Jana Žáková

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.

MEKC LIF method for analysis of amino acids after on-capillary derivatization by transverse diffusion of laminar flow profiles mixing of reactants for assessing developmental capacity of human embryos after in vitro fertilization

Evaluating the physiological state of an organism is of clinical importance. In assisted reproduction, knowledge of the embryos physiology is crucial for selecting the embryo with the highest developmental capacity to ensure high pregnancy rates. Amino acids (AAs) are involved in many biochemical processes during embryo development, which means that the determination of AA fluctuations in the embryos surroundings can determine the embryos physiological state. Since current embryo selection methods are mainly based on visual assessment, which lacks proper accuracy, a novel method for the analysis of AAs in the embryos surroundings was developed. AAs were analyzed by means of MEKC‐LIF after on‐capillary derivatization by naphthalene‐2,3‐dicarboxaldehyde. The reactants were injected under the three zone arrangement and mixed using the transverse diffusion of laminar flow profiles methodology. The resulting derivatives of all the standard AAs were baseline resolved in the BGE comprised of 35 mM sodium tetraborate, 55 mM SDS, 2.7 M urea, 1 mM BIS‐TRIS propane, and 23 mM NaOH within 50 min. The method was applied on an analysis of spent culture media used in assisted reproduction to culture embryos after in vitro fertilization.

Determination of pyruvate and lactate as potential biomarkers of embryo viability in assisted reproduction by capillary electrophoresis with contactless conductivity detection

Human‐assisted reproduction is increasing in importance due to the constantly rising number of couples suffering from infertility issue. A key step in in vitro fertilization is the proper assessment of embryo viability in order to select the embryo with the highest likelihood of resulting in a pregnancy. This study proposes a method based on CE with contactless conductivity detection for the determination of pyruvate and lactate in spent culture media used in human‐assisted reproduction. A fused‐silica capillary of 64.0 cm total length and 50 μm inner diameter was used. The inner capillary wall was modified by the coating of successive layers of the ionic polymers polybrene and dextran sulfate to reverse EOF. The BGE was composed of 10 mM MES/lithium hydroxide, pH 6.50. The sample was injected by pressure 50 mbar for 18 s, separation voltage was set to −24 kV, and capillary temperature to 15°C. The presented method requires only 2 μL of the culture medium, with LODs for pyruvate and lactate of 0.03 and 0.02 μM, respectively. The results demonstrated the methods suitability for the analysis of spent culture media to support embryo viability assessment by light microscopy, providing information about key metabolites of the energy metabolism of a developing embryo.

Methods for preserving fertility in young women suffering from cancer: some aspects of ovarian tissue cryopreservation.

The purpose of our paper is to review shortly the current methods for preserving fertility in young women suffering from cancer. Our preliminary ultrastructural study shows that the method of cryopreservation of human ovarian tissue causes minimal structural changes in primordial and primary ovarian follicles.

Magnetic-Activated Cell Sorting in Combination with Swim-Up Efficiency Improve Effectivity of Spermatozoa Separation

Abstract Correct selection of spermatozoa before their using for an assisted reproductive techniques is one of the crucial step in therapy of human infertility. It was previously reported that male factor plays a major role in infertility. Basic semen analyses and standard methods for sperm selection in many cases does not eliminate sufficiently proportion of spermatozoa with genetics defects. Magnetic Activated Cell Sorting (MACS) is a selection method which reduce apoptotic sperm and improve sperm and embryo quality. The aim of our study was the comparison of swim-up method and MACS and their combination. We tested swim-up and MACS alone and treatment of spermatozoa in combination when was is first swim-up and second MACS and vice versa. In this study we evaluated sperm concentration, motility and their DNA integrity before and after separtion methods. On the basis of our results we recommend to use swim-up before MACS method. This approach brings better results in the sperm selection - lower proportion of spermatozoa with fragmented DNA and also it brings better gain of total spermatozoa usable for next IVF or ICSI methods.

Current Topics in Assisted Reproduction in the Czech Republic

Abstract Assisted reproduction is a very dynamic part of reproductive gynaecology with fast changes in therapeutic approaches resulting from intensive research supported by private or public companies. This progress brings also new questions regarding ethical, legal or therapeutic issues. At the 26th Czech-Slovak symposium of assisted reproduction in Brno, several interesting topics of assisted reproduction were presented. During two days, more than 350 specialists discussed current topics related to gynaecology, embryology, andrology, genetics and sexology. From this fruitful discussion we chose the following topics that we consider to be the most important: (1) “Soft” versus “standard” stimulation protocols for IVF (2) Use of exogenous and endogenous progestins in IVF stimulation protocols (3) Cryopreservation and transplantation of ovarian tissue - state of the art and situation in the Czech Republic (4) Selection of spermatozoa with fragmented DNA: practical recommendations (5) Artificial activation of oocytes (8) Access of single and lesbian women to assisted reproduction technologies and possibility of their reproduction in the Czech Republic. Panel conclusions were presented at the end of the conference, which had great attendance, invoked lively commentaries and produced some definitive consensus. Certain issues remained inconclusive and these matters will be the subject of further discussion in future. This is a summary of the most important theses from this field in the Czech Republic in 2017.

Study of metabolic activity of human embryos focused on amino acids by capillary electrophoresis with light-emitting diode-induced fluorescence detection

Assisted reproduction is a quickly developing field of reproductive medicine whose importance is growing every year due to the increasing number of patients suffering from infertility. As a result, there is a need for the continuous development and/or improvement of assisted reproductive technologies. This paper presents a new method for the in vitro measurement of the amino acid turnover of developing embryos based on capillary electrophoresis with light‐emitting diode‐induced fluorescence detection. Amino acids were derivatized with naphthalene‐2,3‐dicarboxaldehyde/NaCN, and the resulting fluorescent derivatives were baseline resolved within 25 min in a background electrolyte comprised of 50 mM sodium tetraborate, 73 mM sodium dodecyl sulphate, 5 mM sodium deoxycholate and 2.5 mM (2‐hydroxypropyl)‐β‐cyclodextrin (pH ≈ 9.3). The migration time and the peak area repeatability (n = 10) were below 0.5 and 4.3%, respectively. The limits of detection ranged from 12.6 nM (histidine) to 39.3 nM (taurine). The developed method, which only requires 2 μL of raw sample, was successfully applied for determining the metabolic activity of human embryos exposed to different environmental stress conditions.

Cryopreservation of Human Gametes and Embryos: Current State and Future Perspectives

Cryopreservation of human gametes and embryos is an important and widely used method in most embryology laboratories. During last years, the practice of single embryo transfer was a greater demand for reliable cryostorage of surplus embryos. Currently, there are two basic principally different methods usable for cryopreservation: slow freezing and vitrification. Vitrification is a very promising method with massive use in embryology. Nowadays, this method is also suitable for cryopreservation of human mature oocytes (one of the most problematic cell in cryobiology). This progress in the field of cryopreservation opens new perspectives in assisted reproduction. Recent effective oocyte vitrification systems have a significant impact on clinical practice. This chapter gives a view of human gametes (sperms, oocytes) and embryos cryopreservation application and possibilities. Indications and methods of cryopreservation and thawing are mentioned.