0.005 0.17 0.2Fibrinogen (mg/dL) 0.12 0.3 0.33 0.001 0.08 0.5 0.19 0.2Cholesterol (mg/dL) 0.14 0.2 0.22 0.047 −0.07 0.5 0.18 0.2

⁎ Spearman test.

concentrations throughout the ovarian stimulationwere observed in re-lation to CBP2 Ala147Thr and −2345 2G/1G polymorphisms (Fig. 2).The contemporary presence of more than one CBP2 rare variant didnot contribute to further increase TAFI concentrations.

At baseline, no significant difference in PAI-1 concentrations betweenwomen carrying andnot carrying PAI1 4G rare allelewas evidenced (p=0.7). The presence of 4G allele significantly modulated PAI-1 concentra-tions throughout the ovarian stimulation cycle, whereas homozygosityfor the 5G variant did not influence PAI-1 levels (Fig. 3).

Discussion

This study provides evidence for an impairment of fibrinolysis inhealthy infertile women undergoing controlled ovarian stimulation,as showed by a prolonged CLT and by increased TAFI and PAI-1 con-centrations, and demonstrate the influence of TAFI and PAI-1 genesin modulating changes of fibrinolytic parameters.

Table 1bCorrelations between estradiol (E2 ) levels and fibrinolysis parameters according toovarian stimulation phases.

E2

T0 T1 T2 T3

R p⁎ R p⁎ R p⁎ R p⁎

CLT (min) −0.11 0.4 −0.09 0.4 −0.12 0.3 −0.12 0.4PAI-1 (ng/ml) −0.06 0.8 0.09 0.4 −0.05 0.7 −0.03 0.9TAFI (μg/ml) 0.01 0.8 0.00 0.9 −0.18 0.1 −0.09 0.5

⁎ Spearman test.

Table 2Biohumoral parameters in subgroups of infertile women at baseline (T0) and at T1 ovarian stimulation phase.

Variables CLT⁎ (min) TAFI⁎ (μg/ml) PAI-1⁎ (ng/ml)

T0 T1 p† T0 T1 p† T0 T1 p†

BMI>25 Kg/m2 (n=18) 72 (41–96) 80 (68–147) 0.03 10.2 (6.6–15.2) 10.3 (7.5–16.7) 0.4 15 (7–100) 21 (6–84) 0.3BMIb25 Kg/m2 (n=92) 69 (35–102) 73 (40–118) 0.005 8.9 (5.3–13.9) 9.9 (3.9–14.4) 0.06 12 (4–100) 17 (4–60) 0.02p‡ 0.1 0.001 0.1 0.3 0.1 0.05Age>35 yrs (n=68) 72 (35–102) 75 (40–150) 0.03 9.2 (5.3–15.2) 10.1 (5.9–15.1) 0.02 12 (3–100) 18 (4–148) 0.007Ageb35 yrs (n=42) 70 (46–96) 73 (55–147) 0.004 9.0 (6.2–13.9) 9.7 (3.9–16.7) 0.3 16 (5–80) 18 (6–113) 0.3p‡ 0.6 0.9 0.9 0.2 0.1 0.6Smokers (n=14) 71 (36–94) 74 (62–102) 0.9 9.2 (6.6–12.7) 10.0 (5.9–16.7) 0.5 14 (4–100) 18 (5–46) 0.4Non-smokers (n=96) 70 (35–102) 74 (40–147) 0.001 8.9 (5.3–13.9) 10.3 (3.9–15.1) 0.005 13 (5–100) 19 (6–84) 0.03p‡ 0.9 0.8 0.5 0.8 0.7 0.8

⁎median (range), † T1 vs T0 (Wilcoxon test), ‡ comparison between groups at T0 and T1 (Mann-Whitney test).

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The ovarian stimulation for IVF or ICSI procedure promotes an overallimpairment of coagulative and fibrinolytic processes, able to induce ahypercoagulable status, responsible for a prothrombotic phenotype [1].Controlled ovarian stimulation procedures induce increased serum estra-diol levels, thus determining increased thrombin generation [20]; never-theless no information concerning the relationship between estradiollevels and fibrinolysis is available. In the present study, we did not ob-serve a correlation between estradiol levels and fibrinolysis parameters.

Changes concerning fibrin formation and fibrinolysis have beenobserved throughout different female life phases, from oral contra-ceptive use [21] to pregnancy [3,21] and pregnancy-related negativeoutcomes [3,22,23], possibly due to changes of hormonal environment.In particular, hypofibrinolysis has been reported to occur in pregnancy[24],mainly due to increased levels of plasminogen activators inhibitors(PAI-1 and 2), and changes in fibrin clot structure have been related torecurrent miscarriages [3]. The contribution of TAFI to the impairment

Fig. 2. TAFI concentrations (median, range) according to rare and wild-type alleles of three Cstimulation cycle (T0–T3) are shown. In the table below, p for comparisons between (Wilwild-type alleles of CBP2 polymorphisms are reported.

of fibrinolysis associated with adverse pregnancy outcome has also beendemonstrated [22].

In infertile womenwe demonstrated that a reduced fibrinolytic activ-ity takes place after hormonal treatment, as evidenced by a prolongedCLT andhigher TAFI andPAI-1 concentrations inwomenundergoing con-trolled ovarian stimulation.

Interestingly, data from literature provide evidence for the rela-tionship between reduced plasma fibrinolytic potential and recurrentimplantation failure [4,25]. A prolonged CLT and increased TAFI con-centrations in women with recurrent implantation failure followingembryo transfer [4] have been demonstrated, whereas conflictingdata concerning the influence of ovarian stimulation on fibrinolysisin women undergoing assisted reproductive procedures are available[5–7].

Our results, which show increased PAI-1 concentrations andprolonged CLT during the ovarian stimulation, are in line with those

BP2 polymorphisms (Ala147Thr, −2345 2G/1G and Thr325Ile) at all phases of ovariancoxon test) and among (Freedman test) stimulation phases (T0–T3) for both rare and

Fig. 3. PAI-1 concentrations (median, range) according to PAI1 4G and 5G alleles at all phases of ovarian stimulation cycle (T0–T3) are shown . In the table below, p for comparisonsbetween (Wilcoxon test) and among (Freedman test) stimulation phases (T0–T3) for both 4G and 5G alleles of PAI1 polymorphism are reported.

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from Aune B et al., who demonstrated a reduced fibrinolytic activityevidenced by a prolonged CLT [5], and at variancewith Rice andMagnani,who observed a decrease in PAI-1 levels in women undergoing ovulationinduction [6,7]. These conflicting resultsmay be related to the small num-ber of women investigated, and possibly to the different ovarian stimula-tion protocols.

Potential mechanisms responsible for impaired fibrinolysis duringovarian stimulationmay be both endothelial dysfunction and inflamma-tion, and conditions such as overweight, smoking habit and age mayfurther contribute toworsening in the fibrinolytic response. Our findingsdemonstrated aworsening in fibrinolysis after hormone therapy in over-weight women, thus stressing for weight control in women undergoingovarian stimulation procedure.

Fibrinolytic process may be genetically influenced, therefore wecould hypothesize a role for fibrinolysis candidate genes in modulatingfibrinolytic response to ovarian stimulation procedure. Our findingsshowed that genetic variants affected CLT, expression of an impaired fi-brinolysis,which ismainly influenced by PAI-1 and TAFI concentrations.

It is known that the 4G/5G polymorphism in PAI1 gene is one of thedeterminants of PAI-1 concentrations [12], and the insertion/deletion(I/D) polymorphism of the angiotensin converting enzyme (ACE) genehas been demonstrated to account for a significant proportion of the var-iability of PAI-1 circulating levels [26].

A great interindividual variability concerning TAFI concentrationshas been reported [27], and it has been demonstrated that TAFI levelswere under genetic controls [11]. Recently, data from literature reportedthat functional polymorphisms in TAFI (CBP2) gene modulated TAFI con-centrations in women with recurrent implantation failure after IVF andembryo transfer [4].

Our findings are novel in providing further information concerningthe influence of PAI14G/5G andCBP2Ala147Thr and−2345 2G/1Gpoly-morphisms on PAI-1 and TAFI levels, as expression of the fibrinolytic re-sponse to ovarian stimulation.

This study suffers from the lack of information concerning tPA,plasminogen and factor XIII concentrations, as intermediate phenotypes

known to influence fibrinolytic process. Moreover, further studiesperformed on larger populations are needed to confirm the relation-ship between fibrinolysis genes polymorphisms and their intermedi-ate phenotype.

In conclusion, the present study demonstrates fibrinolytic alterationsduring the ovarian stimulation procedure, and suggests the contributionof fibrinolysis gene polymorphisms in modulating fibrinolysis activity ininfertile women undergoing assisted reproductive procedures.

As the use of assisted reproductive technology becomes increasinglyprevalent, the knowledge of mechanisms able to modify a quiet milieuinto a prothrombotic environmentmight contribute to perform a betterclinical management of infertile women.

Conflict of interest statement

Authors disclose any conflict of interest.

Authorship contribution statement

E.S: conception and design of the study, analysis and interpreta-tion, writing the article, final approval; I.R: data collection, statisticalanalysis, final approval; A.P.C: data collection, analysis and interpretation,final approval; D.L: statistical analysis, final approval; S.F: analysis and in-terpretation, statistical analysis, final approval; D.P: critical revision, finalapproval; I.N: data collection, final approval; R.A: conception and design,critical revision of the article, final approval; C.F: conception and design,writing the article, critical revision of the article, final approval.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.thromres.2012.07.005.

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