COVID-19, Vaccination, and Female Fertility in the Czech Republic.

The fast-track process to approve vaccines against COVID-19 has raised questions about their safety, especially in relation to fertility. Over the last 2 years, studies have appeared monitoring female fertility, especially from assisted reproduction centers or in animal experiments. However, studies monitoring healthy populations are still limited. The aim of our study was to monitor the relevant parameters of female fertility (sex and other steroids, LH, FSH, SHBG, Antimüllerian hormone and antral follicle count) before and then 2-4 months after the third dose of vaccination against COVID-19 in a group of 25 healthy fertile woman. In addition, anti-SARS-CoV-2 and anti-SARS-CoV-2S antibodies were determined. We did not observe significant changes in the measured parameters before and after the third dose of vaccination. By comparing levels of the analytes with antibodies indicating a prior COVID-19 infection, we found that women who had experienced the disease had statistically lower levels of estrone, estradiol, SHBG and 5α-dihydroprogesterone, and conversely, higher levels of androgen active dehydroepiandrosterone and dihydrotestosterone. Our results confirm that vaccination does not affect female fertility, and that what fertile women should be worried about is not vaccination, but rather COVID-19 infection itself.

An LC-MS/MS method for the simultaneous quantification of 32 steroids in human plasma.

A development of robust and rapid method with simple sample preparation for the analysis of steroids of C18-, C19-, C21- families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C18- steroids (estrone, estradiol), nineteen C19- steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11ß-hydroxyandrostenedione, 11ß-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7ß-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17ß-diol, 5α-androstane-3ß,17ß-diol, 5ß-androstane-3α,17ß-diol, 5ß-androstane-3ß,17ß-diol), and eleven C21- steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva.

Release of Bisphenol A from Milled and 3D-Printed Dental Polycarbonate Materials.

Polycarbonates are polymers of bisphenol A (BPA), a well-known endocrine disruptor. This study evaluated the release of BPA from polycarbonate crowns that were (1) milled from Temp Premium Flexible (ZPF, Zirkonzahn, Italy) or Tizian Blank Polycarbonate (TBP, Schütz Dental, Germany), or (2) 3D-printed (Makrolon 2805, Covestro, Germany). Commercial prefabricated polycarbonate crowns (3M, USA) and milled poly(methyl methacrylate) (PMMA) crowns (Temp Basic, Zirkonzahn, Italy) were included for comparison. The crowns were stored at 37 °C in artificial saliva (AS) or methanol, which represented the worst-case scenario of BPA release. Extracts were collected after 1 day, 1 week, 1 month and 3 months. BPA concentrations were measured using liquid chromatography-tandem mass spectrometry. The amounts of released BPA were expressed in micrograms per gram of material (µg/g). After 1 day, the highest amounts of BPA were measured from milled polycarbonates, TBP (methanol: 32.2 ± 3.8 µg/g, AS: 7.1 ± 0.9 µg/g) and ZPF (methanol 22.8 ± 7.7 µg/g, AS: 0.3 ± 0.03 µg/g), followed by 3D-printed crowns (methanol: 11.1 ± 2.3 µg/g, AS: 0.1 ± 0.1 µg/g) and prefabricated crowns (methanol: 8.0 ± 1.6 µg/g, AS: 0.07 ± 0.02 µg/g). Between 1 week and 3 months, the average daily release of BPA in methanol and AS decreased below 2 µg/g and 0.6 µg/g, respectively. No BPA was released from PMMA in AS, and the cumulative amount released in methanol was 0.2 ± 0.06 µg/g. In conclusion, polycarbonates could be a relevant source of BPA, but the current tolerable daily intake of BPA (4 µg/kg body weight) should not be exceeded.

Determination of Intraprostatic and Intratesticular Androgens.

Androgens represent the main hormones responsible for maintaining hormonal balance and function in the prostate and testis. As they are involved in prostate and testicular carcinogenesis, more detailed information of their active concentration at the site of action is required. Since the introduction of the term intracrinology as the local formation of active steroid hormones from inactive precursors of the adrenal gland, mainly dehydroepiandrosterone (DHEA) and DHEA-S, it is evident that blood circulating levels of sex steroid hormones need not reflect their actual concentrations in the tissue. Here, we review and critically evaluate available methods for the analysis of human intraprostatic and intratesticular steroid concentrations. Since analytical approaches have much in common in both tissues, we discuss them together. Preanalytical steps, including various techniques for separation of the analytes, are compared, followed by the end-point measurement. Advantages and disadvantages of chromatography-mass spectrometry (LC-MS, GC-MS), immunoanalytical methods (IA), and hybrid (LC-IA) are discussed. Finally, the clinical information value of the determined steroid hormones is evaluated concerning differentiating between patients with cancer or benign hyperplasia and between patients with different degrees of infertility. Adrenal-derived 11-oxygenated androgens are mentioned as perspective prognostic markers for these purposes.

Bisphenol A Release from Dental Composites and Resin-Modified Glass Ionomers under Two Polymerization Conditions.

Bisphenol A (BPA)-based monomers are commonly contained in dental resin-based materials. As BPA is an endocrine disruptor, its long-term release from restorative composites and resin-modified glass ionomers (RM-GICs) under two polymerization conditions was measured in this study. Specimens of two conventional composites containing BPA-based monomers, two "BPA-free" composites, and two RM-GICs were polymerized from one side for 20 s at 1300 mW/cm2 or for 5 s at 3000 mW/cm2. The amounts of BPA released in artificial saliva and methanol after 1, 4, 9, 16, 35, 65, 130, and 260 days were measured using liquid chromatography-tandem mass spectrometry. The highest amounts of BPA were released from conventional composites, followed by RM-GICs, while the least was released from "BPA-free" composites. Amounts of released BPA were significantly higher in methanol and decreased gradually after the first day. Fast polymerization (5 s at 3000 mW/cm2) resulted in a significantly higher release of BPA after 1 day, but the effect of polymerization conditions was not significant overall. In conclusion, fast polymerization increased the initial release of BPA, but the released amounts were significantly lower than the current tolerable daily intake (4 µg/kg body weight/day) even in methanol, representing the worst-case scenario of BPA release.

Oxime-based 19-nortestosterone-pheophorbide a conjugate: bimodal controlled release concept for PDT.

Photodynamic therapy has become a feasible direction for the treatment of both malignant and non-malignant diseases. It has been in the spotlight since FDA regulatory approval was granted to several photosensitizers worldwide. Nevertheless, there are still strong limitations in the targeting specificity that is vital to prevent systemic toxicity. Here, we report the synthesis and biological evaluation of a novel bimodal oxime conjugate composed of a photosensitizing drug, red-emitting pheophorbide a, and nandrolone (NT), a steroid specifically binding the androgen receptor (AR) commonly overexpressed in various tumors. We characterized the physico-chemical properties of the NT-pheophorbide a conjugate (NT-Pba) and singlet oxygen generation. Because light-triggered therapies have the potential to provide important advances in the treatment of hormone-sensitive cancer, the biological potential of this novel specifically-targeted photosensitizer was assessed in prostatic cancer cell lines in vitro using an AR-positive (LNCaP) and an AR-negative/positive cell line (PC-3). U-2 OS cells, both with and without stable AR expression, were used as a second cell line model. Interestingly, we found that the NT-Pba conjugate was not only photodynamically active and AR-specific, but also that its phototoxic effect was more pronounced compared to pristine pheophorbide a. We also examined the intracellular localization of NT-Pba. Live-cell fluorescence microscopy provided clear evidence that the NT-Pba conjugate localized in the endoplasmic reticulum and mitochondria. Moreover, we performed a competitive localization study with the excess of nonfluorescent NT, which was able to displace fluorescent NT-Pba from the cell interior, thereby further confirming the binding specificity. The oxime ether bond degradation was assayed in living cells by both real-time microscopy and a steroid receptor reporter assay using AR U-2 OS cells. Thus, NT-Pba is a promising candidate for both the selective targeting and eradication of AR-positive malignant cells by photodynamic therapy.

Exposure to bisphenols and parabens during pregnancy and relations to steroid changes.

BACKGROUND: The harmful effects of endocrine disrupting compounds (EDCs) on human health are generally well-known, and exposure during fetal development may have lasting effects. Fetal exposure to bisphenol A (BPA) has been recently relatively well-studied; however, less is known about alternatives such as bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF), which have started to appear in consumer products. Parabens are another widespread group of EDCs, with confirmed transplacental passage. The usage of many cosmetic, pharmaceutical and consumer products during the pregnancy that may contain parabens and bisphenols has led to the need for investigation. OBJECTIVES: To shed more light into the transplacental transport of BPA, its alternatives, and parabens, and to study their relation to fetal steroidogenesis. METHODS: BPA, BPS, BPF, BPAF, methylparaben, ethylparaben, propylparaben, butylparaben, benzylparaben and 15 steroids including estrogens, corticoids, androgens and immunomodulatory ones were determined in 27 maternal (37th week of pregnancy) and cord plasma samples using liquid chromatography - tandem mass spectrometry methods. RESULTS: In cord blood, significantly higher BPA levels (p=0.0455) were observed compared to maternal plasma. The results from multiple regression models showed that in cord blood, methylparaben (ß=-0.027, p=0.027), propylparaben (ß=-0.025, p=0.03) and the sum of all measured parabens (ß=-0.037, p=0.015) were inversely associated with testosterone levels. CONCLUSION: To the best of our knowledge, this is the first study reporting the simultaneous detection of BPA, alternative bisphenols, parabens and steroids in maternal and cord plasma. Our study confirmed the transplacental transport of BPA, with likely accumulation in the fetal compartment. The negative association of cord blood parabens and testosterone levels points to possible risks with respect to importance of testosterone for prenatal male development.

Determination of selected bisphenols, parabens and estrogens in human plasma using LC-MS/MS.

In this study, a novel liquid chromatography - tandem mass spectrometry method for the simultaneous determination of bisphenols (BPA, BPS, BPF, BPAF), parabens (methyl-, ethyl-, propyl-, butyl-, benzyl-paraben) and estrogens (estrone, estradiol, estriol) in human plasma is presented. Since all analytes possess the phenolic group, dansyl chloride derivatization was applied in order to gain high sensitivity. The method was validated according to FDA guidelines, and all validation requirements were satisfactory. The lower limits of quantifications were 41.6, 54.9, 43.5 and 150.8pg/mL for BPA, BPS, BPF and BPAF; 172, 149, 171, 134 and 202pg/mL for methyl-, ethyl-, propyl-, butyl- and benzyl-paraben; 10.5, 6.7 and 9.4pg/mL for estrone, estradiol and estriol, respectively. This is the first method allowing the determination of plasma bisphenols, parabens and estrogens in one run, and also the first determination of BPF levels in human plasma. The method was used to examine the plasma levels of healthy normospermic men, where three times higher plasma levels of BPF than BPA were found.

Immunoassay for determination of trilobolide.

Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin bring promising prospects for Tb to be used in the development of new anti-cancer drugs. As long as there are still unanswered questions regarding its investigation, a need for novel analytical tools emerge. Since immunoassays serve as one of powerful tools within the investigation of natural products, the development of indirect competitive enzyme-linked immunosorbent assay (ELISA) utilizing coating based on avidin-biotin technology is described. In our set-up of ELISA, newly synthesized biotinylated Tb served as immobilized competitor. Tb-carboxymethyloxime-bovine serum albumin (BSA) and Tb-succinoyl-BSA conjugates were used separately for immunization of rabbits. Two sets of polyclonal antibodies (RAbs) were obtained. Antibodies against Tb-succinoyl-BSA conjugate (RAb No. 206) were chosen as the best. Under optimized conditions, limit of detection and 50% intercept of our ELISA were 849pg/mL and 8.89ng/mL, respectively. The cross-reactivity (CR) was tested on 10 structurally related compounds and CR did not exceed 6.1%. The reproducibility of the system is expressed as intra- and inter-assay coefficients of variation (9.7% and 11.4%, respectively). Based on conducted experiments, we proposed the use of ELISA for quantification of Tb in complex biological matrices such as plant extracts. A method was applied to analyze three extracts obtained from different parts of L. trilobum. Data obtained were compared to those acquired by UHPLC-MS/MS. The concordance between the methods (103-87%) showed the ability of ELISA to quantify Tb.