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.
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.
High grade serous carcinoma of the ovary, fallopian tube, and peritoneum (HGSC) is the deadliest gynecological disease which results in a five-year survival rate of 30% or less. HGSC is characterized ...by the early and rapid development of metastases accompanied by a high frequency of ascites
the pathological accumulation of fluid in peritoneum. Ascites constitute a complex tumor microenvironment and contribute to disease progression by largely unknown mechanisms.
Malignant ascites obtained from HGSC patients who had undergone cytoreductive surgery were tested for their ability to induce WNT signaling in the Kuramochi cell line, a novel and clinically relevant
model of HGSC. Next, cancer spheroids (the main form of metastatic cancer cells in ascites) were evaluated with respect to WNT signaling. Kuramochi cells were used to determine the role of individual WNT signaling branches in the adoption of metastatic stem cell-like behavior by HGSC cells. Furthermore, we analyzed genomic and transcriptomic data on WNT/Planar Cell Polarity (PCP) components retrieved from public cancer databases and corroborated with primary patient samples and validated antibodies on the protein level.
We have shown that ascites are capable of inducing WNT signaling in primary HGSC cells and HGSC cell line, Kuramochi. Importantly, patients whose ascites cannot activate WNT pathway present with less aggressive disease and a considerably better outcome including overall survival (OS). Functionally, the activation of non-canonical WNT/PCP signaling by WNT5A (and not canonical WNT/β-catenin signaling by WNT3A) promoted the metastatic stem-cell (metSC) like behavior (
self-renewal, migration, and invasion) of HGSC cells. The pharmacological inhibition of casein kinase 1 (CK1) as well as genetic ablation (dishevelled 3 knock out) of the pathway blocked the WNT5A-induced effect. Additionally, WNT/PCP pathway components were differentially expressed between healthy and tumor tissue as well as between the primary tumor and metastases. Additionally, ascites which activated WNT/PCP signaling contained the typical WNT/PCP ligand WNT5A and interestingly, patients with high levels of WNT5A protein in their ascites exhibited poor progression-free survival (PFS) and OS in comparison to patients with low or undetectable ascitic WNT5A. Together, our results suggest the existence of a positive feedback loop between tumor cells producing WNT ligands and ascites that distribute WNT activity to cancer cells in the peritoneum, in order to promote their pro-metastatic features and drive HGSC progression.
Our results highlight the role of WNT/PCP signaling in ovarian cancerogenesis, indicate a possible therapeutic potential of CK1 inhibitors for HGSC, and strongly suggest that the detection of WNT pathway inducing activity ascites (or WNT5A levels in ascites as a surrogate marker) could be a novel prognostic tool for HGSC patients.
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
and
proteins, combined detections of these two bring a diagnostic output with a high sensitivity and specificity. Similar results were observed for combined detection of
and
. The effective using of specific biomarkers from seminal plasma can significantly improve the existing approaches to diagnosis of the causes of male infertility.
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 (≤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.
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.
Abstract Endocrine disruptors (EDs) are chemical substances that affect physiological processes in the organism via hormonal regulation. The EDs are present in the environment and objects of everyday ...use. They are often detected in food, particularly released from packaging of canned food, but also from plastic water bottles, and they are also found in cosmetics and fertilizers. They are commonly detected in children's toys, banknotes, receipts and many more objects. Permanent and long-term utilization of EDs has harmful effects on human reproductive health mainly by interference with sex hormone synthesis and mechanism of action. The endocrine disruptors show many negative effects on male reproductive system. Any change during synthesis or activity of sex hormones can cause abnormal reproduction, including developmental anomalies of the sexual system, disruption of testicular development or deterioration of sperm quality. Mainly the impact on the development of testicles in prenatal and early postnatal period can be crucial for reproductive health in males. This review provides an overview of the EDs and their possible impact on reproductive health in males with focus on sperm quality and development of testicles.
High‐grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in ...the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients’ ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of ‘core ascitic EV‐associated proteins’ but also defined their subset unique to HGSC ascites. Using single‐cell RNA sequencing data, we mapped the origin of HGSC‐specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non‐malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type‐specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics.
We performed the first in‐depth proteomics study of precisely isolated EVs from ascites of HGSC patients and identified core ascitic EV‐associated proteins as well as HGSC‐specific EV‐associated proteins. We provide evidence that by the combination of EV proteomics and single‐cell methods, we can assess the contribution of cells forming the tumour and its environment to EV pool in the ascites. Surprisingly, analysis of cell‐type specific EV markers showed a stronger predictive potential for HGSC patient survival than analysis of ascitic cells.
The vitrification of human embryos is more and more frequently being utilized as a method of assisted reproduction. For this technique, gentle treatment of the embryos after thawing is crucial. In ...this study, the balance of amino acids released to/consumed from the cultivation media surrounding the warmed embryos was observed in the context of a cultivation environment, which was with the atmospheric oxygen concentration ≈20% or with a regulated oxygen level—hysiological (5%). It is the first time that total amino acid turnover in human embryos after their freezing at post compaction stages has been evaluated. During this study, progressive embryos (developed to blastocyst stage) and stagnant embryos (without developmental progression) were analyzed. It was observed that the embryos cultivated in conditions of physiological oxygen levels (5% oxygen) showed a significantly lower consumption of amino acids from the cultivation media. Progressively developing embryos also had significantly lower total amino acid turnovers (consumption and production of amino acids) when cultured in conditions with physiological oxygen levels. Based on these results it seems that a cultivation environment with a reduced oxygen concentration decreases the risk of degenerative changes in the embryos after thawing. Therefore, the cultivation of thawed embryos in an environment with physiological oxygen levels may preclude embryonal stagnation, and can support the further development of human embryos after their thawing.
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.
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