The Endocannabinoid System and Its Relationship to Human Reproduction.

The endocannabinoid system is among the most important regulators of human reproduction. It already applies at the level of the sperm and the egg, plays an important role in the fertilization of the egg, its implantation, regulates the function of the placenta and participates in childbirth. The aim of this work is to summarize the knowledge accumulated so far and to show that the endocannabinoid system must be perfectly regulated in order to maintain a physiological pregnancy from implantation to delivery. Only an exceptional interplay of enzymes such as NAPE-PDL or FAAH, endogenous cannabinoids and cannabinoid receptors CB1 and CB2 can ensure the proper functioning of the reproductive organs and thus lead to delivery on time. Changes in the endocannabinoid system can lead to a number of pathological conditions, e.g., during blastocyst implantation, retardation of embryo development, impaired placental function or miscarriage. Soon, we can expect not only an understanding of all the regulatory events associated with the endocannabinoid system and other regulatory systems that participate in reproduction, but also several possibilities for pharmacotherapeutic interventions that can modify the formation, degradation and effect of endocannabinoids. It cannot be ruled out that some components of the endocannabinoid system could become a marker for monitoring pregnancy and childbirth.

The Effects of Different Types of Diets on Steroid Hormone Concentrations.

The great popularity of various diets in recent years has led us to reflect on their suitability for our health. The aim of this communication is to review current knowledge on the influence of the most well-known diets on the concentrations of the main steroids and to consider possible mechanisms. The influence of diet on hormone concentrations is expected, but the literature data on this topic are inconsistent and yield conflicting results. The main problem in evaluating these influences is the change in weight that a change in diet induces. This effect needs to be filtered out in order to discover interesting associations between diet and steroid hormones. This is illustrated by the example of the effects of ketogenic diets on testosterone levels in men, where the direct effect of the diet is to reduce testosterone levels, but a number of papers have described increases that are due to diet-related weight loss and the modification of obesity-induced changes. A second major driver is the change in circadian rhythm, and it is necessary to assess hormonal changes induced by changing the time of day of the diet. Such shifts within the circadian rhythm rather than due to a particular type of diet itself are documented by changes in the circadian rhythm of cortisol.

The Endocannabinoid System - The Prediction of Spontaneous Preterm Birth in High-Risk Women: Protocol of a Study.

Spontaneous preterm birth (sPTB) is a major cause of perinatal morbidity and mortality, even in developed countries. Prediction of sPTB is therefore a valuable tool to reduce the associated risks. The current standard for the prediction of sPTB consists, in addition to anamnestic data, of previous sPTB and previous second trimester miscarriage, measurement of cervical length by transvaginal ultrasound (TVU CL) together with assessment of fetal fibronectin levels in cervicovaginal fluid. Other evaluation parameters, such as the level of endocannabinoids in the pregnant woman's blood, could increase the sensitivity of this management. Endocannabinoids (eCBs) are a part of the endocannabinoid system (ECS); out of them anandamide (arachidonoyl-ethanolamide, AEA), in particular, plays an important role in the regulation of pregnancy and childbirth. We present the protocol for an open, non-randomized study to evaluate concentrations of AEA and other endocannabinoids: 2 linoleoylglycerol (2-AG), 2 linoleoylglycerol (2-LG), 2 oleoylglycerol (2-OG), and 2 arachidonoyldopamine (2-ADOPA or also NADA) in the blood of pregnant women as potential predictors of sPTB. In a total of 230 women with a history of sPTB or miscarriage, eCBs levels between 22 and 28 weeks of gestation will be assessed from maternal blood, in addition to the standard procedure. The aim of the study is to determine the relationship between blood concentrations of the endocannabinoids tested and the risk of sPTB. The results of this study will describe the prognostic significance of maternal blood eCBs levels for sPTB, and could subsequently enable improved screening programs for early identification of sPTB.

Derivatized versus non-derivatized LC-MS/MS techniques for the analysis of estrogens and estrogen-like endocrine disruptors in human plasma.

Bisphenols, parabens, alkylphenols and triclosan are anthropogenic substances with a phenolic group that have been introduced to the environment in recent decades. As they possess hormone-like effects, they have been termed endocrine disruptors (EDs), and can interfere with steroid pathways in organisms. To evaluate the potential impact of EDs on steroid biosynthesis and metabolism, sensitive and robust methods enabling the concurrent measurement of EDs and steroids in plasma are needed. Of crucial importance is the analysis of unconjugated EDs, which possess biological activity. The aim of the study was to develop and validate LC-MS/MS methods with and without a derivatization step for the analysis of unconjugated steroids (estrone-E1, estradiol-E2, estriol-E3, aldosterone-ALDO) and different groups of EDs (bisphenols, parabens, nonylphenol-NP and triclosan-TCS), and compare these methods on a set of 24 human plasma samples using Passing-Bablok regression analysis. Both methods were validated according to FDA and EMA guidelines. The method with dansyl chloride derivatization allowed 17 compounds to be measured: estrogens (E1, E2, E3), bisphenols (bisphenol A-BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methylparaben-MP, ethylparaben-EP, propylparaben-PP, butylparaben-BP, benzylparaben-BenzylP), TCS and NP, with lower limits of quantification (LLOQs) between 4 and 125 pg/mL. The method without derivatization enabled 15 compounds to be analyzed: estrogens (E1, E2, E3), ALDO, bisphenols (BPA, BPS, BPF, BPAF, BPAP, BPZ), parabens (MP, EP, PP, BP, BenzylP) with LLOQs between 2 and 63 pg/mL, and NP and BPP in semiquantitative mode. Adding 6 mM ammonium fluoride post column into mobile phases in the method without derivatization achieved similar or even better LLOQs than the method with the derivatization step. The uniqueness of the methods lies in the simultaneous determination of different classes of unconjugated (bioactive) fraction of EDs together with selected steroids (estrogens + ALDO in the method without derivatization), which provides a useful tool for evaluating the relationships between EDs and steroid metabolism.

Androgens in SARS-CoV-2 coronavirus infections.

Recent molecular biology findings have shown that for the penetration of the SARS-CoV-2 coronavirus into host cells, a key role is played by protease serine 2, the activity of which is dependent on androgens. The important role of androgens is also evidenced by clinical observations that men in some age categories are infected by this novel coronavirus up to two times more frequently than women. In addition, men with androgenic alopecia tend to have more serious clinical courses, while men with androgen deprivation as a result of prostate cancer treatments tend to have milder courses. This is in line with the fact that preadolescent children are only rarely sickened with serious forms of SARS-CoV-2 infections. Even though these observations may be explained by other factors, many authors have hypothesized that lowered androgen levels and blocking their activity using anti-androgen medication may moderate the course of the viral infection in intermediately- to critically-affected cases. Clearly, it would be important for androgen deprivation to block not just gonadal androgens, but also adrenal androgens. On the other hand, low androgen levels are considered to be a risk factor for the course of SARS-CoV-2 infections, either because low androgen levels have a general effect on anabolic-catabolic equilibrium and energy metabolism, or because of the ability of testosterone to modify the immune system. It is not yet clear if infection with this novel coronavirus might induce hypogonadism, leading to undesirable side effects on male fertility.

What is a hormone?

Starling's original definition of a hormone from 1905 was "a hormone is a substance produced by glands with internal secretion, which serve to carry signals through the blood to target organs". Today, this definition is understood to be lacking, but newer definitions also do not encompass the entire meaning of hormones as specific carriers of information. One main problem is that there is no delineation between hormones and other signaling molecules such as cytokines, growth factors or autacoid compounds. It seems that a precise definition is not even possible, since some cytokines and growth factors, such as the cytokines erythropoietin, lipocalin-2 and asprosin or fibroblast growth factor 23, act as hormones under certain conditions.

11-Keto-testosterone and other androgens of adrenal origin.

The adrenal glands produce significant amounts of steroid hormones and their metabolites, with various levels of androgenic activities. Until recently, the androgenic potency of these adrenal-derived compounds were not well known, but some recent studies have shown that the production of 11-oxo- and 11beta-hydroxy-derived testosterone and dihydrotestosterone evidently have high androgenic activity. This fact has clinical importance, for instance, in various types of congenital adrenal hyperplasia with androgenization or polycystic ovarian syndrome, and laboratory determinations of these substances could help to better evaluate the total androgen pressure in patients with these disorders. Another area of concern is the treatment of prostate cancer with androgen deprivation, which loses effectiveness after a certain time. The concurrent blocking of the secretion of adrenal C(19)-steroids, whether using corticoids or adrenostatics, could increase the effectiveness of androgen-deprivation therapy.

Steroid diagnostics of 21st century in the light of their new roles and analytical tools.

The determination of steroid hormones and subsequent interpretation of results is accompanied by a range of difficulties. The amount of information that current technology can provide on the circulating concentrations of more than a hundred various steroid compounds can lead to problems with interpretation. The aim of this study is to help provide orientation in this maze of data on steroid hormones. First we focus on specific aspects arising from the pre-analytical phase of steroid determination that need to be considered when planning sampling, whether for diagnostics or research. Then, we provide a brief summary of the characteristics and diagnostic relevance of several steroid hormones and/or their metabolites: pregnenolone, 17alpha-hydroxy-pregnenolone, dehydroepiandrosterone, hydroxyderivatives of dehydroepiandrosterone, androstenedione, testosterone, estrone, estradiol, estriol, cortisol, cortisone, which in our institute are determined with validated LC-MS/MS methods. For these steroids, we also provide newly calculated reference values in fertile women according to the phase of their menstrual cycle.

Estradiol, obesity and hypogonadism.

Obesity increases the incidence of hypogonadism in men, and hypogonadism in turn plays a role in obesity. One of the first mechanisms proposed to explain this was a hypothesis based on the principle that obese men have higher estrogen levels, and that increased estrogens provide feedback to the hypothalamic-pituitary-testicular axis, reducing the secretion of gonadotropins and leading to a decrease of overall testosterone levels. This concept has since been questioned, though never completely disproven. In this study we compared hormone levels in three groups of men with differing BMI levels (between 18-25, 25-29, and 30-39), and found correlations between lowering overall testosterone, SHBG and increased BMI. At the same time, there were no significant changes to levels of free androgens, estradiol or the gonadotropins LH and FSH. These findings are in line with the idea that estrogen production in overweight and obese men with BMI up to 39 kg/m(2) does not significantly influence endocrine testicular function.

Endocrine disruptors, obesity, and cytokines - how relevant are they to PCOS?

As environmental and genetic components contribute to the PCOS expression, we compared levels of endocrine disruptors, steroid hormones, cytokines, and metabolic parameters in twenty healthy, nine normal-weight PCOS women, and ten obese PCOS women. Steroid hormones, bisphenols (BPA, BPS, BPF, BPAF) and parabens (methyl-, ethyl-, propyl-, butyl-, benzyl-parabens) were measured by liquid chromatography-tandem mass spectrometry. Differences between the groups were assessed using the Mann-Whitney U test. Spearman correlation coefficients were calculated for the individual parameters relationship. Significantly higher levels of BPA, anti-Müllerain hormone, lutropine, lutropine/folitropine ratio, testosterone, androstenedione, 7beta-OH-epiandrosterone, and cytokines (IL-6, VEGF, PDGF-bb), were found in normal-weight PCOS women compared to controls. Between normal-weight and obese PCOS women, there were no differences in hormonal, but in metabolic parameters. Obese PCOS women had significantly higher insulin resistance, fatty-liver index, triglycerides, cytokines (IL-2, IL-13, IFN-gamma). In healthy, but not in PCOS, women, there was a positive correlation of BPA with testosterone, SHBG with lutropine, and folitropine, while testosterone negatively correlated with SHBG. In obese women with PCOS, insulin resistance negatively correlated with SHBG and estradiol. No differences were observed in the paraben exposure. Levels of BPA were higher in PCOS women, indicating its role in the etiology. Obesity significantly worsens the symptoms.

Dental composites - a low-dose source of bisphenol A?

Dental composite materials often contain monomers with bisphenol A (BPA) structure in their molecules, e.g. bisphenol-A glycidyl dimethacrylate (Bis-GMA). In this study, it was examined whether dental restorative composites could be a low-dose source of BPA or alternative bisphenols, which are known to have endocrine-disrupting effects. Bis-GMA-containing composites Charisma Classic (CC) and Filtek Ultimate Universal Restorative (FU) and "BPA-free" Charisma Diamond (CD) and Admira Fusion (AF) were examined. Specimens (diameter 6 mm, height 2 mm, n=5) were light-cured from one side for 20 s and stored at 37 °C in methanol which was periodically changed over 130 days to determine the kinetics of BPA release. BPA concentrations were measured using a dansyl chloride derivatization method with liquid chromatography - tandem mass spectrometry detection. The amounts of BPA were expressed in nanograms per gram of composite (ng/g). BPA release from Bis-GMA-containing CC and FU was significantly higher compared to "BPA-free" CD and AF. The highest 1-day release was detected with FU (15.4+/-0.8 ng/g), followed by CC (9.1+/-1.1 ng/g), AF (2.1+/-1.3 ng/g), and CD (1.6+/-0.8 ng/g), and the release gradually decreased over the examined period. Detected values were several orders of magnitude below the tolerable daily intake (4 microg/kg body weight/day). Alternative bisphenols were not detected. BPA was released even from "BPA-free" composites, although in significantly lower amounts than from Bis-GMA-containing composites. Despite incubation in methanol, detected amounts of BPA were substantially lower than current limits suggesting that dental composites should not pose a health risk if adequately polymerized.

Midazolam and its effect on vital signs and behavior in children under conscious sedation in dentistry.

Midazolam is a short acting sedative with small number of adverse effects. Administered orally, it is currently the most common form of conscious sedation in children. The objective of this paper is to describe effect of midazolam administered to children during dental treatment on their vital signs, and to monitor changes in children's behavior. We described values of vital signs and behavior in 418 sedations conducted in 272 children between 1-12 years of age. To achieve the following results, we used data from 272 all first-time sedations. After administration of midazolam arterial blood pressure and blood oxygen saturation decreased by values which were not clinically significant. The heart rate increased, with values staying within the limits of physiological range. The speed of onset of midazolam's clinical effects depends on age and dose. The lower age and dose correlated with the higher behavior score. The effectiveness of midazolam treatment is 97.8 %. Unwillingness of child to receive midazolam is predictor for disruptive behavior during sedation. 1.8 % of all sedation cases showed paradoxical reactions. The administration of midazolam in dose of 0.5 mg per 1 kg of child's body weight is safe and could be recommended for dental treatment in pediatric dentistry.

Are there sex differences in the reaction of undercarboxylated osteocalcin to hypoglycemia?

There has been increasing evidence in recent years for the hypothesis of bones as endocrine organs. Osteocalcin, long considered just a marker of new bone formation, is now seen as the first hormone produced by bones, and seems to be associated with regulating glucose metabolism and reproduction. The aim of this work was to monitor changes of osteocalcin in reaction to hypoglycemia, and determine if there are differences in such reactions between the sexes. The study included 61 healthy probands with physiological calciophosphate metabolism (30 men and 31 women). We applied to each of them an insulin tolerance test, and then monitored levels of undercarboxylated osteocalcin and reactions to hypoglycemia at regular time intervals. We found differences in the reaction to hypoglycemia between the sexes. In men there was a significant decline in undercarboxylated osteocalcin between the 30 and 40 min (p<0.0015), which reflects a reaction to a glycemic decline between 25-30 min, followed by reversal. Low undercarboxylated osteocalcin in men lasted up to 90 min, after which they returned to levels before the test. In women we did not find any significant changes in undercarboxylated osteocalcin levels. Changes in undercarboxylated osteocalcin induced by hypoglycemia indicate a relationship between bones and glucose metabolism. There was an interesting difference between the sexes. However, a definitive conclusion about the role of osteocalcin in human metabolism will require numerous future studies.

A method for determination of one hundred endogenous steroids in human serum by gas chromatography-tandem mass spectrometry.

Steroid profiling helps various pathologies to be rapidly diagnosed. Results from analyses investigating steroidogenic pathways may be used as a tool for uncovering pathology causations and proposals of new therapeutic approaches. The purpose of this study was to address still underutilized application of the advanced GC-MS/MS platform for the multicomponent quantification of endogenous steroids. We developed and validated a GC-MS/MS method for the quantification of 58 unconjugated steroids and 42 polar conjugates of steroids (after hydrolysis) in human blood. The present method was validated not only for blood of men and non-pregnant women but also for blood of pregnant women and for mixed umbilical cord blood. The spectrum of analytes includes common hormones operating via nuclear receptors as well as other bioactive substances like immunomodulatory and neuroactive steroids. Our present results are comparable with those from our previously published GC-MS method as well as the results of others. The present method was extended for corticoids and 17alpha-hydroxylated 5alpha/ß-reduced pregnanes, which are useful for the investigation of alternative "backdoor" pathway. When comparing the analytical characteristics of the present and previous method, the first exhibit by far higher selectivity, and generally higher sensitivity and better precision particularly for 17alpha-hydroxysteroids.

Androgens in women - critical evaluation of the methods for their determination in diagnostics of endocrine disorders.

The androgens dehydroepiandrosterone sulfate, dehydro-epiandrosterone, androstenedione and testosterone are routinely assessed in women, and circulating levels of these androgens reflect their production. These androgens are measured in most laboratories using various immuno-analytical methods. Recently, however, androgen assays have begun to be performed using gas or liquid chromatography combined with mass spectrometry. To better understand the difficulties and issues of androgen laboratory diagnostics, it is important to assess each of the methods used, how and why they were introduced into practice, and their advantages, limits, historic milestones and current status. It is also necessary to understand how reference ranges are determined and specifics arising from the physiology of individual androgens. Here we present a summary and discussion of these issues.

Links between the circadian rhythm, obesity and the microbiome.

Obesity is linked to a wide range of serious illnesses. In addition to the important impact on the health of the individual, obesity also has a substantial impact on the economy. Disruption of physiological day-night cycles could contribute to the increased incidence of obesity. According to the American National Sleep Federation, the percentage of the people who reported a sleep duration of six hours or less increased from 12 to 37 % over ten years. Insufficient sleep leads not only to an increase of the total calorie intake but changes the meal preference in favor of palatable foods and meals with high carbohydrate content. A decrease of leptin and increase of ghrelin levels caused by sleep deficiency can also play a role. In addition to the higher caloric intake, the timing of food consumption should be taken into account. The same meal eaten during the night versus the day is associated with increased postprandial glucose and triglyceride levels. The gut microbiome has also been recently understood as an endocrine system, with links between the gut microbiome and circadian rhythm changes possibly influencing increased obesity.

The evolution of taste and perinatal programming of taste preferences.

Taste is important for food intake. The fetus first experiences taste through amniotic fluid, and later via mother's milk. Early human experience with taste has a key importance for later acceptance of food. Dietary behavior is determined by the interaction of many different factors. The development of the olfactory and taste receptors begins at 7-8 weeks of gestation. An early sensitive period probably exists when flavor preference is established. Sweet taste is preferred in early childhood; this is the reason why children are at increased risk of over-consuming saccharides. Gustatory sensitivity declines with age. The threshold for the perception of each basic taste differs, and is established genetically. In this review, we summarize published data on taste preferences and its development and changes during life.

Genetic predictors of the development and recurrence of Graves' disease.

Graves' disease affects approximately 3 % of women and 0.5 % of men. The first-choice therapy is based on the administration of thyrostatic drugs. However, approximately half of patients relapse within two years of discontinuation. These patients must then decide whether to re-initiate thyrostatics, which may have serious side effects, or to undergo surgery or radioiodine treatment. Familial forms of Graves' disease indicate a significant genetic component, with twin studies demonstrating a contribution of genetic factors up to 70-80 %. The autoimmune nature of the disease involves the human leukocyte antigen (HLA) complex, which has a decisive impact on each individual's immune response. Within HLA, some variants of the DRB1, DQA1 and DQB1 genes appear to be possible predictors of the development and recurrence of Graves' disease. Outside the HLA region, many variants of immunocompetent genes have also been identified as potential Graves' disease predictors. Apart from the immune system, some thyroid-specific genes have been described in relation to the disease. Here, we present current knowledge regarding the genetic components involved in the development and recurrence of Graves' disease. Further, we present original pilot results from a cohort of Czech Graves' disease patients regarding the HLA variants.

Parabens and their relation to obesity.

Parabens are a group of chemicals used as preservatives in the food, cosmetic and pharmaceutical industries. They are known to possess estrogenic effects, and therefore have been classified as endocrine disruptors. In addition to the classical endocrine organs, other tissues have endocrine activity, including adipose tissue. Several chemicals are known to cause obesogenic effects, and parabens are currently being studied in this context. The aim of this study was to investigate the possible connections of paraben exposure and obesity. Blood plasma from 27 healthy women was collected during their menstrual cycle. Basal anthropometric measures, levels of parabens (methylparaben, ethylparaben and propylparaben), adipokines (adiponectin, adipsin, leptin, resistin and visfatin) and hormones affecting energy balance and metabolic health (c-peptide, ghreline, GIP, GLP-1, glucagon, insulin, PAI-1) were measured. A Kolmogorov-Smirnov test showed higher methylparaben and propylparaben levels in women with BMI 25-34.9 compared to those with BMI 18.5-24.9. Plasma levels of methylparaben as well as the sum of parabens were positively associated with the plasma adipsin levels. Negative associations for methylparaben were found for glucagon, leptin and PAI-1. In accordance with other experimental studies we observed important associations of methylparaben and hormones affecting energy balance and metabolic health, indicating its obesogenic potential.

Endocrine disruptors of the bisphenol and paraben families and bone metabolism.

After menopause, when estrogen levels decrease, there is room for the activity of anthropogenic substances with estrogenic properties - endocrine disruptors (EDs) - that can interfere with bone remodeling and changes in calcium-phosphate metabolism. Selected unconjugated EDs of the bisphenol group - BPA, BPS, BPF, BPAF, and the paraben family - methyl-, ethyl-, propyl-, butyl-, and benzyl-parabens - were measured by high performance liquid chromatography-tandem mass spectrometry in the plasma of 24 postmenopausal women. Parameters of calcium-phosphate metabolism and bone mineral density were assessed. Osteoporosis was classified in 14 women, and 10 women were put into the control group. The impact of EDs on calcium-phosphate metabolism was evaluated by multiple linear regressions. In women with osteoporosis, concentrations of BPA ranged from the lower limit of quantification (LLOQ) - 104 pg/ml and methyl paraben (MP) from LLOQ - 1120 pg/ml. The alternative bisphenols BPS, BPF and BPAF were all under the LLOQ. Except for MP, no further parabens were detected in the majority of samples. The multiple linear regression model found a positive association of BPA (beta=0.07, p<0.05) on calcium (Ca) concentrations. Furthermore, MP (beta=-0.232, p<0.05) was negatively associated with C-terminal telopeptide. These preliminary results suggest that these EDs may have effects on calcium-phosphate metabolism.