Steroid profiling in pregnancy: A focus on the human fetus Hill, Martin; Pašková, Andrea; Kančeva, Radmila ...
The Journal of steroid biochemistry and molecular biology,
January 2014, 2014-Jan, 2014-1-00, 20140101, Letnik:
139
Journal Article
Recenzirano
•Steroid profiling and physiological relevance of bioactive steroids in pregnancy.•Risk of reproductive disorders, misbalance of HPA in further stages of human life.•Insulin insensitivity and ...cancer-risk assessment.•Changes in fetal steroid levels during pregnancy.
In this review we focused on steroid metabolomics in human fetuses and newborns and its role in the physiology and pathophysiology of human pregnancy and subsequent stages of human life, and on the physiological relevance of steroids influencing the nervous systems with regards to their concentrations in the fetus. Steroid profiling provides valuable data for the diagnostics of diseases related to altered steroidogenesis in the fetal and maternal compartments and placenta. We outlined a potential use of steroid metabolomics for the prediction of reproductive disorders, misbalance of hypothalamic–pituitary–adrenal axis, and impaired insulin sensitivity in subsequent stages of human life. A possible role of steroids exhibiting a non-genomic effect in the development of gestational diabetes and in the neuroprotection via negative modulation of AMPA/kainate receptors was also indicated. Increasing progesterone synthesis and catabolism, declining production of tocolytic 5β-pregnane steroids, and rising activities of steroid sulfotransferases with the approaching term may be of importance in sustaining pregnancy. An increasing trend was demonstrated with advancing gestation toward the production of ketones (and 3β-hydroxyl groups in the case of 3α-hydroxy-steroids) was demonstrated in the fetus on the expense of 3α-hydroxy-, 17β-hydroxy-, and 20α-hydroxy-groups weakening in the sequence C17, C3, and C20. There was higher production of active progestogen but lower production of active estrogen and GABAergic steroids with the approaching term. Rising activities of placental CYP19A1 and oxidative isoforms of HSD17B, and of fetal CYP3A7 with advancing gestation may protect the fetus from hyperestrogenization.
This article is part of a Special Issue entitled ‘Pregnancy and Steroids’.
Despite the extensive research during the last six decades the fundamental questions concerning the role of steroids in the initiation of human parturition and origin and function of some steroids in ...pregnancy were not definitely answered. Based on steroid metabolomic data found in the literature and our so far unpublished results, we attempted to bring new insights concerning the role of steroids in the sustaining and termination of human pregnancy, and predictive value of these substances for estimation of term. We also aimed to explain enigmas concerning the biosynthesis of progesterone and its bioactive catabolites considering the conjunctions between placental production of CRH, synthesis of bioactive steroids produced by fetal adrenal, localization of placental oxidoreductases and sustaining of human pregnancy. Evaluation of data available in the literature, including our recent findings as well as our new unpublished data indicates increasing progesterone synthesis and its concurrently increasing catabolism with approaching parturition, confirms declining production of pregnancy sustaining 5β-pregnane steroids providing uterine quiescence in late pregnancy, increased sulfation of further neuroinhibiting and pregnancy sustaining steroids. In contrast to the established concept considering LDL cholesterol as the primary substrate for progesterone synthesis in pregnancy, our data demonstrates the functioning of alternative mechanism for progesterone synthesis, which is based on the utilization of fetal pregnenolone sulfate for progesterone production in placenta. Close relationships were found between localization of placental oxidoreductases and consistently higher levels of sex hormones, neuroactive steroids and their metabolites in the oxidized form in the fetus and in the reduced form in the maternal compartment.
The boost in placental production of CRH in late pregnancy is specific for human. CRH receptors are expressed in the fetal zone of the fetal adrenal (FZFA). Hence, we evaluated the associations ...between the steroid metabolome and gestational age (GA). The levels of 69 steroids and steroid polar conjugates such as 3β-hydroxy-5-ene steroids (3βOH5S), 3-oxo-4-ene steroids (3O4S), progesterone 5α/β-reduced metabolites, 20α-hydroxy-metabolites of C21 steroids, C19 5α/β-reduced metabolites, 7α/β-hydroxy-metabolites of 3βOH5S, estrogens and 16α-hydroxy-metabolites of 3βOH5S and 3O4S, were measured by GC–MS in plasma from the umbilical artery (UA), umbilical vein (UV), and maternal cubital vein (MV) and in amniotic fluid (AF) in 12 women at normal labor and 38 women at preterm labor due to pathologies unrelated to steroid status. Using multivariate regression, prediction models for GA were completed for the individual body fluids. The conjugated 3βOH5S (the key products of the FZFA), estrogens, some polar conjugates of progesterone 5α/β-reduced metabolites and some steroid 7α/β- and 16α-hydroxy-metabolites showed strong positive correlations with the GA. The predictivity decreased in the following sequence UV (R=0.950), UA (R=0.945), MV (R=0.895), and AF (R=0.891). Although the predictivity of steroids in maternal blood was slightly less effective when compared with the UV and UA, it was the best solution for further practice.
Polarization microscopy, possibly together with some contrast techniques (dark field and color phase contrast), was used to study the periphyton (microbiome) growing on filamentous green algae. The ...material containing filamentous algae with periphyton on the surface was collected in the villages of Sýkořice and Zbečno (Křivoklátsko Protected Landscape Area). The objects were studied in a LOMO MIN-8 St. Petersburg polarizing microscope and a Carl Zeiss Jena NfpK laboratory microscope equipped with an In Ph 160 basic body with variable dark field or color phase contrast and a Nikon D70 DSLR digital camera. Cells of filamentous algae of the genera Cladophora, Vaucheria, and Oedogonium were studied and the periphyton attached to them formed by cyanobacteria of the genera Chamaesiphon and Pleurocapsa and algae of the genera Characium, including diatoms of the genera Eunotia and Synedra. In all cases, the cell walls of the host algae showed a very strong birefringence. In contrast, the walls of cyanobacteria of the genera Chamaesiphon and Pleurocapsa were characterized by a much weaker birefringence (Pleurocapsa somewhat thicker), and the diatom frustules of the genera Eunotia and Synedra were almost without a birefringence. Strongly birefringent granules were found in the cytoplasm of the green alga of the genus Characium, which forms periphyton on the filamentous green algae of the genus Vaucheria. The periphyton on the filamentous alga of the genus Oedogonium, formed by cyanobacteria of the genus Pleurocapsa and diatoms of the genera Eunotia and Synedra, deposited in a massive layer of mucus containing birefringent crystals, showed a particularly strong birefringence. At the end of the vegetation of filamentous algae, their parts and remnants of periphyton (diatom frustules and crystals) became part of the detritus at the bottom of the culture vessel. The use of polarization microscopy in the study of filamentous algae with periphyton on the surface allows us not only to determine the birefringence of the observed structures, but also to partially deduce their chemical composition, or regular arrangement of particles, so-called shape birefringence.
Simultaneous application of polarization microscopy and dark field techniques has been used to study the internal structure of microbial cells. The dark field technique displays subtle cell ...structures like glowing objects on a dark background. In the polarizing microscope, cross polarizing filters along with the first-order quartz compensator and a rotary table show the maximum birefringence of the individual structures. The material containing microorganisms was collected in the villages of Sýkořice and Zbečno (Křivoklátsko Protected Landscape Area). The objects were studied in a laboratory microscope Carl Zeiss Jena type NfpK equipped with In Ph 160 basic body with variable dark field, special condenser with interchangeable diaphragm apertures, a rotary table, Meopta Praha polarizer, analyzer, first-order quartz compensator from LOMO Sankt Petersburg, and a digital Nikon D 70 DSLR camera. Three orders of microorganisms were studied: Siphonocladales, Chlorococcales, and Peritricha. Anisotropic structures in different amounts and sizes (e.g., granules and microfibrils) or in different configurations (e.g., cell walls or pellicle) have been found in all Protista organisms under study. Filamentous algae of the genus
Cladophora
(Cladophoraceae, Siphonocladales, Ulvophyceae) featured a strongly birefringent cell wall (shape birefringence) surrounded by less birefringent periphyton (microbial biofilm), at the edges of which cyanobacterial fibers could be recognized—a very important finding. The coccal algae of the genus
Scenedesmus
(Scenedesmataceae, Chlorococcales, Chlorophyceae) exhibited not only strongly birefringent granules, but also strongly birefringent microfibrils in the cytoplasm outside the strongly birefringent cell walls—very important finding. Of all the studied microorganisms, the weakest birefringence was shown in the surface membrane (pellicle) of the
Vorticella
(Vorticellidae, Peritricha, Ciliata). On the other hand, the ring of cilia on the top of the body had a somewhat stronger birefringence—an important finding. In conclusion, the dark field technique provides a high contrast image in the microscope and, if supplemented simultaneously by polarization microscopy, will allow us to partially infer the composition of the examined structures.
Simultaneous application of polarization microscopy and Interphako interference contrast has been used to study the internal structure of algal cells. The interference contrast technique showed fine ...cell structures (important is the selection of interference colors according to the Mach-Zehnder interferometer setting). In a polarization microscope, the crossed polarization filters together with the first-order quartz compensator mounted turntable showed the maximum birefringence of the individual structures. Material containing green algae was collected in the villages Sýkořice and Zbečno, Protected Landscape Area (PLA) Křivoklátsko. The objects were studied in a Carl Zeiss Jena NfpK laboratory microscope equipped with an In 160 base body with an Interphako In contrast interference module including a Mach-Zehnder interferometer with variable phase contrast, a special condenser with interchangeable aperture plates, a turntable, a Meopta Praha polarizer, a LOMO Sankt Petersburg analyzer, and a quartz compensator with first-order red and the digital camera DSLR Nikon D 70. Green algae of three orders were studied: Siphonocladales, Zygnematales, and Desmidiales. Anisotropic structures were found in all studied representatives of the green algae of the phylum Chlorophyta. Especially their cell walls showed strong birefringence (in all representatives of these orders). On the other hand, a representative of the order Siphonocladales (the genus
Cladophora
, Cladophoraceae, Ulvophyceae) was rarely found to display weak birefringent granules of storage substances due to the setting of the Mach-Zehnder interferometer and the use of the first-order compensator (interference colors are intensified). In addition, a very weak birefringence of periphyton cells (microbial biofilm) was found. In the study of the second algae of the genus
Spirogyra
(Zygnemataceae, Zygnematales, Conjugatophyceae), a strongly birefringent connecting wall between algal cells was found in contrast to the weaker birefringence of the peripheral wall. It was the use of Interphako interference contrast together with polarization filters and a first-order quartz compensator that particularly emphasized the central part of the connecting wall. In the study of the twinned
Pleurotaenium
algae (Desmidiaceae, Desmidiales, Conjugatophyceae), a strongly birefringent wall was found along the periphery of the cell with a nucleus in the middle part (isthmus). In this narrowing in the center of the cell, a sharply delimited birefringent edge of the cell wall is visible, especially when using Interphako interference contrast along with crossed polarization filters and a first-order quartz compensator. In conclusion, Interphako interference contrast provides a high degree of image contrast in a microscope and, if suitably simultaneously complemented by polarization microscopy (including a first-order quartz compensator), it will allow us to infer some of the composition of the investigated structures. However, working with Interphako interference contrast is considerably more difficult (setting Mach-Zehnder interferometer) than using other contrast techniques (positive and negative phase contrast, color contrast, relief contrast, and dark field).
A simultaneous application of negative phase contrast and polarization microscopy was used to study the internal structure of microbial cells. Negative phase contrast allowed us to display the fine ...cell structures with a refractive index of light approaching that of the environment, e.g., the cytoplasm, and converted an invisible phase image to a visible amplitude one. In the polarizing microscope, cross-polarizing filters, together with first-order quartz compensator and a turntable, showed maximum birefringence of individual structures. Material containing algae was collected in ponds in the villages Sýkořice and Zbečno (Protected Landscape Area Křivoklátsko). Objects were studied in a laboratory microscope (Carl Zeiss Jena, type NfpK), equipped with a basic body In Ph 160 with an exchangeable module Ph, LOMO St. Petersburg turntable mounted on a centering holder of our own construction and a Nikon D 70 digital SLR camera. Anisotropic granules were found only in the members of two orders of algae (Euglenales, Euglenophyceae and Chlorococcales, Chlorophyceae). They always showed strong birefringence and differed in both number and size. An important finding concerned thin pellicles in genus
Euglena
(Euglenales, Euglenophyceae) which exhibited weak birefringence. In genus
Pediastrum
(Chlorococcales, Chlorophyceae), these granules were found only in living coenobium cells. In contrast, dead coenobium cells contained many granules without birefringence—an important finding. Another important finding included birefringent lamellar structure of the transverse cell wall and weak birefringence of pyrenoids in filamentous algae of genus
Spirogyra
(Zygnematales, Conjugatophyceae). It was clearly displayed by the negative phase contrast and has not been documented by other methods. This method can also record the very weak birefringence of the frustule of a diatom of genus
Pinnularia
(Naviculales, Bacillariophyceae), which was further reinforced by the use of quartz compensator—an important finding. Simultaneous use of negative phase contrast and polarization microscopy allowed us to study not only birefringent granules of storage substances in microorganisms, but also the individual lamellae of the cell walls of filamentous algae and very thin frustule walls in diatoms. These can be visualized only by this contrast method, which provides a higher resolution (subjective opinion only) than other methods such as positive phase contrast or relief contrast.
Polarization microscopy has been used to study the internal structures of microbial cells and in terms of the birefringence of these structures and its possible relation to the cell function and ...composition. Cyanobacteria of the genus Phormidium were found to contain no anisotropic structures, while other microorganisms were found to contain them, albeit to a different extent, size, and number. The flagellate Euglena was found to contain two large anisotropic bodies, whereas the flagellate of the genus Phacus belonging to the same systematic group Euglenales was observed to contain only one large anisotropic body (storage substances—paramylon). On the other hand, green algae of the genus Scenedesmus, whose cells form four-celled coenobia, contained clusters of small anisotropic granules composed also of storage substances (volutin). Minute anisotropic granules (storage substances) in two smaller clusters were found also in diatoms of the genus Navicula, whereas the green alga of the genus Mougeotia was revealed to contain, in addition to minute anisotropic granules (storage substances) occurring in low numbers in the cytoplasm, also a strongly birefringent cell wall (shape birefringence). Cells of the amoeba of the genus Naegleria and heliozoans of the genus Heterophrys were observed to contain only isolated tiny anisotropic granules (storage substances).
Four simple sets for digital microphotography are described that have been tested with the Carl Zeiss Jena, Meopta Prague, Lambda Prague, and LOMO Sankt Petersburg microscopes and with DSLR Nikon D ...70 and Nikon D 300 cameras. They permit precise image focusing in the camera using a prism Zeiss. The sets make use of commonly available extensions Zeiss, Praktica and reductions Nikon—Praktica manufactured by ROWI (without a lens) or HAMA (with a lens). An extension has further been designed and manufactured for connecting the DSLR Nikon D 300 camera fitted with the HAMA reduction (only with a lens) and a focusing extensible prism with Zeiss Jena light measurement. It permits a precise image focusing of low light intensity objects (autofluorescence or low-contrast or moving objects when using positive or negative phase contrast). The sets are applicable to all microscopes constructed according to German DIN industrial standards.
