Impact of cardiovascular burden on coagulation pathway in apparently healthy women planning oocyte donation

Abstract

BACKGROUND:

Hormonal fertility treatments are associated with increased coagulation factors inducing procoagulant milieu and possibly thrombotic risk.

OBJECTIVE:

To assess coagulation by ROTEM and coagulation tests in apparently healthy infertile women before oocyte donation procedure.

METHODS:

We enrolled 51 women (Assisted Reproductive Technology Centre, Florence). ROTEM and coagulation parameters were assessed before the start of infertility treatment.

RESULTS:

We divided women in 3 groups according to the number of cardiovascular risk factors: Group A (n = 10), Group B (n = 16), and Group C (n = 25). By considering ROTEM Extem test, a significantly increased of MCF, TPI, and G were observed in groups B (p = 0.005, p = 0.03, and p = 0.007) and C (p = 0.01, p = 0.05, and p = 0.005) in comparison to group A. As regards ROTEM Intem test, the TPI and G values were significantly higher in groups B and C in comparison to group A (p < 0.01). MCF by Fibtem test significantly increased in groups B and C than in group A (p = 0.004 and p = 0.002, respectively). FVIII, vWF:Ag and D-dimer values significantly increase according to the presence of≥2 risk factors.

CONCLUSIONS:

Data from coagulative assessment permit to sensitively identify women with potentially procoagulable state, which represents a risk factor of thromboembolic event during hormone treatment.

Keywords

Oocyte donation assisted reproductive techniques blood coagulation tests hemostasis women’s health

1 Introduction

Over the years, Assisted Reproductive Technologies (ARTs) offer to infertile women the opportunity to get pregnant. Hormonal treatment in ART acts as a cardiometabolic stress, which may increase the risk of short and long-term cardiovascular events [1]. This may represent a sex-specific atherothrombotic risk factor, which affects especially women undergoing oocyte donation, older than 40 years and with high prevalence of cardiometabolic risk factors [2].

Hormonal fertility treatments are associated with increased coagulation factors, reducing fibrinolysis and altering rheological parameters, inducing procoagulant milieu [3, 4]. It is noteworthy that Assisted Reproduction provides a risk of venous thromboembolism, possibly related to the estrogen surge, which exerts direct effects on several hemostatic variables [5].

Rotational thromboelastometry (ROTEM) assay allows to measure global blood coagulation and clot formation process [6] and data from literature investigated changes in hemostasis during In Vitro Fertilization (IVF) by the thromboelastography (TEG) [7, 8], but information is no available concerning women undergoing oocyte donation.

Aim of the study was to assess coagulation by ROTEM and conventional coagulation tests in apparently healthy infertile women before the start of oocyte donation procedure.

2 Materials and methods

The entire study population comprised 51 women [median age 42 yrs, (range 33–48)] referred to the Internal Medicine Clinic from February 2019 to September 2019 at the Assisted Reproductive Technology Centre, Division of Obstetrics and Gynaecology, University Hospital, Careggi, Florence, Italy.

Exclusion criteria were reported in Fig. 1. Information concerning cardiometabolic parameters derived from clinical reports, and cardiovascular risk factors were investigated during the clinical evaluation.

Fig. 1

Study flow chart.

According to the World Health Organization criteria, underweight was defined as BMI values < 18.5 and overweight was defined as BMI values 25–29.99 Kg/m².

All anthropometric parameters were also measured; waist circumference was measured midway between the inferior margin of the lowest rib and the iliac crest in the horizontal plane at the end of normal expiration; hip circumference was measured at the widest point over the buttocks. A value ≥80 cm was considered a marker of increased cardiovascular risk according to Alberti KG. et al. [9]; WHR was obtained by dividing the waist circumference by hip circumference, and a value > 0.80 was considered a marker of increased cardiovascular risk [10]. Dyslipidemia was defined according to ESC guidelines [11]. Physical activity grade was investigated and sedentary behaviour was defined as absent or light (ie, either occasional walking or recreational activity only).

Informed written consent for anonymous data analysis was obtained from all women. The investigation conforms to the principles outlined in the Declaration of Helsinki. The original study was approved by the Regional Ethical Committee (29 November 2016, CEAVC 10189, amendment 16 May 2018, 2018-017 CINECA 10189).

2.1 Coagulation parameters

Blood samples were collected from the antecubital vein into 0.109 M trisodium citrate tubes (Vacutainer). Plasma samples were obtained by centrifuging blood at 2,000 g for 15 minutes at room temperature for Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, von Willebrand Factor (vWF:Ag), Factor VIII (FVIII) and D-dimer.

Determination of APTT and PT times were measured using commercially available reagents (Pathromtin SL and Thromborel® S) on the Sysmex CS-2100i coagulation analyzer (Siemens, Marburg, Germany).

Fibrinogen was assessed by Clauss clotting method (Siemens) (r.v. 156–400mg/dL). FVIII activity was determined by a coagulation-based assay, with deficient plasma in the presence of Pathromtin (Coagulation Factor VIII, Siemens) (r.v. 50–150%); vWF:Ag levels were detected by a turbidimetric assay (Siemens) (r.v. 40–160%). D-dimer levels were assessed with the quantitative assay HemosIL -dimer HS (Instrumentation Laboratory, Milan, Italy), on an ACL TOP instrument (Instrumentation Laboratory, Milan, Italy) (r.v. > 500μg/L).

2.2 Rotational thromboelastometry (ROTEM)

Thromboelastometric analysis was performed by using the ROTEM® delta system (Werfen Group, Barcelona, Spain) that records viscoelastic clotting changes in a typical graphic [12]. ROTEM tests were obtained according to the manufacturer’s protocol. Extrinsic and intrinsic coagulation cascade were studied with Extem and Intem tests. The influence of fibrinogen on clot firmness was estimated with the platelet-inactivating Fibtem test and the effect of fibrinolytic activity was assessed by fibrinolysis inactivating test Aptem. The following ROTEM parameters were analyzed according to previously reported [13]: I) Clotting Time (CT, sec), corresponding to the time from the beginning of the coagulation analysis until an increase in amplitude of 2 mm; II) Clot Formation Time (CFT, sec), the time between an increase in amplitude of thromboelastogram from 2 to 20 mm, which reflects the speed of the clotting process; III) Maximum Clot Firmness (MCF, mm), the maximum amplitude in millimeters reached in thromboelastogram, which reflects the maximum tensile strength of the coagulum; IV) maximum lysis (ML, %) that quantifies the percentage reduction of MCF; V) G, or Shear Elastic Modulus Strength, is a calculated parameter: it increases exponentially as compared to the amplitude and allows a more sensitive resolution at high amplitudes; VI) Thrombodynamic Potential Index (TPI) is a calculated parameter that describes the global coagulation. Moreover, we considered parameters by the first derivative of the thromboelastogram: Maximum Velocity (MaxV, mm/min), that is the peak of the curve and the Area Under the Curve (AUC, mm^*100) [13].

2.3 Statistical analysis

Statistical analysis was performed by using the SPSS (Statistical Package for Social Sciences, Chicago, USA) software for Windows (Version 26.0). Continuous variables were expressed as median and range. The categorical variables were expressed as frequencies and percentages.

In order to evaluate the weight of cardiovascular risk factors (age, overweight or waist≥80 cm, smoking habit, migraine with aura, dyslipidemia, Primary Ovarian Insufficiency (POI), sedentary behaviour, Recurrent spontaneous miscarriage/ART failure≥2, Family history of cardiovascular disease) in hemostatic pathway, we divided women in 3 groups according to the number of risk factors: Group A (n = 10) (women without or with one risk factor), Group B (n = 16) (women with 2 or 3 risk factors), and Group C (n = 25) (women with more than 3 risk factors).

The differences between groups are tested with Kruskall-Wallis tests for continuous variables.

A p-value < 0.05 was considered to indicate statistical significance. Gerotziafas GT et al. [8] previously documented FVIII, vWF:Ag and D-dimer levels in healthy women. Post hoc sample size calculation indicated that at least 47 women were sufficient to detect, with a statistical power of 90% (β) and a significance value of 0.05 (α), differences in FVIII, vWF:Ag and D-dimer levels in women planning oocyte donation procedures.

3 Results

In Table 1a clinical characteristics of study population are reported.

Table 1a
Clinical characteristics of study population

Variables 51 infertile women

Age≥40 yrs 41 (80.4)

BMI 25–29.99 Kg/m², n (%) 13 (25.5)

WAIST≥80 cm, n % 17 (33.3)

WHR > 0.80, n (%) 18 (35.3)

Smoking habit (current), n (%) 9 (17.6)

Dyslipidemia, n (%) 20 (39.2)

Sedentary behaviour, n (%) 35 (68.6)

POI, n (%) 4 (7.8)

Migraine with aura, n (%) 2 (3.9)

Recurrent spontaneous miscarriage/ART failure≥2, n (%) 27 (52.9)

Family history of CV disease, n (%) 13 (25.5)

Variables	51 infertile women
Age≥40 yrs	41 (80.4)
BMI 25–29.99 Kg/m², n (%)	13 (25.5)
WAIST≥80 cm, n %	17 (33.3)
WHR > 0.80, n (%)	18 (35.3)
Smoking habit (current), n (%)	9 (17.6)
Dyslipidemia, n (%)	20 (39.2)
Sedentary behaviour, n (%)	35 (68.6)
POI, n (%)	4 (7.8)
Migraine with aura, n (%)	2 (3.9)
Recurrent spontaneous miscarriage/ART failure≥2, n (%)	27 (52.9)
Family history of CV disease, n (%)	13 (25.5)

The evaluation of cardiovascular risk factors evidenced a prevalence comparable with those observed in healthy population; in particular, we observed dyslipidemia in 39.2% of women, and overweight in 25.5%, whereas smoking habit was present in 17.6% of women investigated. Of relevance, sedentary behaviour was present in 68.6% and more than 80% of women had age≥40 yrs. The evaluation of anthropometric measures, such as waist circumference and WHR, showed that values of waist circumference≥80 cm, a measure of visceral adiposity, were present in 33.3% and WHR value > 0.80 in 35.3% of women investigated.

Family history of CV disease was present in 25.5% of ART women and migraine with aura in 3.9%. By analysing personal negative obstetric history, we found that more than 50% women suffered from recurrent spontaneous miscarriage and/or ART failures (≥2).

3.1 Coagulation assessment

In Table 1b are reported coagulation parameters detected by ROTEM and in Table 2 are reported traditional coagulation assay; we found that all variables remained within normal limits.

Table 1b
Coagulation parameters detected by ROTEM of study population

Variables 51 infertile women

MCFextem, mm 64 (56–74)

Gextem 8951 (6381–14077)

Li30extem, % 100 (98–100)

MAXVextem, mm/min 17 (11–29)

MAXVTextem, s 109 (52–156)

AUCextem, mm x min 6394 (5567–7335)

TPIextem, s ^–1 69 (31–168)

TPIintem, s ^–1 69 (20–159)

Gintem 7820 (4722–12767)

MCFfibtem, mm 16 (7–46)

Variables	51 infertile women
MCFextem, mm	64 (56–74)
Gextem	8951 (6381–14077)
Li30extem, %	100 (98–100)
MAXVextem, mm/min	17 (11–29)
MAXVTextem, s	109 (52–156)
AUCextem, mm x min	6394 (5567–7335)
TPIextem, s ^–1	69 (31–168)
TPIintem, s ^–1	69 (20–159)
Gintem	7820 (4722–12767)
MCFfibtem, mm	16 (7–46)

Values are reported as median (range) or n (%). BMI (Body Mass Index); WHR (waist to hip ratio); POI (premature ovary insufficiency); ART (Assisted Reproductive Technology); CV (cardiovascular); Maximum Clot Firmness (MCF); Shear Elastic Modulus Strength (G); Lysis Index (LI); Maximum Velocity (MAXV); Area Under the Curve (AUC); Thrombodynamic Potential Index (TPI).

Table 2

Traditional coagulation assay according to three groups

Variables	Entire study population (n = 51)	Group A (n = 10)	Group B (n = 16)	p ^*	Group C (n = 25)	p ^**
PT, %	99 (78–115)	99 (91–115)	99.7 (84–113)	–	97 (78–114)	–
aPTT, s	29.3 (20–36.1)	29.4 (27–36.1)	29.5 (22.3–34.4)	–	28.6 (20–34.1)	–
FVIII, %	99 (64–225)	90 (64–99)	102 (88–225)^*	0.01	116 (69–187)^**	0.02
vWF:Ag, %	100 (31–347)	68 (31–100)	103 (47–150)^*	0.01	108 (71–347)^**	0.001
D-dimer, μg/L	257 (39–2053)	143 (56–789)	342 (128–2053)^*	0.02	281 (39–859)^**	0.049
Fibrinogen, mg/dL	292 (199–489)	303 (233–346)	282 (200–442)	–	308 (199–489)	–

Values are reported as median (range). p^*= Group A vs. B; p^**= Group A vs. C. Factor VIII (FVIII); von Willebrand Factor (vWF:Ag).

By considering coagulation parameters detected by ROTEM Extem test between the groups, a significantly increased of MCF and G were observed in groups B (p = 0.005 and p = 0.007, respectively) and C (p = 0.01 and p = 0.005, respectively) in comparison to group A. G is a calculated parameter that increases exponentially as compared to the amplitude and allows a more sensitive resolution at high amplitudes. The same behaviour was observed for TPI, that describes the global coagulation; a significantly increased was observed between groups B and C with respect to group A (p = 0.03 and p = 0.05, respectively). The derivative values obtained by ROTEM clotting curve reaction, AUC, was significantly increased (B and C groups p = 0.005) in women with multiple risk factors respect to one or no risk factor (Group A) (Fig. 2).

Fig. 2

Median values of parameters detected by ROTEM according to the number of cardiovascular risk factors (Group A, Group B, Group C). p^*= Group A vs. B; p^**= Group A vs. C. Maximum Clot Firmness (MCF), Shear Elastic Modulus Strength (G), Thrombodynamic Potential Index (TPI), Area Under the Curve (AUC).

As regards parameters obtained by ROTEM Intem test, the TPI and G values were significantly higher in groups B and C in comparison to those observed in group A (p < 0.01) (Fig. 2).

In addition, we evaluated also MCF parameter by Fibtem test, which measures the influence of fibrinogen on the strength of clot firmness achieved by platelet-inactivating, and we found a significant increase in groups B and C in comparison to group A (p = 0.004 and p = 0.002, respectively).

Traditional coagulation assay according to three groups was also investigated; FVIII, vWF:Ag and D-dimer values, but not fibrinogen, progressively and significantly increase according to the presence of two or more risk factors (Table 2).

4 Discussion

This study investigates the clot formation using thromboelastometry in apparently healthy infertile women eligible for oocyte donation. Although all hemostatic parameters remained within normal limits, the contemporary presence of cardiovascular risk factors unmasked a tendency toward a hypercoagulable status.

Women’s reproductive lifespan can be artificially prolonged with hormone therapy and oocyte donation. These approaches may induce medical and obstetrical complications, particularly in women older than 40 years [14, 15]. Age may represent a weighing needle in assessing risk profile, and women undergoing oocyte donation are usually older and suffered from more comorbidities, thus representing a clinical model of increased atherothrombotic risk [2].

It is well known that ovarian stimulation for autologous procedure with gonadotropins causes a rapid increase in estrogens levels and may induce a procoagulable status related to increase in coagulation factors [16, 17].

At the best of our knowledge no data are available concerning the relationship between short-term hormonal therapy and coagulability in oocyte donation.

To date, elevated exogenous estrogen levels are associated with hypercoagulability by increasing levels of coagulation factors such as FVIII, vWF:Ag and fibrinogen and reducing fibrinolysis [16 , 18–20]; Fibrinogen concentration is also associated with an activation of thrombocytes and of platelet aggregation. Both might also contribute to a hypercoagulable state [21, 22]. Moreover, erythrocytes’ membrane damages and increased platelet activation are shown in women under hormonal therapy [23]. These changes in coagulation pathway might cause a shift in hemostasis to a prothrombotic status.

Our findings evidenced that FVIII, vWF:Ag, and D-dimer were increased in women carrying more than one cardiovascular risk factors. Therefore, in these women we can hypothesize that hormonal therapy related to oocyte donation may trigger a procoagulant response.

Data from clinical studies investigated [7, 8] changes in hemostasis during In Vitro Fertilization (IVF) by the TEG, but information is unavailable concerning women undergoing oocyte donation, who are at risk of thromboembolic diseases. In the present study we explored hemostasis by ROTEM, which evolved from TEG, in apparently healthy women before oocyte donation procedure.

Findings from this test evidenced slightly increase in coagulation parameters according to the presence of cardiovascular risk factors, even if most of them remained within the normal range. Although fibrinogen levels did not differ between groups, MCF Fibtem significantly increased in women carrying more than one cardiovascular risk factors. It has been demonstrated a good correlation between the fibrinogen level and the Fibtem assay [24, 25]. In our opinion the MCF parameter obtained by Fibtem test, should not only be considered as a measurement of fibrinogen concentration, but also, a measurement of mechanical properties of the fibrin clot [26]. According to Campello et al. [13] we can hypothesize that in women with multiple risk factors fibrin monomer polymerization, detected by high Fibtem MCF, was improved even though fibrinogen levels remained within the normal range.

This study has several strengths. First, it is novel in investigating whole blood viscoelastic hemostasis in apparently healthy women before oocyte donation procedure. Moreover, the group of oocyte donation women was homogeneous regarding procedure indication. Finally, the current research represents a comprehensive analysis of multiple domains of women’s health, thus adding, with special regards, accurate and detail information on coagulation profile in women undergoing oocyte donation.

On the other hand, our study has some potential limitations. One is represented by the cross-sectional nature of the research itself; moreover, the lack of information on changes in hemostasis during hormone treatment. Finally, another limitation is the small number of subjects investigated.

In conclusion, data from ROTEM and coagulative assessment performed in oocyte donation women permit to sensitively identify women with potentially procoagulable state. This may represent a risk factor of thromboembolic event during hormone treatment.

The key message of our study lies in underlying that oocyte donation women risk might be of paramount concern, and that opportunity exists to build awareness among women of the benefits of addressing modifiable confounders, before ART pregnancy, thus preventing and controlling women’s vascular and pregnancy risk. If confirmed in future prospective studies, our findings could help identifying women who could benefit from prophylaxis to control thrombotic risk.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

None declared.

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