La neoangiogenesi nel reimpianto autologo di tessuto ovarico in pazienti a rischio di fallimento ovarico precoce: ottimizzazione dei metodi di crioconservazione di corticle ovarica

Introduzione L’efficacia dei chemio/radioterapici ha aumentato notevolmente l’aspettativa di vita delle pazienti oncologiche, tuttavia, questi trattamenti possono compromettere la funzionalità ovarica. La crioconservazione di tessuto ovarico, con il successivo reimpianto, ha lo scopo di preservare la fertilità delle pazienti a rischio di fallimento ovarico precoce. Scopo dello studio Definire la migliore procedura di crioconservazione e reimpianto in grado di ottenere la neovascolarizzazione del tessuto reimpiantato nel minor tempo possibile al fine di diminuire la perdita follicolare causata dall’ischemia durante la procedura. Materiali e metodi Per ciascuna paziente (3) le biopsie ovariche, sono state prelevate laparoscopicamente e crioconservate secondo il protocollo di congelamento lento/scongelamento rapido. Campioni di corticale ovarica sono stati processati per l’analisi istologica, ultrastrutturale, immuistochimica e confocale per valutare la preservazione morfologiaca del tessuto. Le fettine di corticale ovarica sono state scongelate e reimpiantate ortotopicamente (2), nelle ovaia e in due tasche peritoneali, o eterotopicamente (1), in due tasche create nel sottocute sovrapubico. Risultati Le analisi di microscopia hanno mostrato il mantenimento di una discreta morfologia dello stroma, e dei vasi criopreservati e un lieve ma non significativo danneggiamento dei follicoli scongelati. Tutte le pazienti hanno mostrato la ripresa della funzionalità endocrina rispettivamente dopo 2/4 mesi dal reimpianto. Il color-doppler, inoltre ha rivelato un significativo aumento della vascolarizzazione ovarica rispetto alla quasi totale assenza di vascolarizzazione prima del reimpianto, quando le pazienti mostravano una conclamata menopausa. Conclusioni Lo studio ha confermato la ripresa della vascolarizzazione dell’ovaio in seguito a reimpianto avascolare di fettine di corticale, senza l’impiego di fattori esogeni o meccanici aggiuntivi, in tempi concordanti con i dati della letteratura. I risultati sono incoraggianti e l’avanzare degli studi e della ricerca potranno contribuire allo sviluppo di nuove metodologie di reimpianto che possano avere un successo clinico ed una sicurezza superiori a quelle finora ottenute.

Effect of antioxidant supplementation on pig and horse gamete storage

According to recent studies, antioxidant supplementation on gamete processing and/or storage solutions improvesgamete quality parameters, after cooling or storage at sub zero temperature. The aim of the present study was to investigate the effects of antioxidant supplementation on pig and horse gamete storage. The first study aimed to determine the effects of resveratrol (RESV) on the apoptotic status of porcine oocytes vitrified by Cryotop method, evaluating phosphatidylserine (PS) exteriorization and caspases activation. RESV(2µM) was added during: IVM (A); 2 h post-warming incubation (B); vitrification/warming and 2 h post-warming incubation (C); all previous phases (D). The obtained data demonstrate that RESV supplementation in the various steps of IVM and vitrification/warming procedure can modulate the apoptotic process, improving the resistance of porcine oocytes to cryopreservation-induced damage. In the second work different concentrations of RESV (10, 20, 40, and 80µM) were added during liquid storage of stallion sperm for 24 hours at either 10°C or 4°C, under anaerobic conditions. Our findings demonstrate that RESV supplementation does not enhance sperm quality of stallion semen after 24 hours of storage. Moreover, the highest RESV concentrations tested (40 and 80µM) could damage sperm functional status, probably acting as pro-oxidant. Finally, in the third work other two antioxidants, ascorbic acid (AA) (100 µM) and glutathione (GSH) (5mM) were added on boar freezing and/or thawing solutions. In our study different sperm parameters were evaluated before freezing and at 30 and 240 minutes after thawing. Our results showed that GSH and AA significantly improved boar sperm cryotolerance, especially when supplemented together to both freezing and thawing media. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels.

Effect of season and cryopreservation on oxidative status of stallion spermatozoa

The aim of this study was to investigate 1) the effect of different ROS and lipid peroxidation on sperm quality, and 2) differences in ROS between non-breeding and breeding seasons. Eighteen ejaculates from six stallions were collected in January and July (N = 36), processed for freezing. After 90’ of cooling, some straws were not frozen (unfrozen), some were frozen (frozen). Rapid sperm (RAP, CASA), membrane-acrosome integrity (MAI), high mitochondrial membrane potential (Mpos), intracellular Ca2+ (Fneg), lipid peroxidation (BODIPY), ROS (DCFH, MitoSOX) and chromatin fragmentation (DFI%) were evaluated by flow cytometry during incubation at +37°C at T0 (after 90 min at +4°C and after thawing), 3, 6, 12 and 24h. In winter, ROS and BODIPY were higher and faster (P < 0.0001) in frozen than unfrozen; DFI% was similar at 0h (P > 0.05) but higher in frozen after 3h of incubation (P < 0.0001). RAP, PMAI, Mpos and Fneg were lower in frozen compared to unfrozen (P < 0.0001). Summer and winter data were compared. Overall, ROS concentrations and BODIPY were higher and faster (P < 0.001) in winter, DFI% was lower in winter (P < 0.001), but similar between the two groups within seasons after thawing. Differences were found at 3h and 12h for DFI%, and for DCFH and MitoSOX at 0h and 12h of incubation in winter and summer respectively. A moderate positive correlations was found between DFI% and MitoSOX, DCFH, BODIPY, whereas a negative correlation, stronger in winter, between RAP, PMAI, Mpos, Fneg and BODIPY, DCFH, MitoSOX. DFI was not different in unfrozen and frozen, despite a significant higher ROS level in winter, and incubation allowed to asses differences in DFI, suggesting that incubation should be included when evaluating stallion frozen semen. Higher level of ROS and BODIPY in winter was less detrimental than freezing-thawing.