Mitochondria-targeted antioxidants have great potential to counterbalance the generated reactive oxygen species (ROS) because they cross the inner membrane of the mitochondria. Still, their use was not reported in vitrified human spermatozoa. Our laboratory has successfully vitrified spermatozoa without the use of permeable cryoprotectants, but subcellular-level evidence was missing. Therefore, this study aimed to improve spermatozoa vitrification using a mitochondria-targeted antioxidant (mitoquinone, MitoQ), reveal ultrastructural changes in the spermatozoa due to the use of a permeable cryoprotectant, and report alterations of functional proteins during the spermatozoa vitrification process. For this, each of 20 swim-up-prepared ejaculates was divided into seven aliquots and diluted with a vitrification medium supplemented with varying concentrations of MitoQ (0.02 and 0.2 μM), glycerol (1, 4, and 6%), and a combination of MitoQ and glycerol. All aliquots were vitrified by the aseptic capillary method developed in our laboratory. The spermatozoa function assays revealed that the addition of either MitoQ (0.02 μM), glycerol (1%), or a combination of MitoQ (0.02 μM) and glycerol (1%) in the vitrification medium results in better or equivalent spermatozoa quality relative to the control. Transmission electron microscopy revealed that MitoQ protects the spermatozoa from undergoing ultrastructural alterations, but glycerol induced ultrastructural alterations during the vitrification process. Next, we performed label-free quantitative proteomics and identified 1,759 proteins, of which 69, 60, 90, and 81 were altered in the basal medium, 0.02 μM MitoQ, 1% glycerol, and Mito-glycerol groups, respectively. Actin, tubulins, and outer dense fiber proteins were not affected during the vitrification process. Some of the identified ubiquitinating enzymes were affected during spermatozoa vitrification. Only a few proteins responsible for phosphorylation were altered during vitrification. Similarly, several proteins involved in spermatozoa–egg fusion and fertilization (IZUMO1 and Tektin) were not affected during the vitrification process. In conclusion, MitoQ attenuates the vitrification-induced ultrastructural changes and alterations in the key proteins involved in spermatozoa functions and fertilization.

Introduction: The in vitro culture of primordial follicles is the only available option for preserving fertility in prepubertal girls with malignant tumors. The cultivation of primordial follicles in scaffolds as artificial ovaries is a promising approach for this. Methods: Dissociated follicles were placed into an artificial ovarian scaffold composed of fibrinogen and thrombin. The follicles were cultured in a dish dedicated to live cell imaging and observed for growth using immunofluorescence and development via optical microscopy. The morphology of the follicles in the scaffold was three-dimensionally reconstructed using the Imaris software. Growth and development were also quantified. Results: The morphology of artificial ovaries began to degrade over time. Within approximately 7 days, primordial follicles were activated and grew into secondary follicles. A comparison of optical and confocal microscopy results revealed the superior detection of live cells using confocal microscopy. The three-dimensional reconstruction of the confocal microscopy data enabled the automatic enumeration and evaluation of the overall morphology of many follicles. Conclusions: The novel artificial ovary-enabled primordial follicles to enter the growth cycle after activation and grow into secondary follicles. The use of a fibrin scaffold as a carrier preserves the developmental potential of primordial germ cells and is a potentially effective method for preserving fertility in prepubertal girls.

Introduction: Spermatozoa cryopreservation is an important technique to preserve fertility for males. This study aimed at exploring the stability of epigenetics information in human spermatozoa, manipulated by two different technologies, freezing and vitrification. Methods: Spermatozoa samples were distributed into three groups: 1. Fresh spermatozoa (control group), 2. Frozen spermatozoa, 3. Vitrified spermatozoa. Epigenetic differences of fresh and cryopreserved spermatozoa were evaluated using high-throughput RNA sequencing. Results: Differentially expressed genes (DEGs) in frozen (1103 genes) and vitrified (333 genes) spermatozoa were evaluated. The bioinformatical analysis identified 8 and 15 significant pathways in groups of frozen and vitrified spermatozoa, respectively. The majority of these pathways are most relevant to immune and infectious diseases. The DEGs of the fertilization process are not detected during vitrification. The freezing process induces more down-regulation of genes and is relevant to apoptosis changes and immune response. Conclusion: Cryopreservation of human spermatozoa is an epigenetically safe method for male fertility preservation. Cryoprotectant-free vitrification can induce more minor biological changes in human spermatozoa, in comparison with conventional freezing.

The molecular mechanisms of normal cervical squamous epithelium advancing to cervical intraepithelial neoplasia (CIN) and eventually to cervical squamous cell carcinoma (CSCC) are largely unknown. This study explored abnormal expression of Yin Yang 1 (YY1) in cervical cancer and its correlation with the expression of E-cadherin and human papillomavirus (HPV) 16 E6. YY1, E-cadherin and HPV16 E6 expression were detected by immunohistochemistry in 90 cervical tissue specimens collected from 30 patients with hysteromyoma, 15 patients with CIN I, 15 patients with CIN II-III, and 30 patients with CSCC. The H-score method was employed to measure the expression of YY1, E-cadherin and HPV16 E6. Increased expression of YY1 and HPV16 E6, and the decreased expression levels of E-cadherin were strongly associated with malignant transformation of the cervical epithelium and the histological progression of CSCC. The expression of YY1 in cervical tissues was inversely correlated with E-cadherin expression, and positively correlated with HPV16 E6 expression. Expression of YY1 in CSCC tissues was not significantly correlated with tumor differentiation, but was significantly correlated with an advanced clinical stage of CSCC. These results suggest that up-regulation of YY1 is closely associated with the progression of CSCC, and YY1 may play an important role in the pathogenesis of cervical cancer by modulating the expression of E-cadherin and HPV16 E6.