Testicular Tissue Vitrification: a Promising Strategy for Male Fertility Preservation.

Destruction of spermatogonial stem cells in juvenile men survivors of pediatric cancers leads to infertility as a side effect of gonadotoxic therapies. Sperm freezing before cancer treatment is commonly used in the clinic for fertility preservation, but this method is not applicable for prepubertal boys due to the lack of mature sperm. In these cases, cryopreservation of testicular tissues is the only option for fertility preservation. Although controlled slow freezing (CSF) is the most common procedure for testicular tissue cryopreservation, vitrification can be used as an alternative method. Controlled vitrification has prevented cell damage and formation of ice crystals. Procedures were done easily and quickly with a brief exposure time to high concentration of cryoprotectants without expensive equipment. Different studies used vitrification of testicular tissues and they assessed the morphology of seminiferous tubules, apoptosis, and viability of spermatogonial cells. Transplantation of vitrified testicular tissue into infertile recipient mice as well as in vitro culture of vitrified tissues was done in previous studies and their findings showed complete spermatogenesis and production of mature sperm. Review articles usually have compared controlled slow freezing with vitrification. In this review, we focused only on the vitrification method and its results. Despite promising results, many studies have been done for finding an optimal cryopreservation protocol in order to successfully preserve fertility in prepubertal boys.

Efficacy of Whole Cancer Stem Cell-Based Vaccines: A Systematic Review of Preclinical and Clinical Studies.

BACKGROUND: Despite the conventional cancer therapeutic, cancer treatment remains a medical challenge due to neoplasm metastasis and cancer recurrence; therefore, new approaches promoting therapeutic strategies are highly desirable. As a new therapy, the use of whole neoplastic stem cells or cancer stem cell (CSC)-based vaccines is one strategy to overcome these obstacles. We investigated the effects of whole CSC-based vaccines on the solid tumor development, metastasis, and survival rate. METHODS: Primary electronic databases (PubMed/MEDLINE, Scopus, Embase, and Web of Science) and a major clinical registry were searched. Interventional studies of whole CSC-based vaccines in rodent cancer models (38 studies) and human cancer patients (11 studies) were included; the vaccine preparation methodologies, effects, and overall outcomes were evaluated. RESULTS: Preclinical studies were divided into 4 groups: CSC-lysates/ inactivated-CSC-based vaccines, CSC-lysate-loaded dendritic cell (CSC-DC) vaccines, cytotoxic T-cell (CTL) vaccines generated with CSC-DC (CSC-DC-CTL), and combinatorial treatments carried out in the prophylactic and therapeutic experimental models. The majority of preclinical studies reported a promising effect on tumor growth, survival rate, and metastasis. Moreover, whole CSC-based vaccines induced several antitumor immune responses. A small number of clinical investigations suggested that the whole CSC-based vaccine treatment is beneficial; however, further research is required. CONCLUSIONS: This comprehensive review provides an overview of the available methods for assessing the efficacy of whole CSC-based vaccines on tumor development, metastasis, and survival rate. In addition, it presents a set of recommendations for designing high-quality clinical studies that may allow to determine the efficacy of whole CSC-based-vaccines in cancer therapy.

Li-Doped Bioactive Ceramics: Promising Biomaterials for Tissue Engineering and Regenerative Medicine.

Lithium (Li) is a metal with critical therapeutic properties ranging from the treatment of bipolar depression to antibacterial, anticancer, antiviral and pro-regenerative effects. This element can be incorporated into the structure of various biomaterials through the inclusion of Li chloride/carbonate into polymeric matrices or being doped in bioceramics. The biocompatibility and multifunctionality of Li-doped bioceramics present many opportunities for biomedical researchers and clinicians. Li-doped bioceramics (capable of immunomodulation) have been used extensively for bone and tooth regeneration, and they have great potential for cartilage/nerve regeneration, osteochondral repair, and wound healing. The synergistic effect of Li in combination with other anticancer drugs as well as the anticancer properties of Li underline the rationale that bioceramics doped with Li may be impactful in cancer treatments. The role of Li in autophagy may explain its impact in regenerative, antiviral, and anticancer research. The combination of Li-doped bioceramics with polymers can provide new biomaterials with suitable flexibility, especially as bio-ink used in 3D printing for clinical applications of tissue engineering. Such Li-doped biomaterials have significant clinical potential in the foreseeable future.

Protective roles of seminal plasma exosomes and microvesicles during human sperm cryopreservation.

RESEARCH QUESTION: Do seminal plasma microvesicles and exosomes, as two subtypes of extracellular vesicles, exert cryoprotective properties in sperm cryopreservation? DESIGN: Microvesicles and exosomes isolated from normozoospermic semen samples (n = 10) by serial ultracentrifugation were determined using scanning electron microscopy, dynamic light scattering and western blot analysis. The interactions between extracellular vesicles and spermatozoa were detected using Dil labelling. Purified spermatozoa from different normozoospermic samples (n = 25) were then treated individually with exosomes or microvesicles for 1 h and subsequently cryopreserved. The effects of extracellular vesicles during cryopreservation were investigated by determining post-thaw sperm motility, morphology, viability, reactive oxygen species (ROS) generation, lipid peroxidation, total antioxidant capacity (TAC), mitochondrial membrane potential (MMP), DNA integrity, and apoptosis rate. RESULTS: Microvesicles and exosomes displayed a round-shape morphology, with about 70% of exosomes ranging from 43-144 nm, microvesicles ranging from 144.5-486 nm and both expressed tetraspanin markers. Fluorescence microscopy showed that exosomes and microvesicles absorbed mainly in the sperm head and less frequently in the neck and tail. The post-thawing results indicated that the diluent with exosomes or microvesicles had improved sperm motility (P = 0.007), morphology (P < 0.001) and viability (P < 0.001) compared with untreated samples. The ROS levels decreased significantly (P = 0.001), with a consequent decrease in DNA damage (P = 0.001). The TAC activity (P = 0.001) and MMP levels (P = 0.001) were also significantly improved; levels of malondialdehyde (MDA) (P = 0.62) and apoptosis rate (P = 1.000) remained unchanged. CONCLUSION: Seminal plasma microvesicles and exosomes could protect spermatozoa from cryopreservation chilling injuries.

Randomized controlled trial of astaxanthin impacts on antioxidant status and assisted reproductive technology outcomes in women with polycystic ovarian syndrome.

PURPOSE: Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women, is typically accompanied by a defective oxidative defense system. Here, we investigated the effect of astaxanthin (AST) as a powerful antioxidant on the oxidative stress (OS) response and assisted reproductive technology (ART) outcomes in PCOS patients. METHODS: In this double-blind, randomized, placebo-controlled trial, PCOS patients were randomly assigned into two groups. The intervention group received 8 mg AST, and the control group received the placebo daily for 40 days. The primary outcomes were the serum and follicular fluid (FF) levels of the OS biomarkers and the expression levels of the specific genes and proteins in the oxidative stress response pathway. The secondary outcomes were considered ART outcomes. RESULTS: According to our findings, a 40-day course of AST supplementation led to significantly higher levels of serum CAT and TAC in the AST group compared to the placebo group. However, there were no significant intergroup differences in the serum MDA and SOD levels, as well as the FF levels of OS markers. The expression of Nrf2, HO-1, and NQ-1 was significantly increased in the granulosa cells (GCs) of the AST group. Moreover, the MII oocyte and high-quality embryo rate were significantly increased in the AST group compared to the placebo group. We found no significant intergroup difference in the chemical and clinical pregnancy rates. CONCLUSION: AST treatment has been shown to increase both serum TAC levels and activation of the Nrf2 axis in PCOS patients' GCs. TRIAL REGISTRATION: ClincialTrials.gov Identifier: NCT03991286.

An overview on tumor treating fields (TTFields) technology as a new potential subsidiary biophysical treatment for COVID-19.

COVID-19 pandemic situation has affected millions of people with tens of thousands of deaths worldwide. Despite all efforts for finding drugs or vaccines, the key role for the survival of patients is still related to the immune system. Therefore, improving the efficacy and the functionality of the immune system of COVID-19 patients is very crucial. The potential new, non-invasive, FDA-approved biophysical technology that could be considered in this regard is tumor treating fields (TTFields) based on an alternating electric field has great biological effects. TTFields have significant effects in improving the functionality of dendritic cell, and cytotoxic T-cells, and these cells have a major role in defense against viral infection. Hence, applying TTFields could help COVID-19 patients against infection. Additionally, TTFields can reduce viral genomic replication, by reducing the expressions of some of the vital members of DNA replication complex genes from the minichromosome maintenance family (MCMs). These genes not only are involved in DNA replication but it has also been proven that they have a crucial role in viral replication. Also, TTFields suppress the formation of the network of tunneling nanotubes (TNTs) which is knows as filamentous (F)-actin-rich tubular structures. TNTs have a critical role in promoting the spread of viruses through improving viral entry and acting as a protective agent for viral components from immune cells and even pharmaceuticals. Moreover, TTFields enhance autophagy which leads to apoptosis of virally infected cells. Thus, it can be speculated that using TTFields may prove to be a promising approach as a subsidiary treatment of COVID-19.

Evaluating the Differential Expression of miR-146a, miR-222, and miR-9 in Matched Serum and Follicular Fluid of Polycystic Ovary Syndrome Patients: Profiling and Predictive Value.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine disorder of women in reproductive age with significant effects on reproductive and metabolic functions. Many molecular players may be involved in PCOS pathology; however, miRNAs possess great ability in gene expression control in normal ovarian function and folliculogenesis. We appraised the relative expression of miR-146a, miR-222, miR-9, and miR-224 in serum and follicular fluid (FF) of PCOS patients compared to control subjects. PCOS (n = 35) and control (n = 30) subjects were recruited in the study during their enrolment in IVF cycles. Serum and FF of human subjects were collected and stored. Total RNA was isolated from samples and cDNA was synthesized using miRNA-specific stem-loop RT primers. Quantitative real-time PCR was used to evaluate the expression of miRNAs relative to U6 expression. The predictive value of miRNAs' expression for discrimination of PCOS patients from control subjects was evaluated by receiver-operating characteristic (ROC) curve analysis. miR-224 was not detected in serum and FF samples. Significantly, higher levels of miR-146a and miR-9 in serum of PCOS group were detected. In contrast, relative expression of miR-146a and miR-9 significantly decreased in FF. In PCOS group, relative expression of all detected miRNAs was elevated in serum in comparison to FF, whereas in control group no change was noticed. Combination of FF miRNAs showed improved predictive value with area under the ROC curve (AUC) of 0.84, 93.8% sensitivity, and 83.3% specificity. Contradicting alternations of miRNAs in serum and FF are indicative of different sources of miRNAs in body fluids. Presumptive target genes of studied miRNAs in signalling pathways may show the potential role of these miRNA in folliculogenesis.

The Potential Relationship Between Different Human Female Reproductive Disorders and Sperm Quality in Female Genital Tract.

Spermatozoa should travel throughout the female reproductive tract to reach its ultimate goal, fertilization of the oocyte. At the ejaculation moment, millions of sperm within a few milliliters of the ejaculate are deposited at the cranial segment of vagina and make their journey to the fertilization site. This is done by means of various factors, such as sperm motility, the uterine and fallopian tubes contractility, and the ciliary movement of the lining cells. During this migration, spermatozoa interact with the female microenvironment both physically and molecularly. In this regard, the quality of the environmental conditions may affect this interaction. Therefore, some alterations in women's genital tract microenvironment, such as conditions that occur in female reproductive disorders, may have detrimental effects on sperm reproductive function. In this review, human sperm migration through the female tract is described, and the potential effects of different reproductive disorders at reproductive organs, such as vagina, uterine cervix, uterus, fallopian tubes, and ovary on sperm survival and quality, are also argued. The understanding of those conditions that may impair sperm fertility in the female genital tract can provide a more accurate diagnosis of the causes of infertility in couples. This can ultimately lead to the discovery of effective treatment approaches.

COVID-19 and varicocele: The possible overlap factors and the common therapeutic approaches.

Varicocele is recognized as one of the main attributable causes of male infertility which can affect spermatogenesis by various pathophysiological mechanisms. Recent studies have identified oxidative stress and reduction in antioxidant, hyperthermia, hypoxia, hormonal dysfunction, and inflammatory conditions as major factors in the pathophysiology of varicocele, all of which have known direct associations with the coronavirus disease 2019 (COVID-19) and can significantly increase the risk of detrimental COVID-19-related outcomes. Emerging data have shown an association between COVID-19 and inflammation, overproduction of cytokine, and other pathophysiological processes. The present review, summarizes the current understanding of the pathophysiology of varicocele and investigates the potential correlation between the severity of COVID-19 and the varicocele disease. In addition, various possible treatments which can be effective in both diseases were examined. Despite numerous challenges associated with the prevalence of COVID-19 in healthcare systems in infected countries, special attention should be given to maintaining a high level of care for complex patients with a pre-existing disease such as varicocele and providing appropriate practical advice for optimal control of the COVID-19 disease.

Antioxidant supplementations ameliorate PCOS complications: a review of RCTs and insights into the underlying mechanisms.

Polycystic ovary syndrome (PCOS) is one of the most important gynecological disorders of women in the age of reproduction. Different hormonal and inflammatory cross-talks may play in the appearance of its eventual complications as a leading cause of infertility. Excessive production of reactive oxygen species over the power of the antioxidant system as oxidative stress is known to contribute to a variety of diseases like PCOS. Thus, the utilization of antioxidants can be efficient in preventing or assistant in treating these diseases. In this review, we describe the clinical trial studies that have examined the efficiency of antioxidant strategies against PCOS and the possible underlying mechanisms. The investigations presented here lead us to consider that targeting oxidative stress pathways is probably a powerful promising therapeutic approach towards PCOS. There is preparatory evidence of the effectiveness of antioxidant interventions in ameliorating some of the PCOS complications, including metabolic and hormonal disorders. Due to limited data and relatively few clinical trials, many of these interventions need further investigation before they can be considered effective agents for routine clinical use.

Anti-IgE monoclonal antibodies as potential treatment in COVID-19.

Coronavirus disease 2019 (COVID-19) is associated with irreversible effects on vital organs, especially the respiratory and cardiac systems. While the immune system plays a key role in the survival of patients to viral infections, in COVID-19, there is a hyperinflammatory immune response evoked by all the immune cells, such as neutrophils, monocytes, and includes release of various cytokines, resulting in an exaggerated immune response, named cytokine storm. This severe, dysregulated immune response causes multi-organ damage, which eventually leads to high mortality. One of the most important components of hypersensitivity is immunoglobulin E (IgE), which plays a major role in susceptibility to respiratory infections and can lead to the activation of mast cells. There is also a negative association between IgE and IFN-α, which can reduce Toll-like receptor (TLR) nine receptor expression and TLR-7 signaling to disrupt IFN production. Moreover, anti-IgE drugs such as omalizumab reduces the severity and duration of COVID-19. In addition to its anti-IgE effect, omalizumab inhibits inflammatory cells such as neutrophils. Hence, blockade of IgE may have clinical utility as an immunotherapy for COVID-19.

Effect of sulforaphane on apoptosis, reactive oxygen species and lipids peroxidation of human sperm during cryopreservation.

Sperm cryopreservation is a common procedure to preserve viable sperm for an indefinite period. This procedure has numerous detrimental effects on sperm function due to increased generation of reactive oxygen species (ROS). During cryopreservation, while ROS increases, antioxidant enzymes level decreases. It has been shown that a relationship exist between lower antioxidant levels and infertility. l-Sulforaphane (SFN) is an isothiocyanate in cruciferous vegetables of the brassica class that has potent protective effects against oxidative stress. The purpose of the present study was to evaluate the effects of SFN supplementation during the freeze-thaw process on different parameters of human spermatozoa which can influence sperm fertilizing ability. Samples were collected from 25 healthy men and each sample was divided into three groups: fresh, control (untreated frozen/thawed samples) and treatment (treated frozen/thawed with SFN) groups. Sperm parameters, ROS production (using flow cytometry), plasma membrane integrity (using flow cytometry), Lipid peroxidation (using ELISA) were evaluated. Our results demonstrated that 5 µM SFN improved all parameters of sperm including viability (P < 0.001), motility, and morphology (P < 0.05) after the freeze-thaw process. Furthermore, SFN reduced the levels of intracellular hydrogen peroxide (P < 0.01) and superoxide anion (P < 0.05). Also, SFN significantly increased the percentage of viable sperm cells with the intact plasma membrane (P < 0.001) and decreased the level of lipid peroxidation after the freeze-thaw process (P < 0.01).Our findings showed that spermatozoa treatment with 5 µM SFN before the freeze-thaw process has protective effects against oxidative stress and could decrease the detrimental effects of this process on sperm quality.

In vitro versus In vivo: Development-, Apoptosis-, and Implantation- Related Gene Expression in Mouse Blastocyst.

BACKGROUND: While mammalian embryos can adapt to their environments, their sensitivity overshadows their adaptability in suboptimal in vitro conditions. Therefore, the environment in which the gametes are fertilized or to which the embryo is exposed can greatly affect the quality of the embryo and consequently its implantation potential. OBJECTIVES: Since providing an optimal culture condition needs a deep understanding of the environmental effects, and regarding the fact that normal morphology fails to be a reliable indicator of natural embryo development, the current study aimed at comparing in vivo- and in vitro-derived blastocysts at the molecular level. MATERIALS AND METHODS: In vivo and in vitro mouse blastocysts were obtained by flushing the uterine horns and in vitro fertilization/culture, respectively. Normal blastocysts of both groups were evaluated in terms of hatching rate and expression of three lineage-differentiation-, apoptosis-, and implantation-related genes. RESULTS: The hatching rate was lower in In vitro fertilization (IVF)-produced blastocysts in comparison with that of the in vivo counterparts. More importantly, the study results indicated significant changes in the expression levels of eight out of ten selected genes, especially Mmp-9 (about -10.7-fold). The expression of Mmp-9 in trophoblast cells is required for successful implantation and trophoblast invasion. CONCLUSIONS: The current study, in addition to confirming that the altered gene expression pattern of in vitro-produced embryos resulted in normal morphology, provided a possible reason for lower implantation rate of in vitro-produced blastocysts regarding the Mmp-9 expression.

Different Origin, Different Response: Gene Expression Pattern in Collapsed Vitrified Blastocyst.

There is a large body of animal experimental data about assisted reproductive techniques that could be applied to improve clinical outcomes. The great part of this information was obtained from research on in vivo-derived embryos. But whether these results are always similar with those we expect from embryos having in vitro origin in the clinical cases is a critical question. The present study was designed to compare the effects of vitrification (VIT) and artificial collapse (AC) as two commonly used techniques on in vivo- and in vitro-derived mouse embryos. In this regard, both origins of blastocysts were produced and randomly divided into three experimental groups, including control (non-vitrified), VIT, and AC-VIT. The survival and hatching rates and the expression of development-related genes were assessed in all groups and compared with their control counterpart. According to our results, although in vivo and in vitro origins followed the same pattern in the hatching rate, the real-time PCR data showed two distinct patterns of gene expression. Compared to the control, vitrification increased the expression of pluripotency genes in in vivo group. While in vitro vitrified blastocysts showed a significant reduction in the transcripts of these genes. More interestingly, although AC resulted in a sharp decrease of Gata6 and Grb2 in post warmed in vivo blastocysts, it could not affect the vitrified IVP ones. In conclusion, it seems that vitrification and artificial collapse techniques have different effects on embryo fate depending on in vivo or in vitro origins of the embryos.

Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts.

Artificial collapse of the blastocoel cavity before vitrification can improve the quality of warmed embryos, yet how reduction of blastocoel fluid impacts formation of the blastocyst cell lineages is not clear. The present study assessed the effect of pre-vitrification blastocoel fluid reduction on the survival, hatching rate, and the expression of genes related to apoptosis (Tp53), pluripotency (Pou5f1, Nanog), and differentiation (Cdx2, Eomes, Gata6) in mouse blastocysts. In vivo-produced blastocysts were randomly divided into three groups: The first group was vitrified and warmed; the second group underwent artificial collapse of the blastocoel cavity prior to vitrification and warming; the third group served as the control, in which neither vitrification or artificial collapse was performed. The survival rate of treatment groups was similar to the control group, whereas the hatching rate of artificial collapse/vitrified blastocysts was significantly higher than vitrified blastocysts. Quantitative reverse-transcription PCR analysis revealed a considerable reduction in the expression of Cdx2, Eomes, Gata6, Grb2, and Tp53 transcripts following artificial collapse/vitrification in comparison to the vitrification-alone group; the abundance of Pou5f1 and Nanog, however, did not change. These results suggest that artificial collapse of the blastocoel cavity before vitrification leads to relatively normal expression of apoptosis and development-related genes plus higher hatching rates. Mol. Reprod. Dev. 83: 735-742, 2016 © 2016 Wiley Periodicals, Inc.