Cross sectional study on exposure to BPA and its analogues and semen parameters in Czech men.

Exposure to bisphenols has been found to have adverse effects on male reproductive function in animals. Human exposure to bisphenols is widespread. Bisphenol A (BPA) and its analogues, including bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF) are utilized in various consumer products such as food contact materials and dental resins. The effects of these compounds on male fertility and spermatogenesis are unclear and findings from human studies are inconsistent. In this cross-sectional study, we evaluated the influence of BPA, BPS, BPF, BPAF (BPs) measured in semen on number of spermatozoa, total motility, progressive motility, morphology, and DNA fragmentation. We also examined the association of bisphenols (BPs) exposure with patients' occupation. A total of 358 patients aged 17-62 years with BMI 18-42 were included in the study from 2019 to 2021. BPs were extracted using solvent extraction followed by preconcentration step and determined by high-performance liquid chromatography and tandem mass spectrometry (LC/MSMS). Bisphenols were detected in 343 from 349 analysed samples (98.3% of all the samples). In 6 samples, the concentration of all BPs was under the limit of detection and in 20 samples under the limit of quantification. We did not find a statistically significant relationship between occupation and BPs. However, we observed significant correlations between the concentration of BPA and a lower motility and normal morphology. For BPS, a significant correlation with a lower ejaculate volume and a lower total sperm count was found. BPF and BPAF were detected only in 14.3% and 23.9% of samples, respectively. For BPF and BPAF, no significant correlations with spermiogram parameters were observed. Our results show that BPs are widespread in the male population (more than 90% of analysed samples), independently of an occupation and in case of BPA and BPS having a negative impact on spermiogram parameters.

Truncated vitronectin with E-cadherin enables the xeno-free derivation of human embryonic stem cells.

Human embryonic stem cells (hESCs) have unique abilities that enable their use in cell therapy, disease modeling, and drug development. Their derivation is usually performed using a feeder layer, which is undefined and can potentially cause a contamination by xeno components, therefore there is a tendency to replace feeders with xeno-free defined substrates in recent years. Three hESC lines were successfully derived on the vitronectin with a truncated N-terminus (VTN-N) in combination with E-cadherin in xeno-free conditions for the first time, and their undifferentiated state, hESC morphology, and standard karyotypes together with their potential to differentiate into three germ layers were confirmed. These results support the conclusion that the VTN-N/E-cadherin is a suitable substrate for the xeno-free derivation of hESCs and can be used for the derivation of hESCs according to good manufacturing practices.

Non-Invasive Diagnostics of Male Spermatogenesis from Seminal Plasma: Seminal Proteins.

The compounds of seminal plasma have great potential as biomarkers of male fertility and can be used as a diagnostic tool for types of azoospermia. Azoospermia occurs in approximately 1% of the male population, and for an effective therapy of this form of male infertility, it is important to distinguish between obstructive and non-obstructive azoospermia. Proteins in seminal plasma can serve as biomarkers for diagnosing azoospermia. Considering the various types of obstructions, a combination of multiple proteins is advisable for diagnostic purposes. In this context, testicular and epididymal proteins are particularly significant, as they are specific to these tissues and typically absent in ejaculate during most obstructions. A combination of multiple biomarkers is more effective than the analysis of a single protein. This group of markers contains TEX101 and ECM1 proteins, combined detections of these two bring a diagnostic output with a high sensitivity and specificity. Similar results were observed for combined detection of TEX101 and SPAG1. The effective using of specific biomarkers from seminal plasma can significantly improve the existing approaches to diagnosis of the causes of male infertility.

Czech Women's Point of Views on Immediate Breast Reconstruction after Mastectomy due to BRCA Gene Mutation or Breast Cancer.

(1) Objective: Breast cancer is the most common cancer in women, and the incidence of the disease continues to increase. The issue of immediate breast reconstruction (IBR) in women with BRCA mutations and breast cancer is highly topical. This study is based on the long-term experience of our workplace with the diagnosis and treatment of breast cancer in women. We use the possibilities of oncoplastic surgery, including IBR. Our effort involves learning about women's awareness of IBR with a mastectomy at the same time. (2) Methods: The method of quantitative research of women's awareness using a structured anonymous questionnaire was chosen. Out of the total number of 84 respondents who already underwent IBR, 36.9% were due to BRCA mutations, and 63.1% were due to breast cancer. (3) Results: All of the respondents learned about the possibility of IBR before treatment or during treatment planning. The information was first obtained mainly from an oncologist. Women obtained the most information regarding IBR from a plastic surgeon. Before the mastectomy, all of the respondents already knew what IBR meant, as well as about the payment of IBR by the health insurance company. All of the respondents would choose the IBR option again. A total of 94.0% of women cited preservation of body integrity as a reason for undergoing IBR, and 88.1% of women knew about the possibility of performing IBR with their own tissues. (4) Conclusions: There are few specialized centers with a team of experts in reconstructive breast surgery in the Czech Republic, especially those that perform IBR. Research has shown that all of the patients were well informed about IBR, but the vast majority only learned about IBR before the surgical procedure was planned. All of the women wished to maintain body integrity. Our study results in the recommendations for patients and for healthcare management.

The bisphenol S contamination level observed in human follicular fluid affects the development of porcine oocytes.

Bisphenol S (BPS), the main replacement for bisphenol A (BPA), is thought to be toxic, but limited information is available on the effects of Bisphenol S on ovarian follicles. In our study, we demonstrated the presence of Bisphenol S in the follicular fluid of women at a concentration of 22.4 nM. The effect of such concentrations of Bisphenol S on oocyte maturation and subsequent embryo development is still unknown. Therefore, we focused on the effect of Bisphenol S on in vitro oocyte maturation, fertilization, and embryo development. As a model, we used porcine oocytes, which show many physiological similarities to human oocytes. Oocytes were exposed to Bisphenol S concentrations similar to those detected in female patients in the ART clinic. We found a decreased ability of oocytes to successfully complete meiotic maturation. Mature oocytes showed an increased frequency of meiotic spindle abnormalities and chromosome misalignment. Alarming associations of oocyte Bisphenol S exposure with the occurrence of aneuploidy and changes in the distribution of mitochondria and mitochondrial proteins were demonstrated for the first time. However, the number and quality of blastocysts derived from oocytes that successfully completed meiotic maturation under the influence of Bisphenol S was not affected.

The contribution of donated human embryos suitable for the production of embryonic stem cells to increase the quality of life: Selection and preparation of embryos in the Czech Republic.

BACKGROUND: Human embryonic stem cells (hESCs) have the unique ability to differentiate into any cell type in the human body and to proliferate indefinitely. Cell therapies involving hESC have shown very promising results for the treatment of certain diseases and confirmed the safety of hESC-derived cells for humans. They are used in cell therapy, mainly in targeted therapy of diseases that are currently incurable. OBJECTIVES: The aim of this study was the derivation of clinical-grade hESCs usable in drug development, non-native medicine and cell therapy. MATERIAL AND METHODS: Embryos were thawed, cultivated to the blastocyst stage if necessary, and assisted hatching was subsequently performed. Embryoblasts were mechanically isolated using narrow needles. Each line was kept as a separate batch. The derived hESCs were cultured under hypoxic culture conditions (5% O2, 5% CO2, 37°C) in a NutriStem® hPSC XF Medium with a daily medium change. RESULTS: From January 2018 to July 2020, 138 selected clients were asked for consent to donate embryos, of whom 52 did not respond, 19 terminated the storage of their embryos and 29 extended the storage. Only 38 clients (27.5%) agreed to donate embryos for the derivation of hESCs. At the same time, personal communication with clients took place and another 17 embryo donors were recruited. A total of 160 embryos from 55 donors aged 26-42 years were collected. The embryos were frozen at the blastocyst (33.1%) or morula (46.3%) stage. After the preparation of 64 embryos, embryoblasts were isolated and cultured. Finally, 7 hESC lines were obtained, 4 research-grade and 3 clinical-grade, the first in the Czech Republic. CONCLUSIONS: We established a current good manufacturing practice (cGMP)-defined xeno-free and feeder-free system for the derivation, culture and banking of clinical-grade hESC lines that are suitable for preclinical and clinical trials. The quality control testing with criteria concerning sterility, safety and characterization according to cGMP ensured the clinical-grade quality of hESC lines.

Proteome Mapping of Cervical Mucus and Its Potential as a Source of Biomarkers in Female Tract Disorders.

Cervical mucus (CM) is a viscous fluid that is produced by the cervical glands and functions as a uterine cervix plug. Its viscosity decreases during ovulation, providing a window for non-invasive sampling. This study focuses on proteomic characterization of CM to evaluate its potential as a non-invasively acquired source of biomarkers and in understanding of molecular (patho)physiology of the female genital tract. The first objective of this work was to optimize experimental workflow for CM processing and the second was to assess differences in the proteomic composition of CM during natural ovulatory cycles obtained from intrauterine insemination (IUI) cycles and in vitro fertilization (IVF) cycles with controlled ovarian hyperstimulation. Proteomic analysis of CM samples revealed 4370 proteins involved in processes including neutrophil degranulation, cellular stress responses, and hemostasis. Differential expression analysis revealed 199 proteins enriched in IUI samples and 422 enriched in IVF. The proteins enriched in IUI were involved in phosphatidic acid synthesis, responses to external stimulus, and neutrophil degranulation, while those enriched in IVF samples were linked to neutrophil degranulation, formation of a cornified envelope and hemostasis. Subsequent analyses clarified the protein composition of the CM and how it is altered by hormonal stimulation of the uterus.

Methods and techniques of fertility preservation in patients with endometriosis.

: Objective: Endometriosis is a chronic disease with a relatively high prevalence in the female population. Both the disease itself and its surgical treatment can adversely affect the fertility of patients. For this reason, endometriosis is offered as a possible indication for fertility preservation by cryopreservation methods. The aim of this paper is to present the current knowledge on the options of fertility preservation in this subpopulation. METHODS: Search of relevant literature in PubMed/Medline, Web of Science and Scopus databases. RESULTS: Fertility preservation by cryopreservation methods has so far been used mainly in the care of women with cancer. With increasing experience, the effectiveness and availability of these methods have increased significantly and the indication spectrum has been extended to selected benign diseases such as endometriosis. Three techniques are currently established in practice: embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation. Oocyte cryopreservation is the most commonly used technique, since it is the most advantageous for the patient and, according to the available data, is an effective way to increase the chances of future pregnancy for patients with endometriosis The purpose is to realize the protection of reproduction before the planned operation. CONCLUSION: The diagnosis of endometriosis negatively affects the fertility of women. For some patients, the solution is fertility preservation by cryopreservation methods. Further clinical studies are needed to define exact, practically applicable indication criteria, potential risks of procedures and their benefits and cost-effectiveness.

Assisted oocyte activation.

OBJECTIVE: Currently, there is a rapid increase in studies on assisted oocyte activation, which can significantly improve the process of in vitro fertilization. Fertilization of oocytes by conventional methods and by intracytoplasmic sperm injection can be affected by insufficient activation of the oocyte. The reason is mainly deviations in the enzymatic equipment of sperm or oocytes or a non-functional activation cascade. In many cases, fertilization can be achieved using artificial oocyte activation by applying calcium ion donors to the oocytes after sperm microinjection. However, opinions on the safety and reliability of this method are not uniform. The aim of the thesis is to present current knowledge about assisted oocyte activation and its impact not only on in vitro fertilization, but also on subsequent embryonic and fetal development. METHODOLOGY: Research of relevant literature in Web of Science, PubMed/Medline and Scopus databases. RESULTS AND CONCLUSIONS: Based on the literature data and the authors' own experience, it follows that this method is effective and safe from the point of view of further development of the embryo, fetus and postnatal development. Extensive meta-analyses focused on this method were carried out, which did not find a negative impact not only on the embryonic and fetal development of the individual, but this method did not have associated with a negative impact on the psychosomatic development of the children.

Selected pathological conditions affecting endometrial receptivity.

OBJECTIVE: A summary of new knowledge on embryo implantation in dependence on quality of the endometrium. METHODS: Literature review from August 2022 of the relevant publications in Web of Science, Scopus and PubMed/Medline databases, focused on "endometrial receptivity", "polycystic ovary syndrome", "endometriosis", "SARS-CoV-2". RESULTS: The receptive state of the endometrium is a result of physiological remodeling and immune system activity modulated by the microbio-me. This balance can be disturbed by myomas, polyps, sactosalpings, adenomyosis, endometriosis, polycystic ovary syndrome, infections. The effect of SARS-CoV-2 infection is being discussed. For a successful implantation, timing of transfer is crucial. The ultrasound examination is used conventionally. In specific cases, hysteroscopy and endometrium bio-psy are recommended. Histological and immunohistochemical evaluation is performed together with examination of microbio-me or transcriptome. To support the implantation, gestagenes are used, or metformin in the patients with polycystic ovary syndrome. In cases of a repeated implantation failure, the intrauterine infusion of mononuclear cells or platelet rich plasma is used, subcutaneous application of granulocyte colony stimulating growth factor, intravenous application of atosiban or intrauterine application of human chorionic gonadotropin. CONCLUSION: Recent research in the field of transcriptomics, proteomics and reproductive immunology uncovers the process of implantation more deeply and opens a new stage of the assisted reproduction.

The Manufacture of Xeno- and Feeder-Free Clinical-Grade Human Embryonic Stem Cell Lines: First Step for Cell Therapy.

Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP.

Assisted reproduction in patients with Klinefelter syndrome.

OBJECTIVE: The goal of this article is to present the current knowledge of Klinefelter syndrome and its impact on male reproductive function as well as the current treatment options. METHODS: PubMed/Medline, WoS and Scopus were searched for articles indexed until November 2021. TEXT: Klinefelter syndrome is a chromosomal aberration with an additional X chromosome in males. This may adversely affect testicular growth and spermatogenesis, thus resulting in male infertility. Recently, new knowledge has appeared about the treatment of male infertility. CONCLUSION: Interdisciplinary approach enables early dia-gnosis and treatment of patients with Klinefelter syndrome. Assisted reproductive technology is essential for infertility treatment in patients with Klinefelter syndrome.

The Bisphenols Found in the Ejaculate of Men Does Not Pass through the Testes.

Exposure to bisphenols is related to negative effects on male reproduction. The bisphenols exposure is associated with several modes of action including negative impact on the blood-testis barrier (BTB) in testes or direct effect on spermatozoa. Bisphenols have been detected in human seminal plasma, but the possible mechanism of seminal transfer of bisphenols is not clear. Some authors consider the transfer through the blood-testis barrier to be crucial. Therefore, in this work, we compared normozoospermic men and men after vasectomy who have interrupted vas deferens and their ejaculate does not contain testicular products. We measured the concentration of bisphenol A (BPA), bisphenol S (BPS) and bisphenol F (BPF) in the urine and seminal plasma of these men using liquid chromatography tandem mass spectrometry (LC/MSMS). We found that the ratio of urinary and seminal plasma content of bisphenols did not differ in normozoospermic men or men after vasectomy. From the obtained data, it can be concluded that the pathways of transport of bisphenols into seminal plasma are not primarily through the testicular tissue, but this pathway is applied similarly to other routes of transmission by a corresponding ejaculate volume ratio. To a much greater extent than through testicular tissue, bisphenols enter the seminal plasma mainly as part of the secretions of the accessory glands.

Overview of the Mechanisms of Action of Selected Bisphenols and Perfluoroalkyl Chemicals on the Male Reproductive Axes.

Male fertility has been deteriorating worldwide for considerable time, with the greatest deterioration recorded mainly in the United States, Europe countries, and Australia. That is, especially in countries where an abundance of chemicals called endocrine disruptors has repeatedly been reported, both in the environment and in human matrices. Human exposure to persistent and non-persistent chemicals is ubiquitous and associated with endocrine-disrupting effects. This group of endocrine disrupting chemicals (EDC) can act as agonists or antagonists of hormone receptors and can thus significantly affect a number of physiological processes. It can even negatively affect human reproduction with an impact on the development of gonads and gametogenesis, fertilization, and the subsequent development of embryos. The negative effects of endocrine disruptors on sperm gametogenesis and male fertility in general have been investigated and repeatedly demonstrated in experimental and epidemiological studies. Male reproduction is affected by endocrine disruptors via their effect on testicular development, impact on estrogen and androgen receptors, potential epigenetic effect, production of reactive oxygen species or direct effect on spermatozoa and other cells of testicular tissue. Emerging scientific evidence suggests that the increasing incidence of male infertility is associated with the exposure to persistent and non-persistent endocrine-disrupting chemicals such as bisphenols and perfluoroalkyl chemicals (PFAS). These chemicals may impact men's fertility through various mechanisms. This study provides an overview of the mechanisms of action common to persistent (PFAS) and nonpersistent (bisphenols) EDC on male fertility.

[Corrigendum] Novel markers of human ovarian granulosa cell differentiation toward osteoblast lineage: A microarray approach.

Following the publication of this paper, the authors have requested that, on p. 4412 of the above article in the Funding section of the Declarations, the acknowledgement to one of the funding sources should be removed from the paper; essentially, the reference to grant no. 2018/31/B/NZ5/02475, formulated by the Polish National Science Centre (grant providing institution), should be removed from the paper. Therefore, the revised version of the Funding section paragraph should read as follows: Funding: The present study was supported by a grant from Poznan University of Medical Sciences (grant no. 502­14­02227367­10694). The authors confirm that there are no further errors in the study, and all the authors agree to this correction. The authors are grateful to the Editor of Molecular Medicine Reports for granting them this opportunity to publish a Corrigendum, and apologize for any inconvenience caused. [the original article was published in Molecular Medicine Reports 20: 4403-4414, 2019, DOI: 10.3892/mmr.2019.10709].

Human Granulosa Cells-Stemness Properties, Molecular Cross-Talk and Follicular Angiogenesis.

The ovarian follicle is the basic functional unit of the ovary, comprising theca cells and granulosa cells (GCs). Two different types of GCs, mural GCs and cumulus cells (CCs), serve different functions during folliculogenesis. Mural GCs produce oestrogen during the follicular phase and progesterone after ovulation, while CCs surround the oocyte tightly and form the cumulus oophurus and corona radiata inner cell layer. CCs are also engaged in bi-directional metabolite exchange with the oocyte, as they form gap-junctions, which are crucial for both the oocyte's proper maturation and GC proliferation. However, the function of both GCs and CCs is dependent on proper follicular angiogenesis. Aside from participating in complex molecular interplay with the oocyte, the ovarian follicular cells exhibit stem-like properties, characteristic of mesenchymal stem cells (MSCs). Both GCs and CCs remain under the influence of various miRNAs, and some of them may contribute to polycystic ovary syndrome (PCOS) or premature ovarian insufficiency (POI) occurrence. Considering increasing female fertility problems worldwide, it is of interest to develop new strategies enhancing assisted reproductive techniques. Therefore, it is important to carefully consider GCs as ovarian stem cells in terms of the cellular features and molecular pathways involved in their development and interactions as well as outline their possible application in translational medicine.

Cryopreservation of sperm before gonadotoxic treatment at the University Hospital Brno in the years 1995-2020

OBJECTIVE: Sperm cryopreservation before gonadotoxic treatment is the basic and mos teffective method of preserving reproduction, which can be used during adolescence. The communication summarizes 26 years of experience in the operation of an oncological sperm bank, analyzes spermiograms of oncological patients, assesses the relationship between sperm pathology and diagnosis, and determines the number of deaths and the use of frozen sperm. METHODS: During the existence of CAR 01 (assisted reproduction center), more than 50,000 spermiograms were performed. From January 1995 to December 2020, a total of 24,729 men were examined within the sperm bank, of which 1,448 (5.9%) had an oncological diagnosis. The spermiograms were evaluated according to current WHO (World Health Organization) manuals. Cryopreservation of sperm has undergone a major development. The rules for the storage of frozen cells have been laid down by Act No. 296/2008 Coll. since 2008. In 2019, the methodology "Cryopreservation of reproductive cells and tissues in patients before cancer treatment" was updated. In all cases, the standard thawing technique was used. The sperms were processed by the swim-up method. As part of the treatment with assisted reproduction methods, oocytes were fertilized by the ICSI (intracytoplasmatic sperm injection) micromanipulation technique. RESULTS: Out of 1,448 examined spermiograms in men with oncological diagnoses, testicular cancer was present in 43.7% of patients and malignant diseases of lymphatic and hematopoietic tissue were found in 24.1%, of which 70,1% included Hodgkin's lymphomas and 29,9% were non-Hodgkin's lymphomas. Leukemia was found in 7.9%, bone and cartilage cancers in 6.8%. The age of the clients of the whole group ranged from 13 to 64 years (27.2 ± 6.8 years). A total of 38.3% of men had normozoospermia, 54.2% of spermiograms showed pathological findings in 1 to 3 evaluated parameters and 7.5% of patients had azoospermia. Severe asthenozoospermia (mobility ≤ 10%) was detected in 57.2% of men and severe oligozoospermia (concentration ≤ 1 × 106 mm3) in 22.3% of patients. The lowest values of the spermiogram were found in men with testicular cancer; the best values were seen in CNS (central nervous system) cancers. The cryopreservation of sperm was performed in 1,340 cases (92.5%). So far, a total of 160 men (11.9%) have used frozen sperm, of which 6.2% in our center. In these 83 cases, the ICSI technique was always used, 38 clinical pregnancies (45.8%) and 32 births were achieved. We have registered 424 completed storages of semen (31.6%), of which 148 (11.0% of all oncology patients) were made due to death and the others at patients' request. Using the sperm of the dead is a specific issue. CONCLUSION: In cancer patients, sperm pathologies occur in high percentage. The lowest spermiogram values were found in men with testicular cancer. It is necessary to take into account long-term storage and fertilization by micromanipulation methods. The number of men who die is significantly higher than the number of those who use sperm to treat infertility. Cryopreservation of sperm should be offered to each patient prior to the therapy leading to the destruction of spermatogenesis.

Adenomyosis - its possible effect on endometrial function and receptivity

OBJECTIVE: There have been many studies on adenomyosis, which can impair the quality of life of a woman. There are various kinds of opinions on the pathogenesis, diagnostics and treatment of adenomyosis. The goal of this article is to present the current knowledge of adenomyosis and its impact on the endometrial function and receptivity. METHODS: PubMed/Medline, Web of Sciences and Scopus were searched for the articles in English indexed until February 2021 with terms of: adenomyosis, endometrial receptivity, and infertility. RESULTS: Recent studies on angiogenesis and epithelial-mesenchymal transition in the endometrium bring new information on the ethiology and pathogenesis of adenomyosis. In clinical practice, the main diagnostic methods of adenomyosis include transvaginal ultrasound, magnetic resonance imaging or hysteroscopy, although the definitive confirmation is set by histopathological examination. The rules of #Enzian classification of endometriosis should be applied for the classification of adenomyosis. The treatment of adenomyosis should consider individual clinical presentation and reproductive plans of a patient and should be performed in centers for the treatment of endometriosis. CONCLUSION: Adenomyosis affects endometrial vascularisation and epithelial-mesenchymal transition/mesenchymal-epithelial transition; thus, it can be the cause of irregular uterine bleeding or embryo implantation failure. The research and analysis of endometrial proteome could lead to the new ways of adenomyosis treatment.

Solved problems of reproductive medicine in the Czech Republic 2020.

INTRODUCTION: During the 30th symposium of assisted reproduction held on November 11, 2020 in Brno, the solved problems in reproductive medicine in the Czech Republic in 2020 were presented. The selected topics have concerned not only current issues in the field of clinical embryology and genetics as well as gynecology, but also legislation and ethics. Discussed topics: 1. How much time does the doctor have in the CAR (centrum of assisted reproduction) outpatient clinic per patient and how does the embryologist communicate with clients? 2. Reproduction and PGT-M in oncology patients and patients at risk with hereditary oncogenic mutations. 3. Non-invasive genetic testing of embryos from culture medium. 4. Genome editing. 5. What is the need to monitor hormonal levels in stimulation protocols? 6. Monitoring and embryo selection for transfer/kryo. 7. Is it time to change the law on donor remuneration? METHODS: The topics were prepared in advance by authorized members of our company with the task of elaborating theses, which they presented in a separate conference block. The presentation and the discussion were broadcast directly from the broadcast studio at Hotel International via an online connection. After the conference, all discussion topics and comments were incorporated. CONCLUSION: The work presents the state of the solved problems of reproductive medicine in the Czech Republic.

Aspects of embryo selection and their preparation for the formation of human embryonic stem cells intended for human therapy.

OBJECTIVE: The work deals with a clinical part of human embryonic stem cell (hESC) research. The aim of the project is the differentiation of somatic cell types, useful in drug development, regenerative medicine and cell therapy. The aim of this work is to enable targeted therapy of yet incurable diseases. The pluripotent hESCs have unlimited self-renewal capacity. This ability is used in therapy to create missing or damaged cells in the human body. It is of interest to develop clinical-grade hESC lines useful in preclinical and clinical studies. METHODS: The derivation of the hESC must respect the legislation of the Czech Republic and the EU. The aim was to develop an informed consent of both donors for donated discarded embryos that are not suitable for treatment by in vitro fertilization according to Directive 2004/23/EC. The FNBs Center for Assisted Reproduction (CAR) participates in oocyte collection, cultivation and cryopreservation of embryos, communication with clients and ensuring the informed consent of embryo donors. A transport protocol and a methodology for handing over the thawed embryos with the original numerical code were developed. Before the embryos are handed over to the ICRC co-authors workplace (CTEF), they are thawed and, if necessary, recultivated to the blastocyst stage; afterwards, assisted hatching is performed. RESULTS: In the period from January 2018 to July 2020, 138 selected suitable clients were asked for donations, with 52 not responding, 19 terminating and 29 extending the embryo storage. Only 38 clients, i.e. 27.5%, agreed with the usage of their embryos for the preparation of hESCs. In the same period, personal communication with suitable CAR clients took place and another 17 embryo donors were obtained. A total of 160 embryos were obtained from 55 donors aged 26 to 42 years. The embryos were most often frozen in the blastocyst (53 embryos - 33.1%) and morula (74 embryos - 46.3%) stages. Of the 29 genetically examined embryos, only 5 are euploid (17.2%), 2 are mosaic and 22 are aneuploid or with translocations or carriers with a monogenic defect. CONCLUSION: We have an informed consent prepared and approved by the Ethics Committee of the Masaryk University and the University Hospital Brno; 160 donated embryos have been selected and secured. A transport protocol and handover methodology are developed. The plan for the transfer of thawed anonymized embryos in the first phase, October - December 2020, includes approximately 5 thawed blastocysts per week with assisted hatching. After their transfer to the CTEF, the embryoblast will be isolated with subsequent cultivation. The established hESCs must meet the specified criteria of safety, stability and pluripotency. We believe that, in accordance with the project plan, we will obtain at least 3 clinical-grade hESC lines, the first created in the Czech Republic, respecting the requirements for Advanced Medicinal Therapy Products   (AMTP).