Vitrification of human immature oocytes before and after in vitro maturation: a review

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations

Pre-implantation mouse embryos cultured In vitro under different oxygen concentrations show altered ultrastructures

Abstract Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O2) concentrations to culture human embryos. Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2

Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes

Purpose: Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration. Methods: Samples were studied by light and transmission electron microscopy. Results: We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed. Conclusions: This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism

Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis

BACKGROUND: Human mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification. METHODS: We used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation. RESULTS: Control, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90-100 mum in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3-4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8-9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied. CONCLUSIONS: Even though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane "recycling" that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardenin

Mancozeb impairs the ultrastructure of mouse granulosa cells in a dose-dependent manner

Mancozeb, an ethylene bis-dithiocarbamate, is widely used as a fungicide and exerts reproductive toxicity in vivo and in vitro in mouse oocytes by altering spindle morphology and impairing the ability to fertilize. Mancozeb also induces a premalignant status in mouse granulosa cells (GCs) cultured in vitro, as indicated by decreased p53 expression and tenuous oxidative stress. However, the presence and extent of ultrastructural alterations induced by mancozeb on GCs in vitro have not yet been reported. Using an in vitro model of reproductive toxicity, comprising parietal GCs from mouse antral follicles cultured with increasing concentrations of mancozeb (0.001-1 µg/ml), we sought to ascertain the in vitro ultrastructural cell toxicity by means of transmission (TEM) and scanning (SEM) electron microscopy. The results showed a dose-dependent toxicity of mancozeb on mouse GCs. Ultrastructural data showed intercellular contact alterations, nuclear membrane irregularities, and chromatin marginalization at lower concentrations, and showed chromatin condensation, membrane blebbing, and cytoplasmic vacuolization at higher concentrations. Morphometric analysis evidenced a reduction of mitochondrial length in GCs exposed to mancozeb 0.01-1 µg/ml and a dose-dependent increase of vacuole dimension. In conclusion, mancozeb induced dose-dependent toxicity against GCs in vitro, including ultrastructural signs of cell degeneration compatible with apoptosis, likely due to the toxic breakdown product ethylenethiourea. These alterations may represent a major cause of reduced/delayed/missed oocyte maturation in cases of infertility associated with exposure to pesticides

The Three-Dimensional Microstructure of the Liver A Review by Scanning Electron Microscopy

The improvement in scanning electron microscopy (SEM) techniques has permitted us to describe the microstructure of the liver. By SEM, the liver peritoneal surface is composed of flat mesothelial cells possessing microvilli and cilia. Hepatic sinusoids connect the portal vessels with the terminal branches of the hepatic vein (central veins). Endothelial cells of the portal space arteries are elongated and arranged longitudinally, while those of the central and portal veins are polygonal and flattened, possessing microvilli. The sinusoidal endothelial cells show both small fenestrations (sieve plates), up to 200 nm in diameter, and large ones, up to 1 m. Within the sinusoids are seen bridging structures, covered by fenestrated endothelium, seeming to have a fibrillar core. Kupffer cells resemble macrophages, showing microvilli, blebs, lamellipodia and filopodia. Within the Space of Disse are seen the fat-storing cells, having laminar dendritic projections. The polyhedral liver cell faces the Space of Disse (vascular pole) or faces an adjacent hepatocyte (biliary pole). Vascular facets are evenly covered by microvilli. Biliary facets show a central longitudinal depression, bordered by microvilli (bile hemicanaliculi). Canaliculoductular junction and bile duct epithelia show blebs, microvilli and cilia. Up to now, fetal liver and liver pathology have been scarcely investigated by SEM: in the future, they can be successfully approached by three-dimensional studies

Autologous Materials in Regenerative Dentistry: Harvested Bone, Platelet Concentrates and Dentin Derivates

The jawbone is a peculiar type of bone tissue, unique for its histological, anatomical and physiological characteristics. Therefore, a defect in the maxilla or in the mandible, because of pathological sequelae is difficult to prevent and to restore. Several biomaterials have been and are currently being developed to respond to the demands of regenerative medicine. A specific group of biomaterials used in regenerative dentistry is represented by the autologous materials. Platelet concentrates harvested bone and dentin derivates are indeed used in an attempt to minimise the alveolar resorption or in vertical ridge augmentation procedures or in sinus lift interventions. The aim of this review is to examine the properties of the above-listed materials, to compare them and to indicate eventual clinical applications

Plasmacytoma in the Maxillary Jaw: A Diagnostic and Therapeutic Challenge

Plasmacytoma is a neoplastic disorder originating from plasma cells, with bone and soft tissue being common sites of manifestation. This report presents the clinical and radiological findings of a 65-year-old female patient who presented with an exophytic lesion in the upper right lateral incisor region. The lesion appeared as a unilocular radiotransparent area in imaging tests. Following an excisional biopsy, histological and immunohistochemical evaluations confirmed the presence of mature plasmacellular elements and small infiltrates of B and T lymphocytes. The patient did not exhibit systemic manifestations of multiple myeloma. Surgical intervention, in the form of enucleation of the lesion combined with root canal treatment and apicoectomy, was performed. This case underscores the rare occurrence of plasmacytoma in the jaw region and highlights the importance of surgical management in cases where structural damage or functional impairment is present. Further research on novel treatment approaches is also mentioned, including targeted therapies, immunomodulatory agents, and monoclonal antibodies. The patient is currently under the care of a hematologist for further investigation and the choice of the most appropriate therapy

The role of innovation technology in teaching and learning strategies in anatomy curricula in dental hygiene school

Purpose. This research aims to assess the diversification of pedagogical and learning methodologies, leveraging advanced technological tools within a dental hygiene educational framework. Methods. Students enrolled in the dental hygiene program were considered as population sample, divided in two groups: The test group (N=16) subjected to the investigation of the virtual dissection table (VDT) and the control group (n=17) who attended lectures using traditional teaching method. The control group’s performance was assessed through a 40-item multiple-choice questionnaire and an open-ended question; the test group was evaluated via a final test consisting of a presentation on an anatomical topic and a 40-item multiple-choice questionnaire. The final grade derived from the mean score of the final tests, and it was assigned on a scale of 30/30. Additionally, the test group’s perceptions toward the use of the VDT were gauged through an evaluative questionnaire comprising 7 questions. Results. The study found a statistically significant difference in failure rates between the control group and the test group. However, the average exam scores did not show a statistically significant difference between the two groups, despite the test group showing some improvement. The evaluation survey results indicated high levels of satisfaction with the use of the VDT, with the quality of the virtual images, anatomical resources, and the user-friendly interface. Additionally, students strongly supported integrating traditional lectures with VDT laboratory sessions, with no negative feedback reported. Conclusions. The VDT represents an innovative anatomy teaching tool, warmly welcomed by healthcare profession students, whose performances were positively affected

The Use of Sonic Frequencies as a Cleaning Agent of Specimens to be Observed by Scanning Electron Microscopy

The presence of mucus and/or cellular debris can obscure the fine morphology of the gastrointestinal or respiratory luminal surface, when observed by scanning electron microscopy. With the intent of obtaining a good cleaning of the mucosal surface without altering the ultrafine morphology of epithelial cells, a new model of sonicator/ultrasonicator is presented. The instrument is supplied with a control system for wave frequency, amplitude and form, and permits a precise regulation of the wave energy. With this instrument it is possible to produce a cleaning effect by using any kind of frequency (either sonic or ultrasonic) and/or amplitude and/or waveform and/or liquid. We report the application of sonic frequencies through water as a fluid for immersion to obtain a gentle and slow removing of mucus and in order to explore the possibility to clean hydrated tissues. With the employment of sonic frequencies (from 5 to 15 kHz modulated by 200 Hz) and water as the immersion fluid, we were able to generate a gentle wave energy which effected an optimal removal of the mucus, with the consequent exposure of a well preserved epithelial surface of rat trachea and small intestine