*5.2.3. Comparative analysis of the prognostic value of a number of factors contributing to the failure of IVF*

100 Enhancing Success of Assisted Reproduction

factor clearly demonstrated.

cycle hypofibrinolysis indicated a sharp increase in EAAF index combined with the decrease in CLT. Interestingly, vases compression led to the significant increase of t-PA activity as well as to the reduction of PAI-1activity at the end of the IVF cycle. It was also discovered that the application of IPC led to increased thrombin generation which Peack thrombin

**Figure 3.** The evolution of laboratory parameters of hemostasis and fibrinolysis in women who require

Combined application of IPC and low molecular weight heparin produced complex beneficial effect on hemostasis and fibrinolysis to be in correspondence with the maximum

In Fig. 3 the dynamics of the main parameters studied in the course of the treatment for the

concomitant therapy, but do not receive it (A) during such therapy (B)

correction of hemostasis and fibrinolysis is highlighted.

increase in the number of IVF successful outcomes (Tables 4, 7, 8 and 12).

This section has recorded and compared the effect of risk factors in the failure of IVF in 1st (observation) group and women of (2nd) group. We studied a wide range of adverse prognostic factors which are well-known in Reproduction and discussed in this publication: markers of thrombogenic risk and blood fibrinolytic activity reduction as well as hyperhomocysteinemia, thrombogenic mutations and polymorphisms carriers, presence of an inflammatory response (Bates et al., 2008; Coulam & Jeyendran, 2009b; Heit, 2007; Qublan & Eid, 2006), "0" blood type negative factor [Canonico et al., 2008; Ohira et al., 2007.)

Assessing the reasons for the failure of pregnancy in IVF cycles in 163 patients of the 1st (observation) group based on the analysis of the odds ratio (OR), 9 out of 27 (33.3% ) factors became the most important adverse factors or symptoms to be rated as fairly significant (Table 13). Importantly, indicators reflecting increased thrombin generation and inhibition of fibrinolytic reactions, respectively, 2nd and 3d entered the adverse factors as well. Hypo fibrinolysis factors with high reliability proved to be at the 5th and 6th adverse factors ranking. Consequently, in addition to well-known factors listed in Section 2, increased ability to thrombosis, in response to the stress estrogen and inhibition of fibrinolytic reactions are among the leading causes of failure of IVF. It is interesting to note that such a well-known and widely used in clinical practice marker of thrombinemia as high D-dimer plasma levels did not vary in frequency of occurrence in impregnate women at all stages of observation (OR 0,99; 0,95% CI 0,42-2,31 – 21 rank).

Carriage of thrombogenic mutations and polymorphisms were identified in the majority of our patients (71.2%). By the rare mutations - Factor V Leiden and prothrombin in only 4 cases (2.4%), we cannot judge the significance of their influence. However, the combination of polymorphisms MTGFR and PAI-1 was found in a slightly larger percentage of cases with unsuccessful IVF (24.3% vs. 16.0%) to be, however, insignificant. Interestingly, blood type "not 0"did not prove to be protrombogen/unfavorable by nature in our observation as well as a number of variants of virus infection carriers and the manifestations of inflammation (fibrinosis, leukocytosis).

The number of factors contributing to the failure of pregnancy in IVF changed dramatically after exposure to therapeutic correction of hemostasis and fibrinolysis, used in the present publication. In accordance with the data in Table 14, traditional reasons for Reproduction are among the leaders: early hyperstimulation, male factor, and others. Hyperhomocysteinemia (OR 3,45; 0,95% CI 1,16-10,2) became one of the hemostatic reasons at the 4th rank to be the result of obvious lack of attention to the problem of metabolic methionine in preparation for IVF protocol. The significance level of manifestation of high thrombin generation shifted from the 2nd and 3rd rank to 8th and 9th rank whereas EAAF index - from 6th to 15th rank to be the further proof of the effectiveness of our methods of applied therapeutic intervention. Please, note that the calculations in this table exclude 40 patients with disorders of hemostasis and fibrinolysis who did not receive treatment for a number of reasons (subgroup 2.2.4).


The Means of Progress in Improving the Results of *in vitro* Fertilization Based on the Identification and Correction of the Pathology of Hemostasis 103

> Odds ratio (0,95% CI)

(0,83-3,79)

(0,72-3,87)

(0,59-3,84)

(0,59-2,27)

(0,38-3,54)

(0,39-2,78)

(0,42-2,31)

(0,41-1,59)

(0,20-2,85)

(0,35-1,58)

(0,31-1,73)

(0,28-1,10)

(0,03-8,45)

P-value

0,148

0,315

0,500

0,735

> 1,00

> 1,00

> 1,00

> 1,00

0,737

> 1,00

0,535

0,113

> 1,00

Success of IVF (n=56)

26 24,3 9 16,0 1,67

42 39,2 20 35,7 1,16

14 13,0 7 12,5 1,05

19 17,7 10 17,8 0,99

23 21,5 15 26,8 0,74

30 28,0 23 41,0 0,55

1 0,9 1 1,7 0,51

Criterion Failure of

16. The combination of

T/T) and PAI-I (5 G/4 G, 4 G/4 G)

G/4 G, 4 G/4 G)

g/l (for 2nd point of

ng/mL (2nd point of

24. Leukocytosis over 11,0×109/l (1st point of

26. Polymorphism of MTHFR

27. Mutation of FV Leiden

observation)

observation)

observation)

(C/T, T/T)

(G/A)

(n = 163)

polymorphisms MTHFR (C/T,

18. Polymorphism of PAI-I (5

20. Fibrinosis greater than 5.0

21. D-dimer levels over 500

IVF (n=107)

15. High dose-protocol 35 32,7 12 21,4 1,78

17. Low ovarian reserve 19 17,7 7 12,5 1,51

19. Cytomegalovirus infection 11 10,3 5 8,9 1,16

22. Herpes type 1 and 2 35 32,7 21 37,5 0,81

23. Hypothyroidism 6 5,6 4 7,1 0,77

25. Blood group - is not "0" 85 79,4 47 83,9 0,74

**Table 13.** Factors contributing to the failure of pregnancy in IVF cycles in the 1 st. observation group

Abs. % Abs. %


1. Hyperstimulation

2. ETP more than 1900 nM/min (2nd point of

3. Peack thrombin more than

(2nd point of observation)

5. Clot lysis time of over 12 minutes (1st point of

6. EAAF index less than 11% (1st point of observation)

8. Insufficient number of embryos transferred

11. Hypoplasia of the

more than 15 mM/l (1st point of observation)

12. Unsuccessful IVF attempt

13. Homocysteine in blood of

endometrium

in history

4. Oligozoospermia (moderate and severe)

(early stage)

observation)

observation)

in IVF cycles

(1-2)

360 nM/L

Criterion Failure of

IVF (n=107)

7. Defective embryo 23 21,4 3 5,3 4,83

9. Mutation FV (G/A, A/A) 3 2,8 0 0 3,78

10. Difficult embryo transfer 32 29,9 6 10,7 3,55

14. Age (36-40 years) 19 17,7 6 10,7 1,79

Abs. % Abs. %

Success of IVF (n=56)

22 20,5 0 0 29,7

73 68,2 4 7,1 27,9

74 69,1 5 8,9 22,8

45 42,0 2 3,5 19,5

69 64,5 5 8,9 18,5

62 57,9 6 10,7 11,5

38 35,5 6 10,7 4,58

11 10,3 3 5,3 2,02

20 18,7 6 10,7 1,91

22 20,5 7 12,5 1,81

Odds ratio (0,95% CI)

(1,76-500)

(9,33-83,4)

(8,36-62,5)

(4,53-84,6)

(6,81-50,3)

(4,53-29,1)

(1,38-16,9)

(1,80-11,6)

(0,19-74,5)

(1,38-9,12)

(0,54-7,57)

(0,72-5,08)

(0,72-4,54)

(0,67-4,80)

P-value

< 0,00001

< 0,00001

< 0,00001

< 0,00001

< 0,000001

0,00005

0,011

0,0007

0,319

0,006

0,383

0,260

0,280

0,262

**Table 13.** Factors contributing to the failure of pregnancy in IVF cycles in the 1 st. observation group (n = 163)


The Means of Progress in Improving the Results of *in vitro* Fertilization Based on the Identification and Correction of the Pathology of Hemostasis 105

> Odds ratio (0,95% CI)

(0,36-4,38) 0,760

(0,54-2,89) 0,673

(0,45-2,98) 0,813

(0,56-2,33) 0,720

(0,47-2,77) 0,824

(0,24-4,33) > 1,00

(0,24-4,33) > 1,00

(0,27-2,23) 0,790

(0,16-0,83) 0,018

P-value

Success of IVF (n=63)

Failure of IVF (n=61)

18. High-dose protocol 6 9,8 5 7,9 1,26

19. Blood group - is not "0" 48 78,6 47 74,6 1,24

20. Low ovarian reserve 11 18,0 10 15,8 1,16

minutes (1st point of observation) 35 57,4 34 53,9 1,14

(1st point of observation) 13 21,3 12 19,0 1,15

endometrium 4 6,5 4 6,3 1,03

24. Hypothyroidism 4 6,5 4 6,3 1,03

25. Herpes type 1 and 2 7 11,4 9 14,3 0,77

T/T) 12 19,6 25 39,6 0,37

**Table 14.** Factors contributing to the failure of pregnancy in IVF cycles in the 2nd group (n = 124)

In our publication we studied a number of women with the known risk factors of IVF failures to be less significant regardless of the ongoing correction of hemostasis and hypo fibrinolysis. We also record such risk factors as manifestations of inflammatory reaction (leukocytosis, fibrinosis) of virus (herpes, cytomegalovirus), the pathology of the thyroid

We are particularly interested in the reasons for the failure of IVF which remained relevant after therapeutic correction aimed at hemostasis and fibrinolysis. The list is given in Table.

As noted earlier (Table 14), in addition to gynecological risk factors, hyperhomocysteinemia, and manifestations of excessive thrombin generation became the causes of the failure of IVF. Consequently, despite treatment some female patients maintained the trend to intravascular coagulation. Earlier, Table 11 demonstrated that isolated course of IPC to treat hypo fibrinolysis and other effects led to increased thrombin generation whereas the combination of IPC with dalteparin did not lead to such a shift (Table 12). Considering that we used three variants of therapeutic intervention our attempt was to determine the frequency of each of them under different outcomes of IVF in women with a high ETP in thrombin generation

Abs % Abs %

Criterion

21. Clot lysis time of over 12

23. Hypoplasia of the

22. Leukocytosis over 11,0×109/l

26. Polymorphism of MTHFR (C/T,

gland and a number of others.

15.

test.


2. Oligozoospermia (moderate and

3. Insufficient number of embryos

4. Homocysteine in blood of more than 15 nM/l (on the 1st point of

8. Peak thrombin more than 360

9. ETP more than 1900 nM/min

10. Unsuccessful IVF attempt in

11. D-dimer levels over 500 ng/mL

13. Polymorphism of PAI-I (5 G/4

15. EAAF index less than 11% (1st

polymorphisms MTHFR (C/T, T/T)

17. Fibrinosis greater than 5.0 g/l

16. The combination of

and PAI-I (5 G/4 G, 4 G/4 G)

mM/l (for 2nd point of

observation)

Failure of IVF (n=61)

1. Hyperstimulation (early stage) 12 19,6 0 0 32,0

severe) 26 42,6 4 6,3 10,9

transferred in IVF cycles (1-2) 23 37,7 8 12,7 4,16

5. Difficult embryo transfer 18 29,5 7 11,1 3,34

6. Mutation FII (G/A, A/A) 1 1,6 0 0 3,14

7. Defective embryo 17 27,8 8 12,7 2,65

(2nd point of observation) 41 67,2 31 49,2 2,11

anamnesis 11 18,0 6 9,5 2,09

(2-nd point of observation) 13 21,3 8 12,7 1,86

12. Age (36-40 years) 8 13,1 5 7,9 1,75

G, 4 G/4 G) 26 42,6 20 31,7 1,59

14. Cytomegalovirus infection 3 4,9 2 3,1 1,57

point of observation) 33 54,0 27 42,8 1,57

(for the 2nd point of observation) 8 13,1 6 9,8 1,43

Abs % Abs %

Success of IVF (n=63)

14 22,9 5 7,9 3,45

43 70,4 32 50,8 2,31

17 27,8 13 20,6 1,48

Odds ratio (0,95% CI)

(1,85-555) < 0,0001

(3,53-34,0) 0,0002

(1,68-10,28) 0,003

(1,16-10,2) 0,043

(1,28-8,74) 0,013

(0,12-78,8) 0,491

(1,04-6,72) 0,044

(1,10-4,84) 0,028

(1,02-4,38) 0,047

(0,72-6,06) 0,198

(0,71-4,87) 0,476

(0,53-5,68) 0,392

(0,76-3,32) 0,265

(0,25-9,78) 1,677

(0,77-3,19) 0,280

(0,64-3,40) 0,706

(0,46-4,40) 0,580

P-value

Criterion

observation)

**Table 14.** Factors contributing to the failure of pregnancy in IVF cycles in the 2nd group (n = 124)

In our publication we studied a number of women with the known risk factors of IVF failures to be less significant regardless of the ongoing correction of hemostasis and hypo fibrinolysis. We also record such risk factors as manifestations of inflammatory reaction (leukocytosis, fibrinosis) of virus (herpes, cytomegalovirus), the pathology of the thyroid gland and a number of others.

We are particularly interested in the reasons for the failure of IVF which remained relevant after therapeutic correction aimed at hemostasis and fibrinolysis. The list is given in Table. 15.

As noted earlier (Table 14), in addition to gynecological risk factors, hyperhomocysteinemia, and manifestations of excessive thrombin generation became the causes of the failure of IVF. Consequently, despite treatment some female patients maintained the trend to intravascular coagulation. Earlier, Table 11 demonstrated that isolated course of IPC to treat hypo fibrinolysis and other effects led to increased thrombin generation whereas the combination of IPC with dalteparin did not lead to such a shift (Table 12). Considering that we used three variants of therapeutic intervention our attempt was to determine the frequency of each of them under different outcomes of IVF in women with a high ETP in thrombin generation test.


The Means of Progress in Improving the Results of *in vitro* Fertilization Based on the Identification and Correction of the Pathology of Hemostasis 107

To sum up the results we note that excessive thrombin generation and a fibrinolytic inhibition fatal reaction (without correction) reduces the effectiveness of IVF and has a comparable value compared to the traditional risk factors of reproduction. Thus, our study proves a number of recent statements devoted to this problem (Martinez-Zamora et al., 2011; Meltzer et al., 2010; Nelson & Greer, 2008; Rova et al., 2012; Westerlund et al., 2012). However, we have made further progress due to suprathreshold values of laboratory parameters which allow to monitor the increased propensity to blood clotting and / or hypo fibrinolysis and therefore to identify patients with high risk of IVF failure in order to conduct therapeutic correction of disorders . In particular, by the calibrated test of thrombography administration of dalteparin was authorized by its indicators (ETP, Peack thrombin) to lead to the effective reduction of thrombin generation as well as the significant

We first proposed and tested method and mode of correction hypo fibrinolysis IPC for women in a cycle of IVF. It was shown that vases compression in these cases leads to the increased activity of t-PA and reduced PAI-1 activity, which is clearly manifested by the sharp increase in the calculated EAAF index , accelerated clotlysis time and the increase in the number of positive outcomes for assisted reproductive technology in 3 times. However, there were no obvious reasons to reduce the dynamic activity of PAI-1 during the course of IPC, even though it appeared to be a favorable result of the non-drug therapy. A negative consequence of the IPC was the phenomenon increasing the generation of thrombin which did not have the prior record. The calculations showed that the IPC in all cases should be combined with heparin prophylaxis to obtain the best clinical results. In this publication, combining vases compression with prophylactic doses of LMWH (low molecular weight heparin) really helped to increase the number of pregnancies in 6.5 times. In our opinion, this non-pharmacological approach to correcting hypo fibrinolysis demonstrates great potential for use in a number of clinical situations, including pregnancy period. In this chapter, we do not include the results of vases compression in women with low fibrinolytic activity after IVF in the first 12 weeks of pregnancy. However, the results are encouraging

The leading role of increased thrombin generation and hypo fibrinolysis in negative consequences of this reproductive technology has been proved in comparative evaluation of the significance of the risk factors of IVF failure. Its value appeared to be comparable with such risk factors as ovarian hyperstimulation syndrome, male factor, poor embryo or small quantities, or hard to bear embryos. In the meantime, the range of risk factors and their significance has changed dramatically after the treatment and correction of disorders of hemostasis and fibrinolysis. In particular, the list of relevant factors was reduced significantly to hypo fibrinolysis (rated by EAAF index) whereas indicators of excessive thrombin generation remained, though in less prominent positions. In addition, risk factors such as male factor, insufficient and difficult embryo transfer, as well as their low quality became more significant. The publication indicates the important role of hyperhomocysteinemia in the failure of IVF. As you know, it refers to the controllable risk

**6. Discussion** 

increase in positive outcomes of IVF (6.4 times).

and will be published later.

**Table 15.** Causes for the failure of IVF under conducted therapeutic correction of hemostasis and fibrinolysis (n = 98)

**Figure 4.** Analysis of the effectiveness of different therapies with excessive thrombin generation (ETP to 1900 nM/min in the 2nd point of observation)

The data in Figure 4 show that the high generation of thrombin and IVF failure is more common in the course of isolating IPC and less - in combination therapy.
