Valutazione dei parametri nemaspermici di spermatozoi umani vitrificati mediante liquido seminale artificiale, in assenza di crioprotettore

Uno dei problemi legati alla crioconservazione del liquido seminale è la riduzione dei parametri vitali quali la vitalità e la motilità. La cri preservazione può inoltre indurre modificazioni della normale morfologia della componente nemaspermica. Attualmente la crioconservazione è applicabile attraverso due metodologie: congelamento lento/scongelamento rapido e vitrificazione/riscaldamento. Il primo metodo tende a prevenire la formazione di ghiaccio intracellulare tramite un lento abbassamento della temperatura: in tal modo la cristallizzazione che avviene all’esterno della cellula dovrebbe non disturbare l’ambiente intracellulare; il secondo invece mira ad evitare la formazione di ghiaccio intracellulare attraverso un istantaneo abbassamento della temperatura facendo in modo che il tempo richiesto per la transizione termica non sia tale da permettere il passaggio di stato. Per crioconservare con successo limitando il danno intracellulare si utilizzano delle sostanze dette crioprotettori, che sono però particolarmente tossici per le cellule. Una questione controversa nel campo della crioconservazione/crioprotezione dei gameti riguarda la presenza o assenza del liquido seminale durante la crioconservazione degli spermatozoi. Il potenziale protettivo del liquido seminale è stato confermato dalla presenza in esso di ioni come zinco e di antiossidanti; esso può però contenere alti livelli, potenzialmente tossici, di specie reattive dell’ossigeno. Lo scopo di questa tesi è stato quello di testare gli effetti di un medium di coltura artificiale simile nella composizione al liquido seminale ma privo degli effetti collaterali negativi e valutare il suo effetto sugli spermatozoi umani andando ad analizzare una serie di parametri prima e dopo la vitrificazione. In particolare mediante analisi microscopica sono stati valutati motilità, vitalità, morfologia e ultrastruttura. Sono stati selezionati 30 campioni di liquido seminale e ogni campione è stato sottoposto a swim-up e successivamente trattato in modo da ottenere 5 gruppi di spermatozoi a fresco e vitrificati in diversi media: HTF a fresco (Human Tubal Fluid), ASF a fresco (Artificial Seminal Fluid), Vit HTF (vitrification in Human Tubal Fluid), Vit SF (vitrification in Seminal Fluid), Vit ASF (vitrification in Artifical Seminal Fluid). Dopo vitrificazione e scongelamento tutti i campioni sono stati sottoposti ad analisi seminale mediante l’utilizzo della Microscopia Ottica, e successivamente sono stati trattati per l’analisi ultrastrutturale mediante l’uso della Microscopia Elettronica a Trasmissione. Nei gruppi a fresco HTF e ASF, tutti i parametri nemaspermici sono risultati simili e paragonabili. Invece, come era prevedibile, i parametri relativi alla morfologia normale, motilità e vitalità sono apparsi significativamente ridotte nei gruppi vitrificati rispetto a quelli a fresco (p<0.001). Andando a paragonare i parametri in condizioni di vitrificazione diverse invece, la motilità progressiva, la vitalità e la normale morfologia sono significativamente più alte in Vit ASF rispetto a Vit HTF, mentre tutti i parametri risultano in generale migliori in Vit ASF rispetto a Vit SF ma solo la vitalità sembra differire in maniera significativa. I tassi di recupero di vitalità e della normale morfologia erano significativamente maggiori nel gruppo Vit ASF rispetto a Vit SF e Vit HTF (p<0.05). I tassi di recupero della motilità progressiva e della normale morfologia erano significativamente maggiori nel gruppo Vit ASF rispetto a Vit HTF (p=0.03). Analizzando le immagini scattate al microscopio elettronico a trasmissione degli spermatozoi a fresco e vitrificati, i campioni a fresco hanno mostrato membrana plasmatica intatta e normali acrosomi. Nonostante si sia notata una migliore preservazione nel gruppo Vit ASF sono stati osservati diversi tipi di danno in tutti i gruppi vitrificati, nei quali l’acrosoma è risultata la parte anatomica più sensibile al crio-danno. La membrana plasmatica ha mostrato molti difetti dopo lo scongelamento come rigonfiamento, rotture, raggrinzimenti. Nei nuclei dei vitrificati è stata osservata una minore elettrodensità rispetto ai controlli a fresco e sono state osservate inclusioni granulari, voluminosi vacuoli apparentemente vuoti e non delimitati da membrana posizionati solitamente al centro del nucleo. I mitocondri sono apparsi tondi con creste poco definite e visibili. In conclusione abbiamo potuto dimostrare che il liquido seminale (SF) può fungere da crioprotettore in soggetti normospermici. Abbiamo dimostrato inoltre che l’ASF può effettivamente meglio preservare tutti i parametri nemaspermici degli spermatozoi in confronto al SF, ma soprattutto in confronto al HTF, incrementato con albumina sierica e saccarosio

Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p &lt; 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p &lt; 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF. © 2016 American Society of Andrology and European Academy of Andrology

Enhanced lipid extraction from unbroken microalgal cells using enzymes

The marine microalga Nannochloropsis sp. was chosen as a model organism to investigate the feasibility of using cell wall-degrading enzymes to enhance the recovery of intracellular lipids. An enzyme cocktail containing galactomannanase, 1,4-β-cellobiosidase and β-glucosidase as main components was prepared from commercial enzyme preparations. The effects of pretreatment time (P), enzyme dosage (D), pH and temperature (T) on the amount of extracted lipids were investigated using response surface methodology. Under the best conditions (P = 90 min, D = 1.3 mg g–1, pH = 5, T = 36°C) over 70% of the lipids present in the microalga were recovered. SEM and TEM characterization of enzyme-treated microalgae showed extensive cell damage with significant disruption of the cell wall and release of algal material. Overall, the results of this study strongly support the use of commercial enzyme preparations to improve lipid recovery from microalgae and provide useful information on the influence of process conditions on the treatment efficiency

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

Ultrastructure of cytoplasmic fragments in human cleavage stage embryos

Purpose: The goal of this study was to evaluate the ultrastructure of cytoplasmic fragments along with the effect of cytoplasmic fragment and perivitelline space coarse granulation removal (cosmetic microsurgery) from embryos before embryo transfer on ART outcomes. Methods: One hundred and fifty intracytoplasmic sperm injection cycles with male factor infertility were included in this prospective study. Patients were divided into three groups of case (n = 50), sham (n = 50), and control (n = 50). Embryos with 10–50 % fragmentation were included in this study. Cosmetic microsurgery and zona assisted hatching were only performed in case and sham groups respectively. Extracted fragments were evaluated ultrastructurally by transmission electron microscopy (TEM). Rates of clinical pregnancy, live birth, miscarriage, multiple pregnancies, and congenital anomaly in the three groups were also compared. Results: Micrographs from TEM showed that mitochondria were the most abundant structures found in the fragments along with mitochondria-vesicle complexes, Golgi apparatus, primary lysosomes, and vacuoles. There were no significant differences in demographic characteristics, laboratory and clinical data, or embryo morphological features between the groups. The rate of clinical pregnancy in control, sham, and case groups had no significant differences (24, 18, and 18 %, respectively). The rates of live birth, miscarriage, multiple pregnancy, and congenital anomaly were also similar between the different groups. Conclusions: Our data demonstrated that cosmetic microsurgery on preimplantation embryos had no beneficial effect on ART outcomes in unselected groups of patients. As mitochondria are the most abundant organelles found in cytoplasmic fragments, fragment removal should be performed with more caution in embryos with moderate fragmentation

Surgical and Bioengineering Integration in the Anatomy Course of Medicine and Surgery High Technology: Knowledge and Perception of Anatomy

The Locomotor System Anatomy (LSA) course, placed in the first semester of the first year of the new Master’s degree in Medicine and Surgery High Technology (MSHT) at the Sapienza University of Rome, was integrated with surgical and bioengineering content. This study investigated the educational value and the students’ perceptions of the effectiveness of these two types of integration, comparing surgical integration (SI) with engineering integration (EI). Anatomy knowledge and students’ opinions attending the LSA course in MSHT degree (n = 30) were compared with those of students (n = 32) attending another medical and surgery course not comprising EI. Data show that students in the MSHT course like in-depth SI much more than in-depth EI. However, those who like in-depth SI also like in-depth EI. Significant differences were in anatomy knowledge between the two groups in the three sections of the test. There was no significant correlation between the three test scores and the levels of liking, while there was a significant correlation between students liking SI and those liking EI. A statistically significant correlation was also found in students who correctly responded to questions on the head and trunk, with students responding correctly to questions on the upper limbs. This study will be important in optimizing the deepening of SI and EI in the LSA course. Keywords: human anatomy; anatomical sciences education; gross anatomy teaching; locomotor system; neurosurgery; orthopedics; surgical integration; bioengineering integration; technical physician; technical medicin

SEM study of incus surface erosion due to cholesteatoma action

Cholesteatoma is a noncancerous cystic lesion derived from an abnormal growth of keratinizing squamous epithelium in the temporal bone (1). It causes significant problems due to its erosive and expansile properties, resulting in the destruction of the ossicles. Over 5 million people worldwide are affected of cholesteatomas and gradually loss hearing. (2). In order to provide a prognostic tool useful during surgical procedures, we are performing a SEM morphological analysis of incus surface erosion due to cholesteatoma action; then we will investigate (on the same samples) the relationship among data from SEM analysis and genetical, proteomical, biochemical and histological data. Up to now we have observed 10 incus from patients with cholesteatoma. Samples were fixed immediately upon recovery in 2.5% glutaraldehyde in PBS at 4°C for 48 h, than they were prepared for scanning electron microscopy. Samples were gently sonicated before sputter coating, to remove excess of keratinizing squamous epithelium, that would have prevented erosion observation. The total surface area of the observed side was measured (3,54±0,21 mm2). The mean distance from the surgical removal point to the bone far end was measured in order to define a ROI (2,37±0,31 mm2). Five consecutive fields at 100X magnification aligned in 3 raws, the first one proximal and the last one distal to surgical removal point were analized. A total of 60 field for each raw were observed. Degree of erosion was classified as: No erosion=0, light =1, mild=2 high=3. Presence of biofilm was also recorded. Our early data suggest that although a gradient proximal to distal exists, looking to the distribution of eroded areas, grade 3 erosion is not limited only to the area proximal to cholesteatoma (first raw) but is also present in raw 2 and sometimes scattered since raw 3. Grade 3 erosion was observed around nutrient foramina of the bone (65%). Biofilm of bacteria was observed in 50% of analyzed fields, this is con- sistent with results reported in literature. Our data suggest that relapse of cholestea- toma is due to erosive activity of cells far from surgical removal point

The effect of postmastectomy radiation therapy on breast implants. Material analysis on silicone and polyurethane prosthesis

The pathogenic mechanism underlying capsular contracture is still unknown. It is certainly a multifactorial process, resulting from human body reaction, biofilm activation, bacteremic seeding, or silicone exposure. The scope of the present article is to investigate the effect of hypofractionated radiotherapy protocol (2.66 Gy × 16 sessions) both on silicone and polyurethane breast implants.Silicone implants and polyurethane underwent irradiation according to a hypofractionated radiotherapy protocol for the treatment of breast cancer. After irradiation implant shells underwent mechanical, chemical, and microstructural evaluation by means of tensile testing, infrared spectra in attenuated total reflectance mode, nuclear magnetic resonance, and field emission scanning electron microscopy.At superficial analysis, irradiated silicone samples show several visible secondary and tertiary blebs. Polyurethane implants showed an open cell structure, which closely resembles a sponge. Morphological observation of struts from treated polyurethane sample shows a more compact structure, with significantly shorter and thicker struts compared with untreated sample. The infrared spectra in attenuated total reflectance mode spectra of irradiated and control samples were compared either for silicon and polyurethane samples. In the case of silicone-based membranes, treated and control specimens showed similar bands, with little differences in the treated one. Nuclear magnetic resonance spectra on the fraction soluble in CDCl3 support these observations. Tensile tests on silicone samples showed a softer behavior of the treated ones. Tensile tests on Polyurethane samples showed no significant differences.Polyurethane implants seem to be more resistant to radiotherapy damage, whereas silicone prosthesis showed more structural, mechanical, and chemical modifications