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UDC 618.2:612.273.2-092.4
THE ROLE OF ACUTE HYPOXIC HYPOXIA IN INCREASING THE CONCENTRATION OF AMNIOTIC LIQUID COMPONENTS IN RABBITS AT LATE TERMS OF PREGNANCY
1 Altai State Medical University, Barnaul
2 State University of New York at Albany, College of Nanoscale Science and Engineering, New York Ye.V. Suzopov1, A.V. Popotseva1, I.A. Lytar'1, L.A. Kinan2, Yu.V. Korenovsky1
Research objective: to assess the influence of 60 minute exposure to acute hypoxic hypoxia (10,0 ± 2,0 % O2) on the amniotic fluid (AF) of first-pregnant rabbits on the 27-28th day of gestation.
Materials and methods: Female rabbits were randomly divided into experimental (n = 9) and control groups (n = 6). Volume of amniotic fluid, osmolality and concentration of electrolytes - Na+, K+, Cl-, non-organic phosphate (P), Ca2+ and organic components (creatinine, urea, protein) in AF were measured.
Results: It was found that acute hypoxic hypoxia causes an increase in volume and osmolality of AF, as well as an increase in AF's electrolytes and organic components. We suppose that changes in AF parameters were caused by an increase of AF formation and an acceleration of water resorption in fetus kidneys or fetus membranes. Conclusion: It was found that an increase in AF osmolality during hypoxia is associated with an increasing concentration of normal components of AF, and it is possible that this phenomenon may increase the concentration of diagnostic biomolecules. We hypothesize that correcting the concentration of diagnostic biomolecules in AF according to it's osmolality (especially in hypoxia) could provide a more precise diagnostic technique than using raw concentrations.
Key words: amniotic fluid, pregnancy, fetus, hypoxic hypoxia, rabbits.
The volume and composition of the amniotic fluid (AF) reflects the homeostasis of the mother and the fetus, therefore, it is useful to monitor it during pregnancy with the definition of a number of important parameters [1]. However, the study of the regulation of the volume and composition of AF is hampered by the presence of a number of sources of its formation and outflow [2].
The regulation of the volume and composition of AF involves several mechanisms: fetal diuresis, intramembrane pathway (transfer of water and electrolytes from AF to fetal vessels), ingestion of AF to the fetus, secretion by the lungs and na-sopharyngeal of the fetus, transmembrane pathway (absorption of liquid through the amnion into the mother's body), percutaneous pathway and absorption through the umbilical cord epithelium [3]. It should be borne in mind that by the 20th day of pregnancy, the skin of the fetus and the surface of the umbilical cord are keratinized, as a result of which the percutaneous and umbilical cord paths do not practically transport AF components [4]. The remaining six paths can be divided into pathways of formation (fetal urine, lung secretions and nasopharyngeal secretions) leading to an increase in AF volume, and outflow pathways (intramembrane pathway, ingestion and transmembrane pathway) leading to its decrease.
Fetal diuresis is the main way of forming AF [5]. There is shown a direct correlation between the speed of the urine flow of the fetus and the volume of the AF [6], which forms approximately three-quarters of the volume of the AF,
being the main source of its ionic and organic composition [2]. It is also known, that the course of pregnancy largely depends on the provision of normal respiratory metabolism between the mother's body and the fetus. Recent studies show that chronic hypoxia does not cause changes in the volume of AF [7].
The purpose of this study was to study the effect of acute hypoxic hypoxia on the volume and electrolyte composition of the amniotic fluid on the 2728th day of pregnancy.
Materials and methods
The study was conducted on primigravida rabbits (n = 15) weighing 4-5 kg at a period of gestation of 27-28 days (with a normal gestational age of 31 days). Fertilization was performed by various randomly selected males, after which the female rabbits were kept in single cages on a free diet. Rabbits were randomly divided into two groups: experimental (n = 9) and control (n = 6).
Rabbits from the experimental group were placed in an individual hermetic flow chamber for 60 minutes, where a gas mixture containing 10 ± 2% oxygen and 90 ± 2% nitrogen was injected with a compressor. The control of the gas composition in the chamber was carried out using a Microlux O2+CO2 gas analyzer (Mikrolux LLC, Yekaterinburg, Russia). Rabbits from the control group were placed in the same chamber for 60 minutes, containing atmospheric air.
After that, animals were killed by the method of cervical dislocation and in 15 minutes exposed
to midline laparotomy, and the uterus was removed. Amniotic bags with fetuses were isolated and removed from the uterine cavity and the maternal and fetal parts of the placenta were separated without disturbing the integrity of the amniotic sac. The amniotic fluid was removed from the am-niotic sac by a single-use syringe.
Criteria for the inclusion of the rabbits in the study: 1) gestational age of 27-28 days; 2) the mass of pregnant rabbits 4-5 kg. The criterion for excluding the fetuses from the study is the weight of the fetus less than 20g. The general characteristics of the rabbits and their fetuses are presented in Table 1.
Table 1
Mass of the fetus and placenta on the 27-28th day of life
Hypoxia (n = 43) Control (n = 40) P
Fetus weight, g 37,8 (30,6-50,3) 38,4 (31,0-41,8) P = 0,280
Mass of the fetal part of placenta, g 3,37 (2,79-3,71) 3,47 (3,10-4,22) P = 0,180
Note: data are presented in the form of - median (25-75%), P - the significance of intergroup differences according to the Mann-Whitney U-test.
Samples of amniotic fluid were obtained from the rabbits of the experimental group (n = 43) and the control group (n = 40). Samples were cen-trifuged for 15 minutes by 1200 g, frozen and stored at a temperature of minus 20 ° C no more than one month before the biochemical study. In samples of amniotic fluid, concentrations of Na+, K+, Cl-ions, inorganic phosphate (Pi) and Ca2+, creatinine, urea, protein and osmolality were determined.
Biochemical studies were performed on an automatic biochemical analyzer Dimension Xpand (Siemens, Germany). The concentration of Na+, K+ and Cl- ions was determined by a potentiometric method using the QuikLyte Integrate Multisensor module (Siemens, cat. No. S600, USA); total concentration of Ca2+ - colorimetric method with CA Calcium Flex reagent cartridge reagent kit (Siemens, cat. No. EA4164, USA); the concentration of Pi - by the colorimetric method using the PHOS Phosphorus Flex reagent cartridge reagent kit (Siemens, cat. no. EA4172, USA). Osmolality of AF was determined using a Vapro osmometer (Wes-cor, United States). The concentration of creati-nine in AF was determined by the modified Jaffe method using CREA Creatinine Flex reagent cartridge reagents (Siemens, cat. No. DA4254, USA); the concentration of urea - kinetic enzymatic (ure-ase) method reagents BUN Urea Nitrogen Flex reagent cartridge ("Siemens", cat. No. EB4309, USA); protein concentration - by a colorimetric method with pyrogallol red using the Belok-PGK-Novo reagent kit (Vector-Best, Cat No. B-8084, Russia) using the Bellur-600 total protein analyzer (Tehnome-dika Research and Production Enterprise, Russia). This work has been approved by the local ethical committee of the Federal State Budgetary Educational Institution of Higher Education of the Altai State Medical University.
Data processing
The statistical data processing was performed using the JMP 7.0 program (SAS Institute, USA). The median, 25 and 75 percentiles, the accu-
racy of intergroup differences were calculated by the Mann-Whitney test, multiple correlation analysis - by the Spearman test. The level of statistical significance was taken as 5% (p <0.05).
Results and discussion
Acute hypoxic hypoxia caused an increase in the osmolality of the AF and the concentration of all the studied inorganic ions and organic components (Table 2).
There was found an increase in the concentration of all the components studied, as well as os-molality of the AF, including an increase in only the filtered components — creatinine and urea. This suggests that filtration in the kidneys of the fetus has increased, but water reabsorption has also increased, either at the stage of the formation of fetuses by the kidneys [7] or in the amniotic sac itself [8]. Considering the existence of two transport systems in the fetal membranes - water transport by aquaporins [9] and transcytosis [10], as well as the fact that the concentration of most of the studied components (except Na+ and Cl- ions) were exceeded by about a third, the most likely is the combination of the activation of reabsorption of AF components in fetal membranes by both of these mechanisms.
The volume of AF correlated with the concentration of creatinine and Ca2+ by acute hypoxic hy-poxia (r = 0,460, p = 0,023; r = 0,445, p = 0,029, respectively), but did not correlate in the control group (r = 0.093, p = 0.597; r = 0.187, p = 0,286, respectively), which indicates an increase in the filtration process in the kidneys of the fetus. In addition, the volume of the amniotic fluid was normally inversely related to the urea concentration (r = -0.481, p = 0.0004), by acute hypoxic exposure, the connection became direct (r = 0.434, p = 0.034).
Conclusion
In accordance with the knowledge of the ways of AF formation (fetal urine, secretion by the lungs and nasopharynx, intramembrane pathway, swal-
lowing, transmembrane pathway), the revealed AF concentration on the background of acute hypoxic hypoxia is likely due to the removal of water from
tration of biomolecules in AF during hypoxia, for example, lactate [11] or interleukin-8 [12], may be associated with an increase in the osmolality of AF. To normalize the concentration of biomolecules in AF (especially during hypoxia), it may be important to calculate the relationship of the studied biomolecules to osmolality or the normal component of fetal urine, for example, creatinine [3].
References
1. Korenovsky Yu.V.£j, Lytar I.A., Buryako-va S.I. Amniotic fluid volume regulation. Obstetrics and gynecology. 2016;2:44-48.
2. Brace RA, Cheung CY. Regulation of amniotic fluid volume: evolving concepts. Adv Exp Med Biol. 2014;814:49-68.
3. Torrance HL, Pistorius L, Voorbij HA, Visser GH. Lactate to creatinine ratio in amniotic fluid: a pilot study. J Matern Fetal Neonatal Med. 2013;26(7):728-730.
4. Maruyama T, Yoshizuka M, Fujimoto S. Light and electron microscopy of fetal rabbit skin with special reference to the role of mesenchymal cells in epidermal differentiation. Acta Anatomica. 1988;133:143-155.
5. Beall MH, van den Wijngaard JP, van Ge-mert MJ, Ross MG. Amniotic fluid water dynamics. Placenta. 2007;28:816-23.
6. Mann SE, Nijland MJ, Ross MG. Ovine adaptations to chronically reduced urine flow: preservation of amniotic fluid volume. J Appl Physiol. 1996;81:2588-2594.
7. Thurlow RW, Brace RA. Swallowing, urine flow and amniotic fluid volume responses to pro-
the resulting primary urine of the fetus or fetal membranes from the amniotic sac.
necol. 2003;189:601-608.
8. Daneshmand SS, Cheung CY, Brace RA. Regulation of amniotic fluid volume by intramem-branous absorption in sheep: role of passive permeability and vascular endothelial growth factor. Am J Obstet Gynecol. 2003;188:786-93.
9. Mann SE, Dvorak N, Gilbert H, Taylor RN. Steadystate levels of aquaporin 1 mRNA expression are increased in idiopathic polyhydramnios. Am J Obstet Gynecol. 2006;194:884-887.
10. Chen J, Braet F, Brodsky S, Weinstein T, Romanov V, Noiri E. et al. VEGF-induced mobilization of caveolae and increase in permeability of endothelial cells. Am J Physiol Cell Physiol. 2002;282:1053-63.
11. Wiberg-Iteel E, Pettersson H, Andolf E, Hansson A, Winbladh B, Akerud H. Lactate concentration in amniotic fluid: a good predictor of labor outcome. Eur J Obstet Gynecol Reprod Biol. 2010;152(1):34-38.
12. Kacerovsky M, Drahosova M, Hornycho-va H, Pliskova L, Bolehovska R, Forstl M, Tosner J, Andrys C. Value of amniotic fluid interleukin-8 for the prediction of histological chorioamnionitis in preterm premature rupture of membranes. Neuro Endocrinol Lett. 2009;30(6):733-8.
Contacts
Corresponding author: Suzopov Yegor Valer-yevich, Laboratory Assistant of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics of ASMU, Barnaul. Tel.: (3852) 241309. Email: [email protected]
Effect of acute hypoxic hypoxia on the volume and composition of amniotic liquid of female rabbits on 27-28 days of pregnancy Table 2
Hypoxia (n = 43) Control (n = 40) P
Amniotic fluid volume, ml 0,36 (0,15-0,88) 0,46 (0,24- 0,78) 0,357
Osmolality, mosmol/ kg 301,0 (295,8-307,3) 237,0 (223,0-245,3) < 0,001
Na+, mmol/l 144,0 (142,0-148,0) 132,0 (129,0-135,3) < 0,001
K+, mmol/l 10,50 (9,80-11,13) 7,40 (6,68-8,93) < 0,001
Cl-, mmol/l 109,0 (107,0-111,0) 102,0 (100,8-106,0) <0,001
Ca2+, mmol/l 2,99 (2,51-3,55) 2,13 (2,05-2,30) < 0,001
Pi, mmol/l 1,32 (1,12-1,55) 1,00 (0,78-1,11) < 0,001
Protein, g/l 5,15 (4,89-5,38) 3,65 (3,15-3,75) < 0,001
Creatinine, mmol/l 0,200 (0,162-0,231) 0,155 (0,136-0,166) < 0,001
Urea, mmol/l 8,20 (7,75-8,70) 6,50 (6,05-6,85) < 0,001
Note: Data are presented in the form test. - median (25-75%); p - the reliability of intergroup differences according to the Mann-Whitney
These data show that an increase in the concen-
longed hypoxia in the ovine fetus. Am J Obstet Gy-
Author information
Popovtseva Anna Valentinovna, Assistant of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics of ASMU, Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: (3852) 24-13-92.
Email: [email protected]
Lytar' irina Aleksandrovna, Assistant of the Department of Pathological Physiology of ASMU, Barnaul.
656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 24-19-71. Email: [email protected]
Kinan Liza Anna, Specialist of State University of New York at Albany, College of Nanoscale Science and Engineering, New York. Tel.: +1 518-437-8686 Email: [email protected]
Korenovsky Yury Vladimirovich, Candidate of Medical Sciences, Associate Professor, Head of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics of ASMU, Barnaul.
Tel.: (3852) 566862. Email: [email protected]
