**5. Оriginal researches**

84 Enhancing Success of Assisted Reproduction

**pathologies within the IVF program** 

(Lisman et al., 2005; Wichers et al., 2009).

within the IVF cycle.

al., 1997; Aune et al., 1991; Kim et al., 1981; Many et al., 2001; Martinez-Zamora et al., 2011; Meltzer et al., 2010; Nelson, 2009; Rice et al., 1993; Sarto et al., 2000). One of them is a decreased activity of tissue plasminogen activator (t-PA), increased level of its inhibitor - plasminogen activator inhibitor-1 (PAI-1), and increased level of thrombin-activatable fibrinolysis inhibitor (TAFI) dependent on the response of vascular endothelium (Bouma & Meijers, 2003; Martnez-

**4. Potential methods for the correction of hemostasis and fibrinolysis** 

Analysis of publications shows that correction of imbalanced homeostatic and fibrinolytic responses may be used in case of hormonal load within the IVF program accompanied by thrombophiia or present thrombogenic risk factors in a patient (Martinez-Zamora et al., 2011; Nelson & Greer, 2008; Rova et al., 2012; Urman et al., 2005). This is reasonable to determine thrombotic readiness, which becomes evident through the increase of general coagulation activity and thrombinemia and/or fibrinolysis suppression identified, for example, with the help of the thrombin generation assay upon detecting markers of thrombinemia and estimating fibrin clot lysis time for the fibrin obtained from euglobulins

The use of heparins may become one of the methodologies aimed at the decrease of thrombogenicity and increase of IVF efficacy (Nelson & Greer, 2008; Urman et al., 2009). Still, there are no clear indications for the selection of women that need heparin prophylaxis

Correction of hypofibrinolysis within IVF cycle also offers some difficulties for there are no published evidence of any successful drug therapy. Moreover, the hypothetic possibility to use pharmaceutical drugs - fibrinolysis activators (streptokinase, urokinase, and tissue plasminogen activator) - cannot be considered due to the absence of acute thrombosis. In the study conducted by Bjornsson et al. (1989), regular intake of aspirin in high doses (650 mg every 12 hours) caused the acceleration of fibrinolysis. But the mechanism of this effect is not absolutely clear, whereas the use of acetylsalicylic acid in high doses is unsafe due to the potential ulcerogenic effect. Nevertheless, it has been known for about 50 years that some stimuli (venous occlusion, physical load, desmopressin) lead to the acceleration of fibrinolytic responses facilitated by the fast increase of t-PA in blood due to its enhanced secretion by vascular endothelium. The effects of intermittent pneumatic compression (IPC) used to decrease the occurrence of postoperative venous thrombosis became our interests(Browse et al., 1977; Jacobs et al., 1996; Januszko et al., 1967; Holemans, 1963; Keber et al., 1979; Tarnay et al., 1980; Turpie et al., 1977; Weitz et al., 1986). Macdonald et al. (2003) published the results of their randomized pilot study demonstrating the efficacy of heparin prophylaxis combined with IPC in the course of neurosurgical invasions. The study conducted by Tarney et al. (1980) showed that intermittent compression of the calf, along with the increase in linear blood velocity and the decrease in venous stasis, increases local and systemic fibrinolytic potential (according to the shortened fibrin clot lysis time) in

Zamora et al., 2010; Martinez-Zamora et al., 2011; Meltzer et al., 2010).

This publication is based on the clinical study carried out to define the role of pathologies in the coagulation and fibrinolysis systems facilitating IVF failures, as well as to estimate the results of their correction.

In the framework of prospective analysis we collected data on 327 women who have been visiting the Center for Saving and Recovering the Reproductive Function, a subdivision of the Clinical Regional Hospital (Barnaul), from 2010 to 2012, to participate in the IVF program due to infertility. This study was approved by the Regional Ethics Committee of the Altai Medical University, and all the participants under study expressed their informed consent.

At the first visit women were interviewed about their obstetric, gynecological, and thrombotic history, possible diabetes, pathologies in the thyroid gland, heart and blood vessels. We have conducted ultrasonography of the genitals in order to detect organic pathologies of the pelvic organs and estimate the ovarian reserve (according to the quantity of antral follicles), aspiration biopsy and histologic examination of the endometrium, as well as to detect infections, including sexually transmitted diseases. We have also conducted general and special laboratory assays, including hormone panel assessment, blood chemistry panel, thrombogenic mutation and polymorphism carriage, coagulation profiles, and homocysteine presence. Then, based on the obtained results, women received professional consultation by obstetrician-gynecologists, physicians, and hematologists.

The study was chronologically conducted in two stages. At the first (observational) stage, we examined a random sampling of 163 women in their IVF cycle. At the second stage, we examined a random sampling of 164 women, 98 of which underwent the correction of the hemostasis and fibrinolysis systems in the presence of relevant indications - increased thrombin generation and/or decreased fibrinolytic activity of blood plasma (controlled group or group with the therapeutic effect on hemostasis and fibrinolysis) (Fig. 1).

The Means of Progress in Improving the Results of *in vitro* Fertilization Based on the Identification and Correction of the Pathology of Hemostasis 87

with ultrosonic guidance using Medison Sonoace X8 machine. After we counted obtained oocytes and estimated their quality with the conventional scale (normal oocytes of good quality - 4-5 points, modified oocytes - less than 4 points), oocytes and embryos were cultivated in 6-well plates in the IVF medium (Vitrolife, Sweden), at 37 degrees Centigrade, in humid atmosphere containing 5% of CO2. Sperm processing was conducted with the Sil-Select Plus medium (FertiPro, Belgium). The ICSI procedure consisted of fertilization with the injection of a single sperm into the oocyte (177 married couples) in case of decreased sperm mobility or irregular sperm morphology. Embryo transfer was conducted with ultrasonic guidance on the 3rd day of cultivation. Embryos with the highest quality rating (A, AB) were selected for the transfer. Pregnancy was diagnosed two weeks after the embryo transfer by means of detecting b-human chorionic gonadotropin (b-hCG). After three weeks we defined

quantity and location of the implanted embryos with ultrasonography (in 124 patients).

Age, years (mean ± SD) 33.7 ± 4.1 33.2 ±3.6 34.4 ± 3.9 > 0.5 BMI > 25 kg/m2, n (%) 61 (18.7) 34 (20.9) 27 (16.5) 0.323

**- Chronic endometritis, n (%) 108 (33.0) 42 (25.8) 66 (40.2) 0.006**  - Endometriosis, n (%) 55 (16.8) 28 (17.2) 27 (16.5) 0.883 - Myoma, n (%) 45 (13.8) 25 (15.3) 20 (12.2) 0.426

history, n (%) 36 (11.0) 19 (11.7) 17 (10.4) 0.727 **Low ovarian reserve, n (%) 55 (16.8) 19 (11.7) 36 (21.9) 0.017** 



male factors, n (%) 41 (12.5) 23 (14.1) 17 (10.7) 0.316

Examination was conducted three times: 1-2 days before the start of controlled ovarian hyperstimulation and an IVF program (1st observation point), 2-3 days before the puncture of ovarian follicles (2nd observation point), and on the 12th-14th day after the embryo transfer (3rd observation point), when the outcome in terms of pregnancy was defined by

P-value\* for within-group comparison (Fisher,s exact test); SD, standart deviation **Table 1.** Clinical characteristic of the patients examined in the IVF cycle.

*Technique of laboratory assays for hemostatic and fibrinolytic profiles.* 

Group 1 (n=163)

Group 2 (n=164)

P-value\*

(n=327)

Indication Total

Genital pathology:

IVF failure registered in the

Combination of female and

estimating the level of b-hCG.

Extragenital pathology:

Infertility causes:

**Figure 1.** Division of women participating in the study into groups and subgroups

The selection criterion for the patients was any form of infertility non-responsive to traditional treatment. The exclusion criteria were somatic diseases serving as contraindications for carrying a pregnancy and delivery, congenital malformations or acquired deformations of the uterine cavity that make embryo implantation or carrying of a pregnancy impossible, ovarian tumors, benign uterine tumors that require operative invasion, malignant neoplasms.

All patients are representatives of the Caucasian race, their age ranged from 21 to 42 years (Table 1)

There was a difference in clinical profiles of women representing the two groups. The second group of patients suffers from chronic endometritis and has lower ovarian reserve. Besides, male infertility factor was more frequent in this group.

We used standard protocols to induce superovulation. In 72.4% (237 patients) of the whole population we used the "prolonged" protocol with diphereline (Ipsen) 0.1 mg or decapeptyl (Ferring) 0.1 mg and gonadotropic preparations - puregon (MSD) 150-250 IU, menopur (Ferring) 225 IU, or gonal (Merck Serono) 225 IU. In 27.6% (90 patients) of the whole population we stimulated superovulation using the same gonadotropic preparations and an antagonist - cetrotide (Merck Serono) 0.25 mg. Transvaginal follicle puncture was conducted with ultrosonic guidance using Medison Sonoace X8 machine. After we counted obtained oocytes and estimated their quality with the conventional scale (normal oocytes of good quality - 4-5 points, modified oocytes - less than 4 points), oocytes and embryos were cultivated in 6-well plates in the IVF medium (Vitrolife, Sweden), at 37 degrees Centigrade, in humid atmosphere containing 5% of CO2. Sperm processing was conducted with the Sil-Select Plus medium (FertiPro, Belgium). The ICSI procedure consisted of fertilization with the injection of a single sperm into the oocyte (177 married couples) in case of decreased sperm mobility or irregular sperm morphology. Embryo transfer was conducted with ultrasonic guidance on the 3rd day of cultivation. Embryos with the highest quality rating (A, AB) were selected for the transfer. Pregnancy was diagnosed two weeks after the embryo transfer by means of detecting b-human chorionic gonadotropin (b-hCG). After three weeks we defined quantity and location of the implanted embryos with ultrasonography (in 124 patients).

86 Enhancing Success of Assisted Reproduction

invasion, malignant neoplasms.

(Table 1)

The study was chronologically conducted in two stages. At the first (observational) stage, we examined a random sampling of 163 women in their IVF cycle. At the second stage, we examined a random sampling of 164 women, 98 of which underwent the correction of the hemostasis and fibrinolysis systems in the presence of relevant indications - increased thrombin generation and/or decreased fibrinolytic activity of blood plasma (controlled

group or group with the therapeutic effect on hemostasis and fibrinolysis) (Fig. 1).

**Figure 1.** Division of women participating in the study into groups and subgroups

Besides, male infertility factor was more frequent in this group.

The selection criterion for the patients was any form of infertility non-responsive to traditional treatment. The exclusion criteria were somatic diseases serving as contraindications for carrying a pregnancy and delivery, congenital malformations or acquired deformations of the uterine cavity that make embryo implantation or carrying of a pregnancy impossible, ovarian tumors, benign uterine tumors that require operative

All patients are representatives of the Caucasian race, their age ranged from 21 to 42 years

There was a difference in clinical profiles of women representing the two groups. The second group of patients suffers from chronic endometritis and has lower ovarian reserve.

We used standard protocols to induce superovulation. In 72.4% (237 patients) of the whole population we used the "prolonged" protocol with diphereline (Ipsen) 0.1 mg or decapeptyl (Ferring) 0.1 mg and gonadotropic preparations - puregon (MSD) 150-250 IU, menopur (Ferring) 225 IU, or gonal (Merck Serono) 225 IU. In 27.6% (90 patients) of the whole population we stimulated superovulation using the same gonadotropic preparations and an antagonist - cetrotide (Merck Serono) 0.25 mg. Transvaginal follicle puncture was conducted


P-value\* for within-group comparison (Fisher,s exact test); SD, standart deviation

**Table 1.** Clinical characteristic of the patients examined in the IVF cycle.

#### *Technique of laboratory assays for hemostatic and fibrinolytic profiles.*

Examination was conducted three times: 1-2 days before the start of controlled ovarian hyperstimulation and an IVF program (1st observation point), 2-3 days before the puncture of ovarian follicles (2nd observation point), and on the 12th-14th day after the embryo transfer (3rd observation point), when the outcome in terms of pregnancy was defined by estimating the level of b-hCG.

Sampling of venous blood was in the cubital vein in VACUETTE test tubes with the buffer solution of sodium citrate with the proportion of 9:1 (9NC Coagulation sodium citrate 3.2%). Blood was centrifuged at 1400 g and room temperature for 15 minutes. Plasma samples were generally studied within the two hours after being obtained. Prior to conducting immune-enzyme assays and estimating the endogenous thrombin potential, plasma was stored at -40 degrees Centigrade for the period of time ranging from 24 hours to 1 month.

The Means of Progress in Improving the Results of *in vitro* Fertilization Based on the Identification and Correction of the Pathology of Hemostasis 89

exogenous t-PA. For our study we use a modified method suggested by Kowarzyk and Buluk (1954) and used kaolin that activates the contact phase of coagulation and starts activation cascade. Factor XIIa → kallikrein → plasmin. Description of this method was given earlier by Barkagan and Momot [Barkagan Z.S., Momot A.P. Diagnosis and controlled therapy of hemostasis pathologies. / M., Publisher: Nyudiamed-AO, 2001. - 296 P.]. Range of

*Definition of the D-dimer concentration in blood plasma* was conducted with the help of reagent set D-dimer Red-700 (Helena Bioscience, UK) and blood coagulation analyzer Sysmex CA-1500 (Sysmex, Japan). This parameter was considered in accordance with conventional conceptions as a global marker for the completion of fibrin generation and fibrinolysis of

*Definition of gene mutations and polymorphisms*, predisposing to thrombosis, was conducted with the method of polymerase chain reaction. Thus, we detected the carriers of factor V Leiden (1691G>A) and factor II (20210G>A) mutations, MTHFR (C677>T) and PAI-1 gene polymorphisms related to potential activation of coagulation or decreased plasmin

Statistical processing of the obtained data was made with the following software: Microsoft Office Excel 2003, Statistica 6.1, and Medcalc 12.2.1. Validity of the differences in mean values was defined with the Student's t-test (t). Group distribution normalcy was estimated with the Shapiro-Wilk test. In cases when the distribution deviated from the norm, we used the non-parametric Mann-Whitney U test for two independent groups and the Spearman's rank correlation coefficient (R). For experimental data presented in percentages or rates the Fisher's exact test was used. Odds ratio (OR) and log-linear rate analysis were calculated as a measure of predictor impact. To estimate the accuracy of obtained values, we defined a 95% confidence interval. Differences P<0.05 were considered statistically significant. We assessed the efficacy of the chosen treatment methods with conventional criteria applied in evidencebased medicine, including Absolute Risk Reduction (ARR), Relative Risk (RR), Relative Risk

generation, as well as to IVF failures and miscarriages (Coulam et al., 2006a).

Reduction (RRR), Number Needed to Treat (NNT), and Confidence Interval (CI).

*therapeutic intervention during the controlled ovarian hyperstimulation* 

*5.2.1. Estimation of hemostatic and fibrinolytic indications in the IVF cycle and definition of threshold values occurring in the assay results for the selection of women who need* 

At the beginning of the study, it was important to investigate shifts in the hemostatic and fibrinolytic systems that may affect IVF outcomes (in terms of the pregnancy rate) and to define their quantitative level that may help in selecting those women who need the correction of disturbed hemostatic and fibrinolytic responses. As a result, we defined that

normal CLT variations in this modification is 8-12 minutes.

stabilized fibrin.

**5.1. Statistical analysis** 

**5.2. Study results** 

*Measuring of the endogenous thrombin potential* was made as a part of the thrombin generation assay (TGA). We believe that this method may be regarded as a historical modification of the two-stage self-coagulogram suggested by Berkarda et al. (1965) and developed to pursue similar goals. It was demonstrated that TGA allows experts to measure the dynamics of thrombin generation and inactivation with high precision (Hemker et al., 2003; Hemker et al., 2006). To perform calibrated automated thrombography, the Fluoroskan Ascent microplate fluorometer was applied (Thermo Fisher Scientific, Finland) equiped with a dispenser with Thrombinoscope 3.0.0.26 software. Coagulation of plasma under study was conducted in the presence of tissue factor (5 μM) and phospholipids (4 μM); thrombin generation was continually registered by measuring the signal of fluorogenic substrate (Z-Gly-Gly-Arg-АМС). The following parameters were considered: endogenous thrombin potential (ETP, nM×min), calculating the area under the thrombin generation curve and taking into account specifics of the enzyme inactivation, and peak thrombin concentration - Peak thrombin (nM/l), maximal thrombin concentration per time unit.

*Activity of t-PA and PAI-1* was defined by means of the immune-enzyme analysis with sets of reagents t-PA Combi Actibind ELISA Kit and Actibind PAI-1 ELISA (Technoclone, Austria), while the collected data was estimated by mutual comparison. Due to the fact that these important participants of fibrinolytic responses are antagonistic to each other and have a common origin (vascular endothelium), we calculated the index of endothelial ability to activate fibrinolysis (EAAF index). To do that we applied the following formula:

$$\text{EAAF index}\_{\prime} \text{ \%} = \frac{\text{Activity of t} - \text{PA}\_{\prime} \text{ un } / \text{ml}}{\text{Activity of PAI} - 1 \text{ } \text{un} / \text{ml}} \times 100\%.$$

*Defining clot lysis time (CLT).* Many authors refer this method to the group of global assays for fibrinolysis assessment. It can be conducted in a variety of ways (Lisman et al., 2005; Martinez-Zamora et al., 2011; Wichers et al., 2009).

Method to define spontaneous lysis time of a fibrin clot obtained from plasma euglobulins was described by Kowarzyk and Buluk (1954). It is based on the precipitation of euglobulin fraction stabilized with sodium citrate in the acid medium with the simultaneous removal of fibrinolysis inhibitors. Then, following this methodology, specialists promote clot formation by recalcifying the reconstituted euglobulin solution and register period for its complete dissolution at fixed temperature (+37 degrees Centigrade). Martinez-Zamora et al. (2011) have recently published original CLT results obtained in women participating in IVF cycles using another method described by Lisman et al. (2002). In particular, this method implies the study of CLT for clots obtained from blood plasma by means of activitaing fibrinolysis with exogenous t-PA. For our study we use a modified method suggested by Kowarzyk and Buluk (1954) and used kaolin that activates the contact phase of coagulation and starts activation cascade. Factor XIIa → kallikrein → plasmin. Description of this method was given earlier by Barkagan and Momot [Barkagan Z.S., Momot A.P. Diagnosis and controlled therapy of hemostasis pathologies. / M., Publisher: Nyudiamed-AO, 2001. - 296 P.]. Range of normal CLT variations in this modification is 8-12 minutes.

*Definition of the D-dimer concentration in blood plasma* was conducted with the help of reagent set D-dimer Red-700 (Helena Bioscience, UK) and blood coagulation analyzer Sysmex CA-1500 (Sysmex, Japan). This parameter was considered in accordance with conventional conceptions as a global marker for the completion of fibrin generation and fibrinolysis of stabilized fibrin.

*Definition of gene mutations and polymorphisms*, predisposing to thrombosis, was conducted with the method of polymerase chain reaction. Thus, we detected the carriers of factor V Leiden (1691G>A) and factor II (20210G>A) mutations, MTHFR (C677>T) and PAI-1 gene polymorphisms related to potential activation of coagulation or decreased plasmin generation, as well as to IVF failures and miscarriages (Coulam et al., 2006a).
