4 Abstract Estrogens are considered to belong to chemicals that negatively affect the endocrine system, even if present at very low concentrations. They are discharged into environment as a result of an increasing application of drugs etc. This work is focused on the separation and quantification of five estrogens, namely estrone (E1), 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2) and estriol (E3) in natural water samples by HPLC-UV method. The chromatographic system consisted of a C18 stationary phase (SunFire® C18, 150 x 4.6 mm, octadecyl bounded to silica gel, particle size 5 µm) and binary mobile phase of acetonitrile/water in various ratios in isocratic separation mode. The effect of acetonitrile content in the mobile phase and flow rate of the mobile phase on retention and separation parameters was tested. Under the optimized separation conditions (acetonitrile/water 40/60 (v/v), 1.3 ml/min), all the compounds were baseline resolved and eluted within 15 min. These experimental conditions were applied to the calibration measurements which were carried out within the concentration range from 0.001 to 1 mg/ml. Limits of detection (LOD) and limits of quantification (LOQ) for the individual estrogens and their mixture (standards dissolved in methanol) were determinated. The detection...

Fertilization is a unique biological event where male and female gametes fuse together to produce a new organism. Before the gametes are able to fuse, however, they must undergo a series of controlled changes. For the male gamete, capacitation and acrosome reaction (AR) must occur, which take place during the sperm migration through the female genital tract. Unfortunately, while the process of capacitation has been known for over half a century, the molecular basis and influential factors behind it are not fully understood. Although estrogens have been considered mainly female reproductive hormones, there is increasing evidence suggesting that these steroids have an important role also in regulation of male reproductive functions. Sperm come into the contact with estrogens during their formation in the male and female genital tract, indicating that the hormone may play an important role in sperm maturation. In this study, we examined the importance of three endogenous estrogens (E1 -estron, E2 - 17β estradiol, E3 - estriol) and one synthetic estrogen (EE2 - 17α ethinylestradiol) on sperm maturation during capacitation and AR. Stimulatory effect were observed with all tested estrogens on both capacitation and zona pellucida induced AR. Moreover, we have determinied that the stimulatory effect on...

There are an increasing amount of compounds in the environment that can have a negative effect on reproductive parameters in both male and female organism. There has been a worldwide decline of sperm quality during past decades and this fact lead to an increase of unnatural ways of conception through assisted reproduction techniques in the specialised centres. Natural estrogens are one of these compounds and they get into waste water after being excluded from the body by the urine. They get back into the human body from drinking water or from the food, and they can interfere with function of endogenous hormones in very low concentrations. For these reasons it is up to date to deal with the influence of these compounds on mammalian sperm. For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the development of germ cells, spermatogenesis and processes leading to successful egg fertilization such as a capacitation or acrosomal reaction. Tyrosine phosphorylation is one of the essential steps for the properly ongoing process of capacitation in sperm followed by a...

Estrogen compounds are a subgroup of the environmental pollutants named Endocrine Disrupting Chemicals. It is a large group of chemicals which are capable of causing hormonal imbalance of an organism and affecting its reproductive, developmental or behavioral functions. In the environment, they are found in very low concentrations (below ng/l). This makes their detection and elimination highly problematic. The Research Centre for Toxic Compounds in the Environment (RECETOX) has been developing an experimental device which employes reverse osmosis (RO) to concentrate the trace amounts of micropollutants (e.g. estrogens). The aim is to lower the detection limit of subsequent analytical methods. RO seems to be a promising alternative to commonly used methods which are more time consuming and costly. The aim of this thesis is to create a mathematical model of RO, which would describe the process of estrogen concentration and therefore could contribute to the optimization of experimental device. The mathematical model was created in MATLAB-simulink and verified by comparison to thirteen experimental results. The solutions used to test the model were NaCl at 0.002–2 g/l and drinking water with 17-ethinylestradiol of 25 ng/l. In order to concentrate the retentate on a flat-sheet RO membrane the model achieves a mean relative error in the range of 0.9–4.9 % for NaCl and drinking water. For the concentration of estrogens the deflection is 3.6 %. Comparing the model and experimental concentration of NaCl on a spiral wound RO-membrane, the deviation is in the range of 5.9–8.7 %. So far, ratio of retentate volume from the beginning to the end of the process has been used to determine the process recovery. However, due to the results obtained in the thesis, this approach appears inaccurate. A more accurate recovery would likely be achieved by using the theoretical concentration value obtained by the proposed mathematical model. However, to increase reliability, the further measurements with estrogens are needed, and also to determine the causes of deflection of model and experimental retentate volume values at the end of the concentration

Patients with Turner syndrome (TS) have increased fracture risk and decreased bone density, patients with severe hemophilia have low bone density. The etiology of these changes hasn't been completely elucidated in any of the two diseases. Our aimes were to assess bone density and geometry at the radius using a new method peripheral quantitative CT densitometry (pQCT) and to describe associations between densitometry parameters and estrogen treatment in TS and laboratory as well as clinical markers of disease severity in haemophilia. Sixty-seven girls with TS (median age 14.3 years, range 6.0-19.4) and 42 boys with haemophilia (median age 12.7 years, range 6.6-19.2) have been measured using peripheral quantitative CT at the radius. The results have been compared to published reference data. Girls with TS had decreased cortical bone density and thinner cortex. These parameters were positively correlated to the length of estrogen treatment. Trabecular bone density was normal in prepubertal girls but it was decreased after puberty. There was no association between trabecular bone density and the estrogen treatment. Boys with haemophilia had decreased trabecular density and low muscle area at the forearm. Densitometry parameters were influenced neither by the clotting factor VIII/IX level nor by the frequency of...

At present, the issue of occurrence of female sex hormones, estrogens and progestogens, in aquatic ecosystems is often discussed by experts and the general public. These substances of steroid structure can be difficult to remove completely by conventional wastewater and drinking water treatment technologies. In given context advanced oxidation processes based on in situ generation of highly reactive hydroxyl radicals can be a suitable technique. This thesis deals with the study of kinetics and degradation products of photocatalytic decomposition of seven female sex hormones (estrone, -estradiol, estriol, ethinylestradiol, diethylstilbestrol, progesterone and norethindrone). Experiments were conducted in a laboratory glass reactor, which was equipped with an energy efficient UV-A LED light source (365 nm emission wavelength) and an immobilised photocatalyst in a form of TiO2 five-layer film deposited on glass. Model samples of water with the initial hormone concentration of 1 mg·L-1 were used and the degradation process was monitored by an HPLC-MS method. In the given system all compounds of interest except estriol had very significant tendency to be adsorb. In the case of estriol the formal rate constant of photocatalytic decomposition was determined based on the Langmuir-Hinshelwood model for two different initial concentrations, 0.5527 hour-1 (1 mg·L-1) and 0.1929 hour-1 (5 mg·L-1), and by comparison of these values it was found that the higher degraded compound concentration, the slower decomposition (fivefold increase of the initial concentration resulted in the constant decrease to almost one-third). Moreover nine degradation products of estriol photocatalytic decomposition were recorded and their structure was designed based on mass spectra. In the second thematic part of the thesis attention was paid to development of a SPE-HPLC-MS method for simultaneous determination of female sex hormones in water ecosystems, with emphasis on an extraction part optimization. The final samples treatment process included besides extraction with Supel™ Select HLB 200 mg SPE cartridges also mechanical impurities removal, hormones extraction from solids trapped on filtration material, sample acidification and extract purification with Supelclean™ ENVI-Florisil® cartridges. Optimised method was used for determination of female sex hormones in two Brno rivers, Svitava and Svratka. In the most cases the concentration was below the detection or quantification limit.

The thesis focuses on the use of passive sampling during an analysis of hormones in drinkable water. In the theoretical part there are described selected passive samplers and the sample of the type of POCIS is described at fuller length. The next chapter engages with steroid hormones and an input of estrogenic hormone into the environment. The thesis contains chapters dealing with possibilities of the determination of hormones in water. It is specialized on the analysis of drinkable and surface water. In the last chapter of the theoretical part there are introduced water works where samples for an experimental assessment were collected from. The experimental part of the thesis presents the specification of the extraction of analytes and an adjustment of used device. In the thesis there are specific calibrations for individual assessment of hormones. The results of the assessment for each collection point are ordered to a well-arranged table. Increased levels of targeted estrogens were not approved in most of sampling points.