**3. Data on the IVF results as affected by pathologies of hemostasis**

As we have already noted, unsuccessful IVF cycles are caused by many factors, including thrombogenicity of the medical technology itself due to the high estrogen-gestagen rate and frequent presence of thrombogenic risk factors and predisposition to intravascular coagulation (thrombophilia) in women that need IVF. According to up-to-date conceptions, the term "thrombophilia" means predisposition to arterial or venous thrombosis as a result of several hereditary or acquired disorders in the systems of blood coagulation, anticoagulation, or fibrinolysis (Bates et al., 2008; Heit, 2007). We use this term in a different sense, which makes most of the described thrombophilias no more than thrombogenic risk factors that may or may not become evident during the lifespan of a human being. According to our conceptions, thrombophilia should be detected in case of everpresent thrombosis risk factors (thromboses) or miscarriage syndrome in the individual medical history. In order to prove it, we note the fact that, according to the guidelines of the International Society on Thrombosis and Haemostasis (ISTH), diagnosis of antiphospholipid syndrome (APS) shall be considered invalid unless at least one or more clinical implications of this pathology match the results of special laboratory assays (lupus anticoagulant effects, antiphospholipid antibodies in the diagnostic titer) (Harris & Pierangeli, 2008).

80 Enhancing Success of Assisted Reproduction

et al., 2005).

20% (Gelbaya et al., 2009).

hyperandrogenism, the suppression of ovarian function takes place, which leads to the development of a number of disorders in the menstrual cycle accompanied by anovulation.

*Diminished endometrial receptivity.* After high-quality embryos have been transferred into the uterine cavity and all evident causes for the failure of the IVF program have been eliminated, the unsuccessful IVF cycle is regarded as a result of disorders that occurred during the embryo implantation stage. A few years ago a new term - "repeated implantation failure" - was introduced (Margalioth et al., 2006; Tan et al., 2005). Recent years have shown that, despite the selection of obviously normal embryos for the transfer, only 20% of human embryos transferred in IVF cycles have been implanted in the uterus (International Committee for Monitoring Assisted Reproductive Technology (ICMART), 2002; Nyboe Andersen et al., 2009). This condition is considered to be based on the endometrial dysfunction occurring on the mollecular-cellular level. Lately, as a result of the tendency to transfer one or two embryos inside of an uterus, the method to determine repeated implantation failure has been modified. Margalioth et al. (2006) concluded that detailed examination should be done after 3 unsuccessful IVF cycles. Thus, the main causes are the factors which diminish endometrial receptivity: anatomical defects in the uterus, chronic endometritis, non-correspondence between the endometrial thickness and the day of embryo transfer, combined gynecologic pathology (adenomyosis, uterine fibroid), somatic diseases (including autoimmune diseases), and thrombophilias (Margalioth et al., 2006; Tan

*Quantity of transferred embryos.* Due to the absence of conventional clinical guidelines for the treatment of infertility with the IVF method, there are on-going discussions regarding the elective transfer of one embryo to patients under 40. In 2009, a mathematical model was drawn to prove that the transfer of only one embryo shall decrease the pregnancy rate by

*Embryo transfer on the stages of cleavage or blastocyst.* The data obtained during the systematic review and meta-analysis (Papanikolaou et al., 2008) of 1654 patients (blastocyst transferred to 815 patients, cleaving embryo transferred to 839 patients) showed that live birth rate was higher with embryos transferred on the blastocyst stage as compared to the rate at the

As we have already noted, unsuccessful IVF cycles are caused by many factors, including thrombogenicity of the medical technology itself due to the high estrogen-gestagen rate and frequent presence of thrombogenic risk factors and predisposition to intravascular coagulation (thrombophilia) in women that need IVF. According to up-to-date conceptions, the term "thrombophilia" means predisposition to arterial or venous thrombosis as a result of several hereditary or acquired disorders in the systems of blood coagulation, anticoagulation, or fibrinolysis (Bates et al., 2008; Heit, 2007). We use this term in a different sense, which makes most of the described thrombophilias no more than thrombogenic risk factors that may or may not become evident during the lifespan of a human being.

cleavage stage. Multiple gestation rates were the same for both study groups.

**3. Data on the IVF results as affected by pathologies of hemostasis** 

Some publications indicate data on typical changes in the system of hemostasis that occurs during the IVF cycle. In particular, demonstration has shown that hormonal stimulation of the ovaries is accompanied by the increased von Willebrand factor, factors V and VIII, fibrinogen, enhanced APC resistance, and the decreased activity of principal physiological anticoagulants - antithrombin, proteins C and S (Andersson, 1997; Biron et al., 1997; Chan & Dixon, 2008; Curvers et al., 2001b; Nelson, 2009). Relationship between the predisposition to intravascular coagulation (thrombophilia) and unsuccessful ART results is actively discussed in current publications; however, mechanisms to produce the impact that increases thrombotic readiness on IVF are not absolutely clear. It is reported that women with thrombophilia may have increased risks for spontaneous abortion, preclinical pregnancy loss and recurrent implantation failure (Christiansen et al., 2006; Coulam et al., 2006b; Curnow et al., 2006; Many et al., 2001; Seghatchian et al., 1996; Stern & Chamley, 2006; Urman et al., 2005; Wichers et al., 2009; Younis et al., 2000). Presently, most studied and prevalent thrombophilias include APS and such risk factors as hereditary antithrombin III deficiency, factor V Leiden mutation, prothrombin mutation, polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene, plasminogen activator inhibitor-1 (PAI-1), fibrinogen, platelet glycoproteins ITGA2, ITGB3, and some others. Beer and Kwak (2000) treated unsuccessful IVF programs as the evident indication for assays capable of detecting hereditary and acquired thrombophilias. In 2004, Azem et al. (2004) demonstrated higher occurrence of hereditary thrombophilia in women with multiple IVF failures as compared to the group of fertile women who became pregnant after the first IVF cycle. In the research conducted by Qublan et al. (2006), 69% of women with recurrent IVF failures had at least one hereditary or acquired thrombogenic risk factor as compared to 25% of women in the group where this reproductive technology was successful. In the publication presented by Grandone et al. (2001), factor V Leiden mutation prevailed (14.4%) in women with recurrent IVF failures as compared to the controls (1%). Recently, Coulam and Jeyendran (2009b) have shown that frequency of genetic polymorphisms has been 1.6 times higher in infertile women with IVF failures as compared to the fertile group; thus, polymorphism of the MTHFR gene has prevailed. It has been also noted that the connection of thrombogenic risk factors with recurrent miscarriages and repeated implantation failures after IVF is mainly evident in the simultaneous carriage of several thrombogenic mutations and polymorphisms (Coulam et al., 2006b).

Mechanisms of hemostasis and implantation pathologies typical of some thrombophilias (carriage of thrombogenic risk factors):


The Means of Progress in Improving the Results of *in vitro* Fertilization Based on the Identification and Correction of the Pathology of Hemostasis 83

investigated. Excessive activation of coagulation, imbalance in the coagulation system, endotheliopathy, local haemorrhages and microthrombi in the area of blastocyst invasion are common elements in the mechanism of implantation failures caused by thrombophilias. Thus, different forms of thrombophilia result in disorders on different stages of the

Analysis of publications demonstrates diverse expert opinions on the role of some mutations, polymorphisms of genes which are part of the hemostasis system and APS role in the development of infertility and ART failures. The American Society of Reproductive Medcine does not find it necessary to examine women who participate in IVF programs in order to detect risk factors for thrombophilia (American Society of Reproductive Medcine .., 2008). On the contrary, approach of the American Society for Reproductive Immunology to treat the same problem is completely opposite (American Society for Reproductive Immunology Antiphospholipid Antibody Committee .., 2000; Gleicher et al., 2002). Inconsistency of the opinions mentioned above may be explained by the absence of vast multicentral studies, use of diverse methodological approaches to diagnosing hemostasis

We believe that the most perspective approach in this area may be found in simultaneous consideration of thrombogenic risk factors and monitoring of the results collected with "global" methods which are capable of detecting disorders in the natural balance of pro- and anticoadulants in blood plasma under controlled ovarian hyperstimulation. This approach offers specialists the potential for the thrombin generation assay (TGA). This assay is known to define the dynamics and intensity of thrombin development; thrombin is the key hemostatic enzyme and relates to the group of integral indexes of the coagulation system (Hemker et al., 2000; Hemker et al., 2003; Hemker et al., 2006; Regnault et al., 2003; Wielders et al., 1997). This methodological approach has been successfully tested as a part of the complex estimation of the hemostasis system during pregnancy (Dargaud et al., 2010), preeclampsia (Macey et al., 2010) and oral contraceptive intake (Tchaikovski et al., 2007). Recent publication presented interesting data on the specifics of thrombin generation in blood plasma within IVF cycle (Westerlund et al., 2012). Shifts detected in 31 women were interpreted as the result of estrogen load and ovarian hyperstimulation syndrome and

Besides, a number of researchers raise a great interest in the decrease of fibrinolytic blood activity, which is often detected in recurrent miscarriages, APS, deep vein thrombosis of lower extremities, oral contraceptive intake, myocardial infarction, and malignant neoplasms (Bertina, 1997; Birkenfeld et al., 1994; Curnow et al., 2006; Dmowski et al., 1995; Egbase et al., 1999; Lisman et al., 2005; Meltzer et al., 2009; Meltzer et al., 2010; Triplett, 1989; Wichers et al., 2009). The system of fibrinolysis, as well as the system of coagulation, is a complex system which gives characteristics to fibrinolytic responses and its central element that plays role in the activation of plasminogen into plasmin. Lately, its pathologies have been treated as new approach that explains the mechanisms of thrombosis pathogenesis (Zorio et al., 2008). The analysis of the fibrinolysis system during the IVF procedure shows the decrease in fibrinolytic responses due to several reasons (Andersson et

coagulation cascade and fibrinolysis.

pathologies and interpretation of examination results.

estimated to be vital for thrombosis prediction and IVF monitoring.


Thus, making a brief summary, different forms of thrombophilia refer to different pathogenesis of implantation failures. Fixed and common conception for all thrombophilias is that pathology mechanisms are revealed in the earliest stages of pregnancy and are caused due to microcirculation and hemostasis disorders, as well as vessel wall pathology. Some researchers deny the impact of mutations of certain genes within the hemostasis system or isolated asymptomatic increase in the level of phospholipid antibodies on implantation. However, combination of several factors is believed to considerably increase individual risk of possible implantation failures and miscarriage. While estimating the occurrence of APS and mutations of genes participating in the hemostasis system in infertile women and women with IVF failures, specialists actively investigate qualitative changes on the level of endometrial structures and vascular endothelium accompanied by thrompophilia (Anteby et al., 2004; Coulam & Roussev, 2009a). Local mechanisms in the basis for implantation failures which occur along with thrombophilias are under investigated. Excessive activation of coagulation, imbalance in the coagulation system, endotheliopathy, local haemorrhages and microthrombi in the area of blastocyst invasion are common elements in the mechanism of implantation failures caused by thrombophilias. Thus, different forms of thrombophilia result in disorders on different stages of the coagulation cascade and fibrinolysis.

82 Enhancing Success of Assisted Reproduction

(Girolami & Vianello, 2000; Poort et al., 1996);

synthesis, and increased platelet aggregation;

decreased depth of blastocyst invasion.

Kim et al., 1997);





Thus, making a brief summary, different forms of thrombophilia refer to different pathogenesis of implantation failures. Fixed and common conception for all thrombophilias is that pathology mechanisms are revealed in the earliest stages of pregnancy and are caused due to microcirculation and hemostasis disorders, as well as vessel wall pathology. Some researchers deny the impact of mutations of certain genes within the hemostasis system or isolated asymptomatic increase in the level of phospholipid antibodies on implantation. However, combination of several factors is believed to considerably increase individual risk of possible implantation failures and miscarriage. While estimating the occurrence of APS and mutations of genes participating in the hemostasis system in infertile women and women with IVF failures, specialists actively investigate qualitative changes on the level of endometrial structures and vascular endothelium accompanied by thrompophilia (Anteby et al., 2004; Coulam & Roussev, 2009a). Local mechanisms in the basis for implantation failures which occur along with thrombophilias are under Analysis of publications demonstrates diverse expert opinions on the role of some mutations, polymorphisms of genes which are part of the hemostasis system and APS role in the development of infertility and ART failures. The American Society of Reproductive Medcine does not find it necessary to examine women who participate in IVF programs in order to detect risk factors for thrombophilia (American Society of Reproductive Medcine .., 2008). On the contrary, approach of the American Society for Reproductive Immunology to treat the same problem is completely opposite (American Society for Reproductive Immunology Antiphospholipid Antibody Committee .., 2000; Gleicher et al., 2002). Inconsistency of the opinions mentioned above may be explained by the absence of vast multicentral studies, use of diverse methodological approaches to diagnosing hemostasis pathologies and interpretation of examination results.

We believe that the most perspective approach in this area may be found in simultaneous consideration of thrombogenic risk factors and monitoring of the results collected with "global" methods which are capable of detecting disorders in the natural balance of pro- and anticoadulants in blood plasma under controlled ovarian hyperstimulation. This approach offers specialists the potential for the thrombin generation assay (TGA). This assay is known to define the dynamics and intensity of thrombin development; thrombin is the key hemostatic enzyme and relates to the group of integral indexes of the coagulation system (Hemker et al., 2000; Hemker et al., 2003; Hemker et al., 2006; Regnault et al., 2003; Wielders et al., 1997). This methodological approach has been successfully tested as a part of the complex estimation of the hemostasis system during pregnancy (Dargaud et al., 2010), preeclampsia (Macey et al., 2010) and oral contraceptive intake (Tchaikovski et al., 2007). Recent publication presented interesting data on the specifics of thrombin generation in blood plasma within IVF cycle (Westerlund et al., 2012). Shifts detected in 31 women were interpreted as the result of estrogen load and ovarian hyperstimulation syndrome and estimated to be vital for thrombosis prediction and IVF monitoring.

Besides, a number of researchers raise a great interest in the decrease of fibrinolytic blood activity, which is often detected in recurrent miscarriages, APS, deep vein thrombosis of lower extremities, oral contraceptive intake, myocardial infarction, and malignant neoplasms (Bertina, 1997; Birkenfeld et al., 1994; Curnow et al., 2006; Dmowski et al., 1995; Egbase et al., 1999; Lisman et al., 2005; Meltzer et al., 2009; Meltzer et al., 2010; Triplett, 1989; Wichers et al., 2009). The system of fibrinolysis, as well as the system of coagulation, is a complex system which gives characteristics to fibrinolytic responses and its central element that plays role in the activation of plasminogen into plasmin. Lately, its pathologies have been treated as new approach that explains the mechanisms of thrombosis pathogenesis (Zorio et al., 2008). The analysis of the fibrinolysis system during the IVF procedure shows the decrease in fibrinolytic responses due to several reasons (Andersson et 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-Zamora et al., 2010; Martinez-Zamora et al., 2011; Meltzer et al., 2010).

The Means of Progress in Improving the Results of *in vitro* Fertilization Based on the Identification and Correction of the Pathology of Hemostasis 85

patients with acute myocardial infarction and prolonged movement disorders. Thus, the larger the volume of the compressed tissue gets, the more apparent became the response. The increase in blood and t-PA fibrinolytic activity after mechanical exposure on blood vessels is supported by the results presented by many authors (Bjornsson et al., 1989; Christen et al., 1997; Jacobs et al., 1996; Pandolfi et al.,1968; Salzman et al., 1987; Tarnay et al., 1980). However, we were not able to find any published data on the dynamics of PAI-1 activity. In the meantime, the correlation of the activities presented by these participants of the fibrinolysis system determines its overall efficacy. Some publications relate the mechanism of the IPC antithrombotic effect to the inhibition of coagulation cascade due to the expression of the tissue factor pathway inhibitor (TFPI) into blood flow and the decrease in the level of factor VIIa (Chouhan et al., 1999; Christen et al., 1997). Currently IPC is used worldwide for thromboprophylaxis in patients with strokes, after arthroplasty and a number of other operative invasions, in medical emergency,and applicable, firstly, in cases when administration of anticoagulants is dangerous due to the development of haemorrhage (Geerts & Selby, 2003; Gordon et al., 2012). As a rule, IPC is performed on the lower extremities, though some publications present positive results for the upper extremities compression (Knight & Dawson, 1976). Despite the fact that legs weigh more than arms, it was proved that forearm veins have considerably more t-PA than leg veins (Pandolfi et al., 1968). In our study this form of IPC was used to activate fibrinolytic

responses within the IVF program and in the presence of relevant indications.

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

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

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.

**5. Оriginal researches** 

results of their correction.

consent.
