INIEZIONE INTRACITOPLASMATICA DI SPERMATOZOI TESTICOLARI CRIOCONSERVATI

2002/2003Lo sviluppo delle tecniche di riproduzione assistita ha rivoluzionato in pochi anni il trattamento della sterilità di coppia. All'inizio degli anni '90, l'introduzione della iniezione intracitoplasmatica dello spermatozoo (ICSI) ha segnato un progresso storico rendendo possibile la gravidanza a coppie con partner maschile con grave oligospermia, che fino ad allora potevano soltanto ricorrere all'adozione o alla fecondazione con seme di donatore. Nel giro di brevissimo tempo, l'impiego di tecniche di prelievo di spermatozoi direttamente dall'apparato genitale maschile, associate alla ICSI, ha permesso il trattamento anche di coppie con partner maschile privo di spermatozoi nell'eiaculato. Molti pazienti azoospermici possono essere sottoposti con successo a prelievo testicolare di spermatozoi e successiva ICSI. Recentemente c'è stato un'incremento dell'interesse per la crioconservazione degli spermatozoi recuperati da tese in quanto questo permette di avere la conferma della presenza degli spermatozoi, in numero sufficiente prima di partire con il ciclo ICSI, inoltre è possibile eseguire più cicli di ICSI con il materiale recuperato e crioconservato da un prelievo evitando di sottoporre il paziente a ripetute biopsie testicolari. Come prerequisito all'utilizzo di spermatozoi testicolari crioconservati è la dimostrazione della comparazione dei risultati con quelli ottenuti utilizzando spermatozoi testicolari freschi. Lo scopo di questo studio è stato quello di analizzare e comparare i risultati, quali grado di fertilizzazione, clivaggio e percentuali di gravidanze, ottenuti mediante m1ez10ne intracitoplasmatica di spermatozoi recuperati da prelievo testicolare, freschi o crioconservati e quelli ottenuti mediante iniezione di spermatozoi eiaculati, freschi o crioconservati. Sono stati pubblicati molti lavori sulla fertilizzazione e successive gravidanze ottenute iniettando spermatozoi testicolari crioconservati. Gianaroli et al. (27) riporta i dati riguardanti la fertilizzazione, lo sviluppo embrionale e il grado di gravidanza ottenuti utilizzando spermatozoi testicolari crioconservati, rispettivamente 64%, 84% e 33%. I risultati del nostro studio riflettono questa stessa abilità nella fertilizzazione (57%), nello sviluppo embrionale (73%) e nelle gravidanze (28%) degli spermatozoi crioconservati dopo tese e si può inoltre notare come non ci siano differenze significative rispetto agli spermatozoi testicolari freschi (fertilizzazione 26%,sviluppo embrionale 100%). Quindi abbiamo visto che il grado di fertilizzazione che si ottiene dalla ICSI effettuata con spermatozoi testicolari crioconservati o freschi è inaspettatamente buono, rispettivamente 57% e 26% ma rimane sempre significativamente più basso rispetto a quello che si ottiene utilizzando spermatozoi eiaculati (70%). Questa differenza potrebbe essere dovuta a un incompleto processo di maturazione degli spermatozoi testicolari anche se però non ci sono differenze significative per quanto riguarda lo sviluppo embrionale e il tasso di gravidanze, rispettivamente 76% e 25% con gli spermatozoi testicolari e 61 % e 21 % con gli spermatozoi eiaculati. I dati che abbiamo ottenuto ci suggeriscono che gli spermatozoi recuperati da biopsia testicolare e poi utilizzati per inseminare gli ovociti mediante la ICSI sono spermatozoi che hanno le stesse caratteristiche, genetiche e morfologiche, degli spermatozoi ottenuti da liquido seminale, anche dopo crioconservazione. In effetti nel caso delle azoospermie ostruttive la spermatogenesi a livello testicolare è normale e gli spermatozoi ottenuti sono normali, nelle azoospermie non ostruttive si ha invece un grave difetto della spermatogenesi ma ci possono essere delle zone in cui questa è normale, in questi casi gli spermatozoi trovati in queste zone sono normali. L'eventuale presenza di un danno genetico solitamente influisce sulla capacità di impianto dell'embrione in quanto le prime divisioni embrionali, fino allo stadio di 8 cellule, sono regolate dalla componente genetica materna. Abbiamo poi avuto dei casi di pazienti oligo-asteno-teratospermici in cui il numero di spermatozoi eiaculati era veramente scarso, inoltre c'era il rischio di un peggioramento della situazione con la conseguente assenza di spermatozoi nel liquido seminale al momento del ciclo ICSI. Per questi pazienti si è proceduto alla crioconservazione del liquido seminale prodotto, dopo accertamento della presenza di spermatozoi e successivamente si è proceduti alla ICSI. I casi trattati in questo modo sono stati pochi ma confrontando i risultati con quelli ottenuti iniettando spermatozoi eiaculati o spermatozoi testicolari abbiamo notato che non ci sono differenze significative. Il grado di fertilizzazione è buono sia utilizzando spermatozoi eiaculati e crioconservati che spermatozoi testicolari ma comunque è sempre più basso rispetto a quello che si ottiene con gli spermatozoi eiaculati freschi. Per quanto riguarda invece lo sviluppo embrionale e il tasso di gravidanza i risultati sono buoni in tutti i casi. Abbiamo visto quindi che è possibile ottenere un normale grado di fertilizzazione, di sviluppo embrionale e tasso di gravidanza utilizzando anche spermatozoi testicolari trattati in modo appropriato, siano essi freschi o crioconservati, in associazione con la ICSI. Il fatto che il grado di fertilizzazione è comunque più basso rispetto a quello che si ottiene utilizzando spermatozoi eiaculati potrebbe essere una conseguenza degli spermatozoi utilizzati. Inoltre abbiamo dimostrato l'efficacia della crioconservazione degli spermatozoi testicolari al momento della prima biopsia diagnostica, procedura utilissima al fine di evitare biopsie ripetute al paziente e stimolazioni inutili alla paziente. Per molte coppie avere dei figli propri è estremamente importante e l'introduzione di queste nuove tecniche ha dato questa possibilità anche a pazienti azoospermici.XVI Ciclo1969Versione digitalizzata della tesi di dottorato cartacea

The application of scanning near field optical imaging to the study of human sperm morphology

BackgroundThe morphology of spermatozoa is a fundamental aspect to consider in fertilization, sperm pathology, assisted reproduction and contraception. Head, neck, midpiece, principal and terminal part of flagellum are the main sperm components to investigate for identifying morphological features and related anomalies. Recently, scanning near-field optical microscopy (SNOM), which belongs to the wide family of nanoscopic techniques, has opened up new routes for the investigation of biological systems. SNOM is the only technique able to provide simultaneously highly resolved topography and optical images with a resolution beyond the diffraction limit, typical of conventional optical microscopy. This offers the advantage to obtain complementary information about cell surface and cytoplasmatic structures.ResultsIn this work human spermatozoa both healthy and with morphological anomalies are analyzed by SNOM, to demonstrate the potentiality of such approach in the visualization of sperm morphological details. The combination of SNOM topography with optical (reflection and transmission) images enables to examine typical topographic features of spermatozoa together with underlying cytoplasmic structures. Indeed the head shape and inner components as acrosome and nucleus, and the organization of mitochondria in the midpiece region are observed. Analogously for principal tract of the tail, the ridges and the columns are detected in the SNOM topography, while their internal arrangement can be observed in the corresponding SNOM optical transmission images, without requiring specific staining procedures or invasive protocols.ConclusionsSuch findings demonstrate that SNOM represents a versatile and powerful tool to describe topographical and inner structural details of spermatozoa simultaneously. This analysis could be helpful for better characterizing several morphological anomalies, often related to sperm infertility, which cannot be examined by conventional techniques all together

Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation

Abstract BACKGROUND: Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation. METHODS: Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p<0.05, Anova). RESULTS: No differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC>SU and 1 was SU>DC. Proteins present in 3 of the differential spots could be identified. One DC>SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC>SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU>DC spot contained pyruvate kinase M1/M2. CONCLUSIONS: The differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential

Effect of seminal leukocytes on in vitro fertilization and intracytoplasmic sperm injection outcomes.

OBJECTIVE: To investigate the influence of seminal leukocytes on conventional IVF and intracytoplasmic sperm injection (ICSI) outcomes, using a flow cytometry method. DESIGN: Prospective study. SETTING: Tertiary infertility center and research institute. PATIENT(S): One hundred sixty-four couples undergoing conventional IVF or ICSI. INTERVENTION(S): Seminal leukocytes were counted by flow cytometry. MAIN OUTCOME MEASURE(S): Correlation between seminal leukocytes concentration and reproductive outcomes in IVF and ICSI cycles. RESULT(S): The median number of oocytes retrieved, the fertilization and cleavage rate, the median number and grade of embryos transferred, the median number of good-quality embryos transferred, and the median percentage of good-quality embryos from total embryos transferred, in leukocytospermic and non-leukocytospermic patients were not statistically different after either IVF or ICSI. Similarly, there were no significant differences between the two groups for implantation rate and clinical pregnancy rate. Multivariate logistic regression analysis showed that the reproductive outcomes were not influenced by adjustment for female age, infertility diagnosis, number of previous attempts, treatment protocol (GnRH agonist or antagonist), assisted reproduction procedure (IVF or ICSI), and leukocytospermia. By profiling the proper Poisson regression models, no leukocytospermia cut-off value was able to identify the subjects at risk for oocyte fertilization or embryo cleavage failure. CONCLUSION(S): Using a flow cytometry method, we demonstrated that leukocytospermia does not significantly influence IVF or ICSI outcomes. The same results were obtained by using lower or higher cut-off values for leukocytospermia (from 0.2 to 2 7 10(6)/mL)

Medical Treatments for Endometriosis-Associated Pelvic Pain

The main sequelae of endometriosis are represented by infertility and chronic pelvic pain. Chronic pelvic pain causes disability and distress with a very high economic impact. In the last decades, an impressive amount of pharmacological agents have been tested for the treatment of endometriosis-associated pelvic pain. However, only a few of these have been introduced into clinical practice. Following the results of the controlled studies available, to date, the first-line treatment for endometriosis associated pain is still represented by oral contraceptives used continuously. Progestins represent an acceptable alternative. In women with rectovaginal lesions or colorectal endometriosis, norethisterone acetate at low dosage should be preferred. GnRH analogues may be used as second-line treatment, but significant side effects should be taken into account. Nonsteroidal anti-inflammatory drugs are widely used, but there is inconclusive evidence for their efficacy in relieving endometriosis-associated pelvic pain. Other agents such as GnRH antagonist, aromatase inhibitors, immunomodulators, selective progesterone receptor modulators, and histone deacetylase inhibitors seem to be very promising, but there is not enough evidence to support their introduction into routine clinical practice. Some other agents, such as peroxisome proliferator activated receptors-γ ligands, antiangiogenic agents, and melatonin have been proven to be efficacious in animal studies, but they have not yet been tested in clinical studies

Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men

A comparative proteomic study of oligoasthenozoospermic and normozoospermic seminal plasmas was conducted to establish differences in protein expression. Oligoasthenozoospermia (when semen presents with a low concentration and reduced motility of spermatozoa) is common in male infertility. Two-dimensional protein maps from seminal plasma samples from 10 men with normozoospermia and 10 men with idiopathic oligoasthenozoospermia were obtained by isoelectric focusing followed by sodium dodecyl-sulphate polyacrylamide electrophoresis. Map images were analysed using dedicated software involving normalization, spot-to-spot volume comparison and statistical treatment of the results to establish the significance of differences between normal and oligoasthenozoospermic samples. Six out of 1028 spots showed over 1.5-fold relative intensity differences (P < 0.05, analysis of variance). Four proteins were identified by nano liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry of their tryptic peptides and database searches. Two proteins were more than three-fold under-expressed in oligoasthenozoospermia, namely epididymal secretory protein E1 and galectin-3-binding protein; the other (lipocalin-1 and a prolactin-inducible protein form) were over-expressed. The identity and differential expression of epididymal secretory protein E1 was verified by Western-blotting. The statistically significant differential expression of these four proteins in oligoasthenozoospermia compared with normozoospermia provides a molecular basis for further investigations into the pathogenic mechanisms underlying idiopathic oligoasthenozoospermia

Investigating the mechanical properties of zona pellucida of whole human oocytes by atomic force spectroscopy

The role of mechanics in numerous biological processes is nowadays recognized, while in others, such as the fertilization process, it is still neglected. In the case of oocytes the description of their mechanical properties could improve the comprehension of the oocyte-spermatozoon interaction and be helpful for application in in vitro fertilization (IVF) clinics. Herein the mechanical properties of whole human oocytes (HOs) immediately after retrieval are investigated by indentation measurements with atomic force spectroscopy under physiological conditions. Measurements are performed on immature (metaphase I - MI) and mature (metaphase II - MII) HOs. According to their morphological characteristics MII-HOs are classified as "suitable" and "rejected"; these latter would be usually rejected for intracytoplasmic sperm injection (ICSI). For all maturation stages we observe that the elastic response of the zona pellucida (ZP) outer layer was different and distinguishable from the rest of the ZP-HO. The elasticity of this ZP outer layer varies with maturation and quality: stiffness decreases from immature MI to good quality MII, up to poor-quality rejected MII. An indirect analysis with IVF outcome indicates that the ZP outer layer of analysed HOs donated by women who achieved pregnancy is stiffer than that of HOs from women with negative outcome. Our findings suggest that mechanical properties can represent important oocyte quality indicators that may be exploited for the design of innovative ICSI dedicated cell sorters

Synchrotron radiation soft X-ray microscopy and low energy X-ray fluorescence to reveal elemental changes in spermatozoa treated with photobiomodulation therapy

Male infertility is a worldwide clinical issue that increases the number of couples resorting to assisted reproductive technologies (ARTs) to achieve pregnancy. Photobiomodulation therapy (PBMT) is a promising technique that can biostimulate cells and tissues and it is currently successfully employed to enhance the sperm motility in vitro. Nevertheless, its use has been so far restricted to the research field. In the present work, we exploited two PBMT protocols at an 800 nm wavelength on sperm derived from infertile individuals, detecting an increase in sperm motility 1 hour after irradiation. Moreover, in order to add new information about the molecular effect of PBMT, the content of some light elements was evaluated using high resolution X-ray fluorescence imaging. Interestingly, an increase in Na content was detected in the irradiated samples, possibly suggesting a role of this element in sperm motility; indeed, a low Na content was previously correlated with a poor sperm quality, low semen volume, and modest fertilization rate. Amplifying the knowledge of PBMT in the ART field will expedite the translational potentiality of the PBMT use in clinical settings

Human \u3b2-defensin 1 in follicular fluid and semen: impact on fertility

\u3b2-defensins are antimicrobial peptides expressed at mucosal level of male and female genito-urinary tract, where they exert protective functions against infections, possibly preserving human health and fertility. In our study, we investigated the possible involvement of \u3b2-defensins in female and male infertility in Italian infertile couples (i) evaluating the presence of human \u3b2-defensin 1 (hBD-1) in follicular fluid (FF) and its correlation with in vitro fertilization (IVF) outcomes; (ii) investigating the relationship between hBD-1 levels in semen and IVF outcomes (comprising correlation with sperm parameters); and (iii) exploring the effect of hBD-1 peptide on spermatozoa motility in vitro