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.

Background: Gene set enrichment analysis (GSEA) was conducted on raw data, and alternative splicing (AS) events were found after mRNA sequencing of human spermatozoa. In this study, we aimed to compare unknown micro-epigenetics alternations in fresh and cryopreserved spermatozoa to evaluate the effectivity of cryopreservation protocols. Methods: Spermatozoa were divided into three groups: fresh spermatozoa (group 1), cryoprotectant-free vitrified spermatozoa (group 2), and conventionally frozen spermatozoa (group 3). Nine RNA samples (three replicates in each group) were detected and were used for library preparation with an Illumina compatible kit and sequencing by the Illumina platform. Results: Three Gene Ontology (GO) terms were found to be enriched in vitrified spermatozoa compared with fresh spermatozoa: mitochondrial tRNA aminoacylation, ATP-dependent microtubule motor activity, and male meiotic nuclear division. In alternative splicing analysis, a number of unknown AS events were found, including functional gene exon skipping (SE), alternative 5′ splice sites (A5SS), alternative 3′ splice sites (A3SS), mutually exclusive exon (MXE), and retained intron (RI). Conclusions: Cryopreservation of spermatozoa from some patients can agitate epigenetic instability, including increased alternative splicing events and changes in crucial mitochondrial functional activities. For fertilization of oocytes, for such patients, it is recommended to use fresh spermatozoa whenever possible; cryopreservation of sperm is recommended to be used only in uncontested situations.