Igor Crha

Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.

Prevention of ovarian function damage by a GnRH analogue during chemotherapy in Hodgkin lymphoma patients

BACKGROUND Frequent negative consequence of chemotherapy (CHT) is ovarian damage and premature ovarian failure (POF). Aim of this prospective case-control study is evaluation of GnRH analogue (GnRH-a) administration to patients with Hodgkin lymphoma (HL) during CHT and prevention of ovarian damage depending upon CHT regimen. METHODS Study group consists of 72 patients in fertile age (18-35 years) with HL diagnosis treated in 2004-2005 by curative CHT together with GnRH analogue (Triptorelin) administration according to a standardized protocol. Patients were divided into three groups according to the stage of disease and treated by three types of CHT regimens (A,B,C) with increased cytotoxicity. Ovarian function of all patients was assessed by gonadotrophin levels (FSH, LH) analysis from peripheral blood before treatment and also 6 and 12 month after it. The number of women with POF after CHT in study group was compared with control group (n = 45, age 18-35 years) of patients treated in 2002-2003 according to the same protocol but without protective GnRH analogue application. RESULTS In study group with GnRH analogue administration during CHT, there was significantly (P < 0.001) fewer cases with POF 6 and 12 month after the end of CHT (37.5% and 20.8%, respectively) than in control group (73.3% and 71.1%, respectively). Comparative analysis depending on cytotoxicity of CHT regimen used showed significant differences in percentage of patient with acquired POF between study and control group only in less aggressive CHT protocols. CONCLUSIONS Study showed a significant reduction of ovarian failure risk in women with HL treated with less aggressive CHT regimens plus a GnRH analogue.

Survival and infertility treatment in male cancer patients after sperm banking

OBJECTIVE To evaluate the relationship between sperm pathology and cancer diagnosis, determine the mortality rate, and evaluate the outcomes of the use of frozen sperm from the sperm bank. DESIGN Prospective study. SETTING University fertility center. PATIENT(S) A total of 619 male patients were referred for sperm freezing before gonadotoxic therapy from 1995 to 2006. INTERVENTION(S) Semen analysis, data verification in the National Oncologic Register, assisted reproduction technologies, and statistical evaluation. MAIN OUTCOME MEASURE(S) Cancer diagnosis and sperm pathology analysis, survival of patients, and infertility treatment success. RESULT(S) Malignant testicular cancer was diagnosed in 43.6% of patients, and malignant neoplasms of the lymphatic and hematopoietic tissues were found in 31.7% of patients. Azoospermia or severe oligospermia (<or=1 million/mL) was detected in 9.7% and 22.6% of patients, respectively. To date, 32 patients (5.2%) sought infertility treatment. Cryopreserved semen was used in 28 couples (87.5%), and 44 intracytoplasmic sperm injection (ICSI) cycles resulted in 13 pregnancies. In total, 74 deaths (11.9%) were reported, 61 of them (82.4%) within 30 months of the cryopreservation of their sperm. CONCLUSION(S) A significant number of patients survived. Intrauterine insemination and ICSI with cryopreserved sperm resulted in deliveries.

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.

Intraindividual variability of homocysteine and related thiols concentrations in follicular fluid

PurposeTo determine intraindividual variability in concentrations of homocysteine and related thiols in follicular fluids of particular follicles after ovarian stimulation and assess the differences between follicles with/or without oocytes.MethodsHPLC-FD analysis of plasma and follicular fluid cysteine, homocysteine, cysteinylglycine and glutathione in women undergoing IVF.ResultsIn blood plasma, the homocysteine, cysteine, and cysteinylglycine concentrations decreased significantly during stimulation with rec FSH (p < 0.001). We found significant differences in concentrations of cysteine and glutathione between follicles with or without retrieved oocytes. High intraindividual variability in concentrations of thiols was determined.ConclusionsThe concentration variability of thiols between single follicles is very high and we recommend mean at least from 3 follicles with/or without oocytes for characterization of each woman. It is the best to examine individual follicles for further research and analysis of fertility outcomes.

The intracellular concentration of homocysteine and related thiols is negatively correlated to sperm quality after highly effective method of sperm lysis

The aims of this study were to determine concentrations of total homocysteine, cysteine, cysteinylglycine and glutathione in spermatozoa, seminal fluid and blood plasma and to analyse their relationships with sperm parameters. For this reason, a new highly effective method of spermatozoa lysis was developed, using methanol, freezing and subsequent thawing in ultrasonic bath. An HPLC‐FD assay was conducted on thiols concentrations in lysed spermatozoa, seminal fluid and blood plasma. Concentrations of thiols in spermatozoa were significantly lower in men with normozoospermia than in samples with pathological semen parameters. Statistical analysis found significant correlations between thiol concentrations in spermatozoa and semen parameters, while the same analysis with thiol concentrations in seminal fluid was substantially less powerful. Only cysteinylglycine concentrations in seminal fluid significantly correlated with pathological semen parameters. No significant differences or correlations were found with blood plasma concentrations.

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.

Analysis of male and female smoking in women pregnant after IVF/ET

Former smokers had significantly lower effects of their infertility treatment using the IVF methods. Treatment of current smokers was compleetely unsuccessful.