Use of Assisted Reproduction Techniques in Male Pathologies: Molecule Based Sperm Selection Methods

Infertility is a widespread problem that is found in over 15% of couples. In 50 % of the cases, the male factor is the key. In order to improve pregnancy rates in assisted reproductive techniques (ART), new methods to select sperm subpopulations are required. These methods are under constant evaluation, and their possible genetic and epigenetic effects on the offspring must be considered. New concepts such as DNA fragmentation and sperm apoptosis are being introduced in andrology and are considered to develop new techniques. Recently, molecule-based methods have been developed for the purpose of overcoming deficiencies in classical selection methods that involve sperm morphology and movement. One of these methods is physiologic intracytoplasmic sperm injection (PICSI), which selects spermatozoa through their property to bind to hyaluronic acid, a component of the cell-oocyte complexes that acts as a natural selector of spermatozoa. But the top rated method is based on the property of the protein annex in A5 (ANXA V) to bind to phosphatidylserine (PS), a marker of apoptosis. Magnetic-Activated Cell Sorting (MACS) conjugated with ANXA V (MACS-ANXA V) is used to remove apoptotic sperm with exposed PS from a semen sample. The use of MACS-ANX V seems to produce good quality sperm when it is evaluated by in vitro assays. But when MACS-ANXA V is used previous to ART, controversies persist regarding the benefits of sperm selection in reproductive outcomes. In this review, we consider the use of MACS-ANXA V, particularly in cases of male pathologies.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Caille, Adriana María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Zumoffen, Carlos María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentin

Bicarbonate is required for migration of sperm epididymal protein DE/CRISP-1 to the equatorial segment and expression of rat sperm fusion ability

Numerous studies have demonstrated that sperm capacitation is a bicarbonate-dependent process. In the rat, capacitation has not been studied as much as in other species, mainly because of the difficulties in carrying out functional assays with this animal model. In the present study, we have examined the influence of bicarbonate in the overall rat sperm capacitation process by analyzing involvement of the anion in 1) protein tyrosine phosphorylation, 2) migration of epididymal protein DE (also known as CRISP-1) from the dorsal region to the equatorial segment of the sperm head that occurs during capacitation, and 3) ability of sperm to fuse with the egg. Incubation of sperm under capacitating conditions produced a time-dependent increase in protein tyrosine phosphorylation. This phosphorylation did not occur in the absence of HCO3- and rapidly increased by either exposure of sperm to HCO3- or replacement of the anion by a cAMP analog (dibutyryl-cAMP) and a phosphodiesterase inhibitor (pentoxifylline). The absence of HCO3- also produced a significant decrease in the percentage of cells showing migration of DE to the equatorial segment. This parameter was completely restored by addition of the anion, but dibutyryl-cAMP and pentoxifylline were not sufficient to overcome the decrease in DE migration. Sperm capacitated in the absence of HCO3- were unable to penetrate zona-free eggs independent of the presence of the anion during gamete coincubation. Exposure of these sperm to bicarbonate, or replacement of the anion by dibutyryl-cAMP and pentoxifylline, only partially restored the sperm fusion ability. Altogether, these results indicate that, in addition to its influence on protein tyrosine phosphorylation, bicarbonate is required to support other rat sperm capacitation- associated events, such as migration of DE to the equatorial segment, and expression of the ability of sperm to fuse with the egg.Fil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Munuce, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Marin Briggiler, Clara Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Busso, Dolores. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Use of Assisted Reproduction Techniques in Male Pathologies: Molecule Based Sperm Selection Methods

Infertility is a widespread problem that is found in over 15% of couples. In 50 % of the cases, the male factor is the key. In order to improve pregnancy rates in assisted reproductive techniques (ART), new methods to select sperm subpopulations are required. These methods are under constant evaluation, and their possible genetic and epigenetic effects on the offspring must be considered. New concepts such as DNA fragmentation and sperm apoptosis are being introduced in andrology and are considered to develop new techniques. Recently, molecule-based methods have been developed for the purpose of overcoming deficiencies in classical selection methods that involve sperm morphology and movement. One of these methods is physiologic intracytoplasmic sperm injection (PICSI), which selects spermatozoa through their property to bind to hyaluronic acid, a component of the cell-oocyte complexes that acts as a natural selector of spermatozoa. But the top rated method is based on the property of the protein annex in A5 (ANXA V) to bind to phosphatidylserine (PS), a marker of apoptosis. Magnetic-Activated Cell Sorting (MACS) conjugated with ANXA V (MACS-ANXA V) is used to remove apoptotic sperm with exposed PS from a semen sample. The use of MACS-ANX V seems to produce good quality sperm when it is evaluated by in vitro assays. But when MACS-ANXA V is used previous to ART, controversies persist regarding the benefits of sperm selection in reproductive outcomes. In this review, we consider the use of MACS-ANXA V, particularly in cases of male pathologies.Fil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Zumoffen, Carlos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Caille, Adriana María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina.Fil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Use of Annexin V based Sperm Selection in Assisted Reproduction

Innovative methods to select sperm subpopulations with the best fertilizing ability are needed in assisted reproductive techniques (ART) in order to improve fertilization and pregnancy rates, while also considering possible epigenetic effects on the offspring. Molecular based selection methods are searched for, under the premise that they could be an improvement over classical selection by morphology and movement. One of these methods sustains the elimination of sperm that can bind to annexin A5 (ANX V), coupled to paramagnetic beads, through the phosphatidyl-serine exposed on their membranes upon apoptosis. Although reports accumulate about the use of this method, controversy persists as to the benefits of ANX V based sperm selection in ART. In this review we consider the arguments in favour and against this method and conclude that to the moment the evidence does not support MACS regular use in ART.Fil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Caille, Adriana María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Zumoffen, Carlos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina

Expression profile and distribution of Annexin A1, A2 and A5 in human semen

Annexins (ANXAs) have been identified in different seminal components mainly by proteomic methods. The presence and distribution of Annexin A1, A2 and A5 (ANXA1, ANXA2, ANXA5) in human semen was analysed and the corresponding mRNAs studied in spermatozoa. All three ANXAs were present in prostasomes and spermatozoa, but only ANXA1 in prostasomes-free seminal plasma. Immunofluorescence showed ANXA1 and ANXA5 in the sperm head, mid-piece and flagellum. The amount of mRNAs corresponding to ANXA1 and A2 decreased with increasing levels of the corresponding proteins indicating a probable regulation of their expression at the translational level during spermatogenesis. Additionally, DNA fragmentation was assessed by the sperm chromatin dispersion test. Lower amounts of ANXA1 and A2 with higher levels of the corresponding mRNAs were noted in poor quality semen samples. ANXA5 was detected in spermatozoa from all semen samples, but no particular trend was noted. The corresponding mRNA were detected both in excellent and poor quality semen samples. Results showed that ANXA1 and A2 expressions appear to be related with DNA fragmentation suggesting their possible use as new biomarkers for sperm DNA quality. ANXA5´s natural presence in spermatozoa suggest that revision of high-quality sperm selection by binding to this protein is needed.Fil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Marini, Patricia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentin

Effects of lactoferrin, a protein present in the female reproductive tract, on parameters of human sperm capacitation and gamete interaction

In a recent study, lactoferrin (LF) was detected in human oviductal secretion. The protein was able to bind to oocytes and sperm, and modulated gamete interaction. The aim of the present study was to investigate the effect of LF on parameters related to human sperm capacitation and sperm-zona pellucida interaction. Semen samples were obtained from healthy normozoospermic donors (n = 7). Human follicular fluids and oocytes were collected from patients undergoing in vitro fertilization. Motile sperm obtained by swim-up were incubated for 6 or 22 h under capacitating conditions with LF (0-100 μg/mL). After incubations, viability, motility, presence of α-d-mannose receptors (using a fluorescent probe on mannose coupled to bovine serum albumin), spontaneous and induced acrosome reaction (assessed with Pisum sativum agglutinin conjugated to fluorescein isothiocyanate), and tyrosine phosphorylation of sperm proteins were evaluated. Sperm-zona pellucida interaction in the presence of LF was investigated using the hemizone assay. The presence of LF did not affect sperm viability or motility, but caused a dose-dependent significant decrease in sperm α-d-mannose-binding sites, and the effect was already significant with the lowest concentration of the protein used after 22 h incubation. Dose-dependent significant increases in both induced acrosome reaction and tyrosine phosphorylation of sperm proteins were observed in the presence of LF. The present data indicate that LF modulates parameters of sperm function. The inhibition of gamete interaction by LF could be partially explained by the decrease in sperm d-mannose-binding sites. The presence of the LF promoted sperm capacitation in vitro.Fil: Zumoffen, Carlos María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Massa, Estefanía María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Caille, A. M.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Ghersevich, Sergio Albino. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

The In Vitro Effect Of Levonorgestrel On The Acrosome Reaction Of Human Spermatozoa From Fertile Men.

The objective of the study was to evaluate the effect of levonorgestrel (LNG) on the occurrence of acrosome reaction (AR) of capacitated spermatozoa from fertile men. A total of 20 semen samples from four fertile men were evaluated. The spermatozoa were separated by swim-up, and subsequently incubated for 20 h under capacitating conditions. Capacitated spermatozoa were exposed to three different concentrations of LNG (200, 400 and 800 ng/mL), follicular fluid (20% v/v), and ethanol or human tubal fluid medium (HTF) as a control. The AR rate and the ratio of live to dead spermatozoa were assessed after 15 and 30 min of incubation at 37 degrees C and 5% CO(2). The different treatments were compared with follicular fluid and HTF medium as positive and negative controls. The main results showed that the AR rate after 15 min of exposure was not affected by LNG and was significantly higher with follicular fluid than with all the other treatments. At 30 min of exposure, the three LNG concentrations induced a greater rate of AR than the HTF and a trend of higher AR rate with greater concentration was observed. Follicular fluid induced a significantly higher rate of AR than the other treatments. In conclusion, the addition of LNG in vitro to capacitated human spermatozoa is associated with a dose-dependent increased rate of AR, but such increase was not as great that induced by follicular fluid.6855-

The In Vitro Effect Of Emergency Contraception Doses Of Levonorgestrel On The Acrosome Reaction Of Human Spermatozoa.

The aim of this study was to evaluate the effect of three concentrations of levonorgestrel (LNG) comparable to the levels found in serum following ingestion of LNG as emergency contraception (EC) on the acrosome reaction (AR) of capacitated and noncapacitated spermatozoa of fertile men. A total of 24 semen samples from three fertile men were evaluated. The spermatozoa were selected by Percoll gradient. Twelve samples were subsequently incubated with human tubal fluid medium supplemented with bovine serum albumin (HTF/BSA) for 20 h under capacitating conditions. The capacitated spermatozoa and the spermatozoa from the remaining 12 samples were exposed to LNG at 1, 10 and 100 ng/mL, to follicular fluid (FF) (20 %v/v) and to HTF medium. The ratio of live to dead spermatozoa was assessed after 1, 2 and 3 h of incubation at 37 degrees C and 5% CO2. After 30 min of exposure to the different LNG concentrations, aliquots were divided into two parts. In the first part, spermatozoa were immediately stained with Hoescht 33258 and fluorescein isothiocyanate-pisum sativum agglutinin (FITC-PSA) in order to assess AR rate and to repeat evaluation of the live-to-dead ratio. After 3 h of incubation, the remaining part of the aliquots were submitted to the same procedures. Each concentration of LNG was then compared with FF and HTF medium as positive and negative controls, respectively. The results showed that in vitro exposure to the three different LNG concentrations did not induce AR. This study failed to show any in vitro effect on AR of LNG concentrations similar to those found in serum following intake of LNG as EC. If this effect exists or if there is any other that influences sperm fertilizing capacity, in vitro experiments are probably not an appropriate way of testing it.72225-