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O. I. Polishchuk, I.P. Polishchuk, N.I. Genyk Hemostasis Condition in Pregnant Women with a Threat of Late Spontaneous Miscarriage Associated with Metrorrhagia and Ascending Infection Ivano-Frankivsk National Medical University, Ivano-Frankivsk, Ukraine Abstract The subject of the research was the condition of hemostasiogram in chronic bleeding and vaginal microbiocenosis in pregnant women with the threat of late spontaneous miscarriage. The objective was to define a condition of vaginal microbiocenosis in pregnant women with the threat of late spontaneous miscarriage associated with chronic metrorrhagia and to investigate hemostasis changes. Materials and methods. Pregnant women with the threat of late spontaneous miscarriage were under the supervision. Clinical, microbiological, hemostasiological, and statistical methods were used in the research. The results. According to the results of the research, the number of lactobacillus (LB) decreases and the growth of obligatory and facultative anaerobes increases in pregnant women with threat of initial late involuntary miscarriage associated with chronic metrorrhagia. Moreover, the vaginal microecology worsens with prolongation of bleeding and the risk of ascending fetal infection and pregnancy loss appears. Conclusions. Local inflammatory response to the arisen infection is manifested in activation of blood coagulation system. Generalized hypercoagulation on the background of blood fibrinolytic properties dicrease leads to chronic DIC. Whereas, the majority of coagulative cascade components stimulate the inflammatory response. Therefore, metrorrhagia during the threat of late spontaneous miscarriage should be considered as a negative factor which causes changes of the vaginal microecology and promotes the ascending placenta and fetal infection and demands specific anti-infectious actions and use of antihemorrhagic preparations. Keywords: miscarriage; metrorrhagia; ascending infection. Problem statement and recent research analysis Numerous recent years researches have considered miscarriage in terms of cause and its elimination [6,7] but there is still no absolute answer about consequences, and especially about interrelation of vaginal microbionomics change [4] and blood rheology [1,3,5,7]. This determined the purpose of our research. Materials and methods 82 patients at the age of 21-35 years with a singleton pregnancy without extragenital pathology were under the supervision. Local or systemic use of antibiotics was not prescribed for the women during the last two weeks. The patients included 52 pregnant women with the threat of late spontaneous miscarriage accompanied by metrorrhagia and 30 pregnant women with physiological pregnancy (PP) (control group). The research was conducted on the 1 st and 7th days from the onset of specified symptoms. Laboratory researchs included microscopic evaluation of vaginal smear according to Gram in Kopeloff modification, bacteriological inoculation of media to determine facultative anaerobic bacteria (FA) and obligate anaerobic bacteria (OA) and to release Gardnerella vaginalis, quantitative analysis of vaginal microflora [2]. Recommendations and charts “Bergey's Manual of Systematic Bacteriology” were laid in the basis of isolated cultures identification. During clinical examination hemostasiogram changes were studied including platelet count; spontaneous platelet aggregation; the determination of Lee-White clotting time; blood plasma recalcification time; activated recalcification time (ART); plasma tolerance to heparin; prothrombin index; active partial thromboplastin time (APTT); amount of fibrinogen, plasminogen, antiplasmin, Antithrombin III; plasma fibrinolytic activity; ethanol gelation test and protamine sulfate test. Statistical processing of results was performed in accordance with generally accepted methods of variation statistics. Results and discussion Vaginal microflora was observed in normal variant in all examined women with physiological pregnancy (PP) (Table 1). Changes in the composition and concentration of types during the research of vaginal microflora in pregnant women with the threat of late spontaneous miscarriage (TLSM) are presented in Table 1.

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Table 1 State of vaginal microbionomics in examined pregnant women Percent, % Concentration, CFU/ml Types

PP (n=30)

Pregnant with TLSM and bleeding on

PP (n=30)

7th day (n=20) L. acidophilus 100.0 97.61 7.14** L. fermentum 70.0 57.14 4.76** 107-108 L.plantarum 55.0 47.61 2.38** L. paracasei 2.38 L. coryneformis 2.38 Corynebacterium spp. 5.0 7.14 14.28* <104 Staph. saprophytics 20.0 21.42 35.71* Streptococcus spp. 7.14* Proteus mirabilis 5.0 4.76 9.52 <103 Gardnerella vaginalis 10.0 14.28 78.57*** E. coli 35.0 38.09 50.0* 103-104 Bifidobacterium spp. 20.0 19.04 11.9 106-107 Peptostreptococcus spp. 15.0 16.67 33.33* 103-104 Peptococcus spp. 10.0 11.9 21.43* Bacteroides spp. 5.0 4.76 22.22** <103 Veillonella spp. 4.76* Actinomycetes spp. 4.76* Mobiluncus spp. 45.24** Eubacterium spp. 11.9* Note: probability * – р1<0.05, ** – р1<0.01, *** – р2<0.001 compared to PP 1st day (n=32)

Pregnant with TLSM and bleeding on 1st day (n=32) 7th day (n=20) 107-108 104-105** <104

106-107** 104-105*

103-104 103-104 106-107 103-104 <10 -

105-106* 106-107** 104-105* 106-107***

3

<103 105-106*

Thus, significant changes were not observed in vaginal microbionomics of pregnant with TLSM compared to those with PP on the 1st day of metrorrhagia. LB were allocated in all patients in both examined groups as associations with a high concentration and high protective ability. However, on the 7 th day of metrorrhagia that had acquired qualities of chronic bleeding LB were preserved only in one-fifth of examined pregnant women in a concentration of 104-105 CFU/ml and were functionally defective (Table 1). FA were found only in monoculture in all examined pregnant women with physiological pregnancy and pregnant with the threat of late spontaneous miscarriage on the 1 st day of bleeding. FA were more common in association of 2 microorganisms in 30 (59.53%) women with the threat of late spontaneous miscarriage, in the association of 3 microorganisms in 7 (16.67%) women, in the association of 4 microorganisms in 5 (11.9%) women (Table 1) at 7th day of metrorrhagia. OA were found only in monoculture in all examined women with physiological pregnancy and in pregnant with TLSM on the 1st day of bleeding. OA were more common in association of 2 microorganisms in 22 (71.42%) women with the threat of late spontaneous miscarriage, in the association of 3 microorganisms in 12 (28.57%) women, in the association of 4 microorganisms in 8 (19.04%) women (Table 1) at 7 th day of metrorrhagia. The research of platelet hemostasis links stated that hyperaggregation (р<0.05) and hyperthrombocytosis (р<0.01) increased in pregnant with the threat of late spontaneous miscarriage along with the increase in metrorrhagia duration. These effects were manifested in thrombocyte aggregation inducted with adrenaline (Table 2). The research found a natural activation of the hemostatic system, which was manifested in blood clotting time reduction, ART, increase in plasma tolerance to heparin during the first phase; levels of prothrombin index increased during the second phase; fibrinogen level increased on the background of APTT falling with the increase in bleeding duration during the third phase (р<0.01) (Table 2). Along with the activation of blood coagulation system on the background of chronic metrorrhagia in pregnant women with TLSM, anticoagulant amount decreased. It was manifested in a reduction of activated partial thromboplastin time and the growth of antithrombin III content (Table 2).

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The research found fibrinolytic system and plasmin activity insufficiency. The progressive reduction in their rates in pregnant with the threat of late spontaneous miscarriage depending on the day of bleeding (p <0.05) only continued (Table 2). Increase in fibrinogen B, ethanol and protamine sulfate tests were observed along with increasing duration of bleeding. Table 2 Changes of hemostasis system indices in pregnant women with TLSM (М±m) Investigated indices

PP (n=30)

Platelets, 109/ml 232.4±5.42 Platelet aggregation index, % 33.79±13.53 Blood clotting time, min 7.1±0.21 Recalcification time, sec 107.2±1.76 ART, sec 59.3±1.23 Plasma tolerance to heparin, min. 6.5±0.62 Prothrombin index, % 102.1±2.01 APTT, sec 39.3±1.25 Fibrinogen, gr/l 4.3±1.52 Antithrombin ІІІ, % 55.4 4.64 Total fibrinolytic activity, % 94.4 5.73 Plasminogen gr/l 153.6 2.42 Plasmin, % 89.7 10.01 Antiplasmin, % 81.3 6.35 Hageman-dependent factor, min 22.4 3.74 Ethanol test -/+ Protamine sulfate test -/+ Fibrinogen В -/+ Note: probability * – р1<0.05, ** – р1<0.01 compared to PP

Metrorrhagia on the 1st day (n=32)

Metrorrhagia on the 7th day (n=20)

218.5±23.72 38.07±11.01 6.5±1.12 104.3±1.73 65.78±3.65 6.1±0.67 105.2±1.56 34.5±0.51 4.4±1.56 56.7±4.75 87.56±3.45 143.45±3.63 92.4±9.13 94.4±5.84 27.5±2.43 -/+ -/+ -/+

171.3±3.42* 57.46±4.92** 3.7±1.43** 92.3±1.73* 83.8±4.09* 3.4±0.56** 118.9±1.93* 27.5±1.04* 6.5±1.39* 80.6 4.52* 79.1 4.71* 139.1 4.24* 111.7±6.33* 129.1 7.91* 39.2±2.31* + + ++

The presence of hypercoagulation with determined positive paracoagulation tests indicated the local DIC development where the degree of its severity increased with the duration of bleeding Conclusions 1. The number of LB decreases and the growth of bacteria’s obligate and facultative anaerobe increases on the background of a chronic metrorrhagia in pregnant with TLSM. Moreover, with bleeding prolongation the vaginal microbionomics only gets worse and the risk of ascending fetal infection appears resulting in pregnancy loss. 2. Local inflammatory response to the infection manifests in activation of blood coagulation system. Generalized hypercoagulation on the background of fibrinolitic properties depression of a blood leads to chronic DIC. In turn, the majority of the coagulation cascade components stimulate the inflammatory response. Prospects for further research involve a study of aninflammatory cytokines in pregnant women with the threat of late spontaneous miscarriage and selection of the most effective means of vaginal dysbiosis pathogenetic correction. References 1. Alekseyeva LA, Ragymov AA. DIC- syndrome. Moscow. GEOTAR-media, 2010; 120. 2. Strizhakov AN, Davydov AI, Ignatko IV [et al.] High-tech methods of the mother and fetus’ condition research: ensuring the health of future generations. Voprosy ginekologii, akusherstva i perynatologii. 2012; 11 (4): 6-12. 3. Makatsaryia AD. Systemic inflammation response syndrome in obstetrics. Moscow. MIA. 2006; 448.

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Gaboriau-Routhiau V, Lécuyer E, Cerf-Bensussan N. Role of microbiota in postnatal maturation of intestinal T-cell responses. Curr Opin Gastroenterol. 2011; 27: 502–508. Kobayashi T. Obstetrical disseminated intravascular coagulation score. Obstet. Gynaecol. Research. 2014; 40 (6): 1500–1506; Rattray DD, O’Connell CM, Baskett TF. Acute disseminated intravascular coagulation in obstetrics: a tertiary centre population review (1980 to 2009). J Obstet Gynaecol Can. 2012; 34 (4): 341–347. Niebyl JR, Galan HL, Jauniaux EМ. [et al.] Obstetrics: normal and problem pregnancies. Elsevier Health Sciences. 2012; 445–446.

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