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).

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.

Sperm cryopreservation before testicular cancer treatment and its subsequent utilization for the treatment of infertility.

AIMS: In this study we report our results with storage of cryopreserved semen intended for preservation and subsequent infertility treatment in men with testicular cancer during the last 18 years. METHODS: Cryopreserved semen of 523 men with testicular cancer was collected between October 1995 and the end of December 2012. Semen of 34 men (6.5%) was used for fertilization of their partners. They underwent 57 treatment cycles with cryopreserved, fresh, and/or donor sperm. RESULTS: A total of 557 men have decided to freeze their semen before cancer treatment. Azoospermia was diagnosed in 34 men (6.1%), and semen was cryopreserved in 532 patients. Seminoma was diagnosed in 283 men (54.1%) and nonseminomatous germ cell tumors in 240 men (45.9%). 34 patients who returned for infertility treatment underwent 46 treatment cycles with cryopreserved sperm. Totally 16 pregnancies were achieved, that is, 34.8% pregnancy rate. CONCLUSION: The testicular cancer survivors have a good chance of fathering a child by using sperm cryopreserved prior to the oncology treatment, even when it contains only limited number of spermatozoa.

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.

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.

Digital holographic microscopy in human sperm imaging.

PURPOSE: The aim of this study was to use digital holographic microscopy (DHM) in human sperm imaging and compare quantitative phase contrast of sperm heads in normozoospermia (NZ) and oligoasthenozoospermia (OAT). METHODS: DHM spermatozoa imaging and repeated quantitative phase shift evaluation were used. Five NZ and 5 OAT samples were examined. Semen samples were examined by semen analysis and processed for DHM. Main outcome measures were maximum phase shift value of the sperm heads. Differences of the phase shift and in NZ and OAT samples were statistically tested. RESULTS: In NZ samples median phase shifts were in the range 2.72-3.21 rad and 2.00-2.15 in OAT samples. Differences among individual samples were statistically significant (p < 0.001) in both groups. Median phase shift according to sperm count was 2.90 rad in NZ samples and 2.00 rad in OAT samples. This difference was statistically significant (p < 0.001). CONCLUSION: Quantitative evaluation of the phase shift by DHM could provide new information on the exact structure and composition of the sperm head. At present, this technique is not established for clinical utility.