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ALTERATIONS OF THE HEMOSTATIC SYSTEM IN THE IVF CYCLE AND THEIR INFLUENCE ON THE PROCEDURE EFFICIENCY
1 Altai State Medical University, Barnaul
2Scientific center of hematology of the Ministry of Health of the Russian Federation, Barnaul 3The Ministry of Health of Altai Krai, Barnaul 4Altai regional clinical perinatal center, Barnaul
A.P.Momot12, I.V. Molchanova1'234, L.P. Tsyvkina1
In vitro fertilization (IVF) is widely used for the solvation of infertility problem. Its failures are connected with a number of reasons including disorders of the hemostatic system. In terms of the current study, there were analyzed risk factors of IVF failures before and after the therapeutic correction of excessive generation of thrombin and hypofibrinolysis.
Key words: extracorporal fertilization, pregnancy, thrombin generation, hypofibrinolysis, heparin prevention, vasocompression.
Today, assisted reproductive technologies (ART) and, first of all, in vitro fertilization (IVF) and embryo transfer (ET), intracytoplasmic sperm injection (ICSI), oocyte donation (OD) have turned from unique techniques for our country to almost routine ones, performed in terms of the program compulsory medical insurance (CMI).
The technique of these procedures is worked out in great detail and is successfully applied in ART centers throughout the country. In 2010, according to the data of the Ministry of Health of the Russian Federation in 2015, 37 664 cycles of ART in the framework of the CMI program were conducted in our country, which is 12 137 cycles more than in 2012. The wide introduction of ART, a sufficiently high level of qualification of reproductive and embryology specialists allowed not only to increase the number of cycles conducted throughout the country, but also to increase their efficiency to 32-33% [3, 4].
However, it should be recognized that an increase in the number of cycles and the accumulation of medical experience led to an avalanche-like increase in information about the problems of ART, including in regional centers, which include the Regional Center for the Preservation and Rehabilitation of Reproductive Function in Barnaul. The Center was established in 2009 with the support of the Governor of Altai Krai A.B. Carlin. The Center made a high-tech way for overcoming infertility available to the population, since its funding was initially provided at the expense of the regional budget. The state form of the work of this center allowed to accumulate invaluable experience and in the future to switch over to work on compulsory medical insurance, having increased volumes 4 times from 300 to 1200 cycles per year. At the same time, despite the annual increase in the effectiveness of ART, this issue is still acute.
It is known that pregnancy largely depends on two components - a functionally complete embryo at the blastocyst stage and endometrial receptivity, which, according to modern concepts, are crucial in achieving optimal implantation conditions, and that many of the mechanisms involved in this process (hormonal interactions, pinopodia formation, local immunity, autoimmune reactions, a complex of molecular and cellular interactions regulated by para- and autocrine factors, the formation of an implantation window), have not yet been studied sufficiently deeply [1, 2, 8, 9, 13.]. It is logical to assume that implantation is the most vulnerable stage of the IVF / ICSI and ET program both from the point of view of pregnancy and its bearing in the early stages. Undoubtedly, in all cases of absence or inferior implantation with further loss of pregnancy, the combined effect of various adverse factors is important. It may sound paradoxical, but the factors affecting the implantation processes and promote early pregnancy loss after IVF are considered by most authors to be the use of drugs - inductors of superovulation, which create prerequisites for abortion along with a high level of follicle-stimulating hormone, a change in the hormonal background, the phenomenon of hypercoagulation as a result of activities aimed at the maturation of follicles [5, 6, 7].
Among the persistent scientific searches for the role of thrombophilia in the formation of obstetric-gynecological pathology, there is no doubt that the stimulation of superovulation in the cycle of IVF is a factor of thrombogenic risk, contributing to the development of hypercoagulable syndrome, which is laboratory confirmed by an increase in von Willebrand factor, V and VIII factors, fibrinogen, Resistance of APC- (activated protein C), against the background of a decrease in the activity of the main physiological anticoagulants -antithrombin, proteins C and S. [10, 11, 12.]. However, scientific studies that determine the influence
of these haemosteziologic changes on the receptive properties of the endometrium and, ultimately, on the outcomes of in vitro fertilization are very few. But even in these works, diagnostic markers are not presented with clear, clinically relevant parameters of shifts in the hemostatic system, which are so important for the practice of the doctors of reproductive centers. Therefore, up to now, the issue of correction of changes in the system of hemosta-sis that have arisen against the background of stimulation of superovulation remains open and controversial, which served as a motive for our own research conducted by the Altai Branch of the Hematology Research Center of the Russian Ministry of Health and the Altai State Medical University.
Materials and methods
There were studied the changes in the coagulation hemostasis and fibrinolytic properties of 163 patients with tubal peritoneal infertility, which were exposed to IVF. Inclusion parameters:
1. Tubal-peritoneal factor of infertility
2. Age under 35 years
3. 1-2 unsuccessful IVF attempts in medical history
4. Identical protocol for the stimulation of superovulation (long-term protocol with the use of diperelin (0.1 mg) or decapeptil (0.1) and gonadotropins (puregon 150-250 IU or 225 IU). All patients received progesterone (dufaston) from the moment of follicular puncture at the dose of 30 mg per day, from the moment of embryo transfer -40 mg per day.
5. Good quality embryos (Class A and B)
Exclusion parameters:
1. Pathology of the reproductive system that changes the receptive properties of the endometri-um (congenital malformations, tumor and hyper-plastic processes, adenomyosis)
2. Severe somatic pathology (systemic, endocrine, autoimmune diseases)
3. Decreased ovarian reserve
4. Co-factor of infertility (male, endocrine)
The patients were young, without any special
health problems, both reproductive and somatic, and each of them had an unsuccessful attempt at IVF in the anamnesis.
By the choice of methods for the study of hemostasis, there were used modern, universally recognized and widely used markers of thrombotic readiness, reflecting the state of both coagulation hemostasis and fibrinolytic activity. However, it was taken into account that different factors of thrombogenic risk lead to violations at various stages of the coagulation cascade and fibrinolysis, and this process is accompanied by the appearance of markers specific for this vector of activation of the hemostasis system, which often makes interpretation of these data difficult.
This circumstance served as a motivation for the use of fundamentally new integral tests in the study, that allow to evaluate the final stage of blood coagulation. These tests include thrombin generation test (TGT) - thrombin peak concentration nmol/l and endogenous thrombin potential (ETP) (nmol x min). Its versatility lies in the fact that it reflects various mechanisms of activation of hemostasis, both coagulative and cellular, which together inevitably lead to thrombinemia.
Fibrinolytic blood activity was assessed using the index of fibrinolysis-activating capacity of the endothelium (FACE index,%), which was calculated from the ratio of the activity of the tissue activator plasminogen and its inhibitor, the lysis time of the clot.
The study of hemostasis was carried out three times, before the stimulation of superovulation, before puncture of the follicles and on the day of HCG.
Results and discussion
As a result of the study, it was found that changes in hemostasis against the background of stimulation of superovulation were detected in 114 patients (70%), low fibrinolytic activity before entry into the cycle - in 36 patients (22%), signs of hyper-coagulation - in 78 patients (48%)
In this case, the data given in Table 1 show, that stimulation of superovulation causes an increase in thrombin generation and practically does not affect the parameters of fibrinolytic activity of the plasma. The current study data fully agree with the findings published in 2012 by Westerlund, who believes that fibrinolytic activity has individual characteristics in each case [14].
When carrying out a correlation analysis of the detected changes in the hemostasis system and the unsuccessful attempts of IVF, it was determined that a high correlation was found between excessive generation of thrombin and low fibrino-lytic activity of the vascular wall with unsuccessful outcomes of IVF. The correlation coefficient (according to Spearman) was 0.88 (P <0.002) and 0.67 (P <0.02), respectively (Table 2).
Further, there were determined the threshold values of these tests, related to the failures of IVF. It turned out that pregnancy does not occur with an increase in endogenous thrombin potential against the background of stimulation of superovulation over 1900 nmol x min and PCC over 360 nmol x l (Table 3).
Failures of IVF are observed in patients with reduced fibrinolytic activity of the vascular wall according to the calculated FACE index less than 11% (Table 3).
Based on the obtained objective laboratory criteria, there were formed the groups of women who need therapeutic correction of hemocoagulation.
In patients with high thrombin generation there were used low molecular weight heparins, in women with impaired fibrinolysis, vasocompression therapy was used. Combined therapy was used by the combination of these changes. In the center for the preservation and restoration of the reproductive function, these therapies were applied in 98 women entering the IVF cycle, and the outcomes of assisted reproductive technologies were compared with a group of 154 women with identical changes in hemostasis not receiving the proposed therapy.
In the course of the study, it was determined that the use of low molecular weight heparin, starting the day after follicle puncture, led to a regular decrease in the intensity of the thrombin generation test, similar to the peak concentration of thrombin.
Similar results were obtained in patients with combined therapy with heparin and vasocom-pression. In women who underwent such therapy,
the parameters reflecting both the rate of fibrin formation and the fibrinolytic activity of blood plasma and the vascular wall were normalized.
As a result, it has been established that all types of treatment undertaken by us reduce the risk of a negative outcome of IVF. Reduction of relative risk (RRR) with the use of heparin therapy was more than 25%, which corresponds to a clear clinical effect. In particular, the reasonable designation of low molecular weight heparin with high thrombin generation helps to reduce the number of unfavorable outcomes of in vitro fertilization by 33%. When combining prophylactic doses of low-molecular heparin with vasocompression in the case of combined hemostasis and fibrinolysis, the relative risk was more than 50%, indicating a pronounced clinical effect. In this clinical group there was a decrease in the number of failures in the cycle of IVF by almost 40%.
Table 1 Dynamics of hemostasis and fibrinolysis (M + SD) by superovulation and embryo transfer to the uterine cavity (n = 163)
Index 1 observation point 2 observation point 3 observation point
ETP, nmol/min 1461,2±81,6 1849,3±89,2 P1-2<0,001 P2-3<0,001 1891,4±53,5 P1-3<0,001
PCC, nmol/l 310,1±23,1 382,4±19,5 P1-2<0,001 P2-3<0,001 375,6±25,2** P1-3<0,001
t-PA, activity, un/ ml 0,30±0,16 0,33±0,15 P1-2>0,5 P2-3>0,5 0,31±0,15 P1-3>0,5
PAI-1, activity, un/ml 2,40±1,13 2,50±1,22 P1-2>0,5 P2-3>0,5 2,44±1,98 P1-3>0,5
FACE index, % 12,5±3,1 13,2±4,2 P1-2>0,5 P2-3>0,5 12,7±3,6 P1-3>0,5
Clot lysis time, min 9,4±3,0 8,7±3,3 P1-2>0,5 P2-3>0,5 10,1±3,5 P1-3>0,5
Note: * - in this table, the significance of differences compared to the control indicators - Pc <0.05; ** - the same, P <0.01. ' c
Table 2 Estimation of informative value of changes in hemostasis parameters to unsuccessful outcomes of IVF
Index Correlation coefficient (P - significance)
at the 1st point of observation at the 2nd point of observation
ETP, nmol/min 0,03 (0,26) 0,79 (0,001)
PCC, nmol/l 0,08 (0,44) 0,88 (0,002)
Level of plasminogen, % - 0,31 (0,23) - 0,24 (0,31)
Clot lysis time, min 0,71 (0,01) 0,66 (0,02)
t-PA, activity, un/ml 0,60 (0,02) 0,52 (0,01)
PAI-1, activity, un/ml 0,51 (0,01) 0,47 (0,02)
FACE index, % 0,67 (0,02) 0,53 (0,01)
Table 3
Factors contributing to the failure of pregnancy in the cycle of IVF (n = 163)
Feature IVF failure (n=107) IVF success (n=56) MIS (0,95% CI*) P
Abs % Abs %
2. ETP over 1900 nmol / 27,9 (9,33-83,4)
min (at the 2nd point of 73 68,2 4 7,1 <0,00001
observation)
3. PCC over 360 nmol / l (at the 2nd point of observation) 74 69,1 5 8,9 22,8 (8,36-62,5) <0,00001
5. Clot lysis time over 12 18,5 (6,81-50,3)
min (at the 1st point of observation) 69 64,5 5 8,9 <0,000001
6. FACE index under 11% (at 62 57,9 6 10,7 11,5 0,00005
the 1st point of observation) (4,53-29,1)
9. FII gene mutation (G/А, А/А) 3 2,8 0 0 3,78 (0,19-74,5) 0,319
11. Endometrial Hypoplasia 11 10,3 3 5,3 2,02 (0,54-7,57) 0,383
12. IVF failure in anamnesis 20 18,7 6 10,7 1,91 0,260
(0,72-5,08)
13. Homocysteine in the blood over 15 mkmol / l (at the 1st point of observation) 22 20,5 7 12,5 1,81 (0,72-4,54) 0,280
16. ombination of polymorphisms MTHFR (G/А, А/А) и PAI-I (G/А, А/А) 26 24,3 9 16,0 1,67 (0,72-3,87) 0,315
18. PAI-I gene polymorphism (G/А, А/А) 42 39,2 20 35,7 1,16 (0,59-2,27) 0,735
20. Hyperfibrinogenemia over 5.0 g / l (at the 2nd point of observation) 14 13,0 7 12,5 1,05 (0,39-2,78) >1,00
21. The level of D-dimers 0,99 (0,42-2,31)
over 500 ng / ml на (at the 2nd point of observation) 19 17,7 10 17,8 >1,00
26. MTHFR gene polymorphism (G/А, А/А) 30 28,0 23 41,0 0,55 (0,28-1,10) 0,113
27. FV Leiden gene mutation (G/A) 1 0,9 1 1,7 0,51 (0,03-8,45) >1,00
Table 4
Influence of used methods of correction of hemostatic and fibrinolytic reactions on the efficacy of IVF
Means of therapeutic effect
Those in need of treatment, but not treated (subgroups 1.2 and 2.2.4, n = 154)
Abs. Pregnant %
Those in need of treatment and treated (subgroups 2.2.1, 2.2.2 and 2.2.3, n = 98)
Abs. Pregnant %
Heparin prophylaxis 64 4 6,2 38 15 39,5
PPK course 48 7 14,6 23 10 43,5
Combined effect 42 3 7,1 37 17 45,9
Effectiveness of various therapies in women in the IVF cycle
Table 5
Means of therapeutic effect Index
ARR NNT OR CI 95% (RRR) CI 95% (OR) RRR%
Heparin prophylaxis 0,27 3,7 0,22 0,52-0,92 0,07-0,69 31
PPK course 0,31 3,2 0,19 0,44-0,94 0,05-0,65 35
Heparin prophylaxis combined with PPK course 0,55 1,8 0,24 0,23-0,60 0,02-0,22 63
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Contacts
Corresponding author: Momot Andrei Pavlov-ich, Doctor of Medical Sciences, Professor, Head of the laboratory of hematology of Central research and development laboratory of ASMU, Barnaul. 656045, Barnaul, ul. Lyapidevskogo, 1. Tel.: (3852) 689-800 Email: [email protected]
