Peritoneal fluid modifies the response of human spermatozoa to follicular fluid

The aim of this study was to elucidate the mechanism involved in the acrosome reaction (AR) induced by follicular fluid (FF) in spermatozoa previously exposed to peritoneal fluid (PF). The influence of progesterone was also investigated. Semen samples were from 18 normozoospermic donors. PF samples were from 13 women with unexplained infertility and from a woman treated with synthetic progestagen. FF samples were collected from six women undergoing IVF/embryo transfer and pooled. Motile spermatozoa were capacitated overnight and a kinetic and inhibition study on the FF-induced AR was performed. Spermatozoa pretreated with PF were challenged with either FF or progesterone. The ability of progesterone- and progestagen-supplemented PF to induce AR was analysed. Enzyme-digested PF was also tested. Pre-incubation with PF for 60 min completely prevented the FF-induced AR; spermatozoa treated with PF were unable to respond to FF or progesterone and this effect was not reversible. Progesterone- and progestagen-supplemented PF stimulated the AR relative to controls. Enzyme-digested PF did not have an inhibitory capacity. These data strongly suggest that there are one or more inhibitory proteins in PF that interact with spermatozoa so as to prevent access of progesterone to its receptor and thus inhibit the occurrence of the AR. The oviduct, or Fallopian tube, provides a place for spermatozoa and egg transport and storage, fertilization and early embryo development. If ovulation has not occurred, spermatozoa may reside in the oviduct for several hours or even a few days, awaiting oocyte arrival. It is assumed that fluids present in the female genital tract may have a role in synchronizing the timing required to guarantee the success of fertilization. We previously observed that the peritoneal fluid that bathes the peritoneal cavity is a suitable medium for sperm survival and we also reported that this fluid could stabilize spermatozoa. In this study we show further evidence that the exposure to peritoneal fluid modifies the response of spermatozoa to oocyte signals.Fil: Caille, Adriana M.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Berta, Cesar L.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Munuce, María J.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; 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: 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

Efecto de la osmolalidad sobre la habilidad fecundante del espermatozoide humano: un modelo de estudio in vitro

Las características funcionales del espermatozoide se modifican a medida que sobrelleva los procesos que lo preparan para adquirir su capacidad fecundante. La composición diferente de los fluidos de los tractos reproductivos masculino y femenino, le imprimirá una característica particular a cada espacio biológico. Como parte del proceso de adquisición de la capacidad fecundante, denominado “capacitación espermática”, se remueven moléculas absorbidas o integradas a la membrana plasmática, se dan cambios en la composición lipídica y aumento de la fluidez y permeabilidad de la membrana. Durante el ascenso hacia el ovocito, el espermatozoide interactúa con la superficie de las células epiteliales, los distintos productos de secreción del tracto y las cubiertas del ovocito. Estas interacciones participan en la modulación de la función espermática, en la adquisición de capacidad fecundante y en la coordinación del transporte y unión de los gametos femenino y masculino. Por otra parte, los espermatozoides pueden ser capacitados y consiguen expresar su habilidad fecundante in vitro. Una característica de los fluidos es su osmolalidad y está dada por la concentración de los componentes orgánicos e inorgánicos presentes en el mismo. Es por esto que la osmolalidad es una variable físico-química, de gran importancia en el metabolismo ntracelular, y depende tanto del tipo como de la concentración de los componentes que conforman a un fluido. Los fluidos fisiológicos, a los cuales están expuestos los espermatozoides en su tránsito hacia el sitio de la ecundación, presentan una osmolalidad variable en un rango importante (280 a 480 mOsmoles/kg). El volumen espermático estaría influenciado por el equilibrio del líquido a través de sus membranas y una falla en dicho equilibrio podría derivar en alteraciones de los mecanismos de capacitación y ecundación. Por otro lado, durante el manejo de los espermatozoides in vitro, ya sea durante las técnicas de reproducción asistida, como en los métodos utilizados para evaluar su capacidad fecundante, se utilizan medios y suplementos que pueden alterar la osmolalidad del medio en contacto con los espermatozoides. Es por todo esto que nuestro objetivo fué estudiar, la asociación entre la osmolalidad del medio al que se exponen los espermatozoides humanos y la adquisición de su capacidad fecundante, mediante un modelo de interacción in vitro

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

Effect of exposure to ulipristal acetate on sperm function

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Objective A pill containing ulipristal acetate (UPA) is used for emergency contraception (EC). Considering that, following its intake, spermatozoa may be exposed to UPA in the female genital tract we intended to evaluate sperm functions after incubation with this compound. Methods Motile spermatozoa were selected by swim-up and were incubated under capacitating conditions with UPA (at concentrations of 1, 10, 100, 1,000, and 10,000 ng/ml) or control medium. The main outcome measures were sperm vitality, sperm protein tyrosine phosphorylation (TyrP), spontaneous acrosomal reaction (AR), and human follicular fluid (hFF)-induced AR. Results Sperm vitality and TyrP pattern were similar between spermatozoa exposed to UPA or control. In addition, spontaneous AR ranged from 14.0. +/- 1.5% to 18.0. +/- 1.9% after exposure to UPA or control medium without significant differences, and UPA did not prevent hFF-induced AR. Conclusions Incubation of sperm with UPA at concentrations around the expected plasma levels after ingestion of this EC pill (similar to 100-200 ng/ml) did not modify the signal transduction of TyrP involved in sperm capacitation. Moreover, UPA showed no agonist effect on progesterone receptors because it did not induce AR. Considering that progesterone in hFF is essential for AR induction, and UPA did not prevent the hFF-induced AR, an antagonist action of UPA on the AR is unlikely.176428437Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Mechanisms involved in the contraceptive effects of ulipristal acetate

The use of emergency contraception (EC) methods is increasing worldwide as it constitutes an effective way to prevent unplanned pregnancy after unprotected sexual intercourse. During the last decade, ulipristal acetate (UPA), a selective progesterone receptor modulator, has emerged as the most effective EC pill, and it is now recommended as first-line hormonal treatment for EC in several countries. Its principal mechanism of action involves inhibition or delay of follicular rupture, but only when administered during the follicular phase before the luteinizing hormone (LH) peak. However, considering the high efficacy of UPA, it is possible that it also exerts contraceptive effects besides ovulation. In the present review, we summarize and discuss the existing evidence obtained on the effect of UPA on sperm function and post-ovulatory events as potential additional mechanisms to prevent pregnancy. The bulk of evidence collected so far indicates that UPA would not affect gamete function; however, it could impair embryo?uterine interaction. Thus, besides the described effects on ovarian function, UPA contraceptive effectiveness might also be attributed to post-ovulatory effects, depending on the moment of the female cycle in which the drug is administered.Fil: Munuce, María José. Universidad Nacional de Rosario; ArgentinaFil: Gómez Elías, Matías Daniel. 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: Caille, Adriana María. Universidad Nacional de Rosario; ArgentinaFil: Bahamondes, Luis. Universidade Estadual de Campinas; BrasilFil: 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; ArgentinaFil: Cohen, Débora J.. 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

Human tubal secretion can modify the affinity of human spermatozoa for the zona pellucida

Objective: To study the effect of the human tubal tissue conditioned medium (CM) on sperm parameters related to sperm-zona pellucida interaction. Design: Controlled experimental laboratory study. Setting: Research laboratory. Subject(s): Semen samples from donors with normozoospermia. Human tubal tissue obtained from women undergoing hysterectomies. Human follicular fluids (hFF) and oocytes collected from patients undergoing IVF-ET. Intervention(s): Incubation of spermatozoa with CM proteins obtained from human tubal tissue culture; sperm binding to the zona pellucida assessment. Main Outcome Measure(s): Explants' viability was assessed by tissue DNA analysis. Sperm ability to interact with zona was tested with use of the whole oocyte test. Expression of d-mannose binding sites was assessed with use of a fluorescent probe on mannose coupled to bovine serum albumin. Human FF-induced acrosome reaction was assessed by the Pisum sativum technique. Result(s): Although treatment with 0.8 μg/μL of CM allowed sperm binding to the zona and the expression of d-mannose binding sites comparable with sperm in control medium, with 3.2 μg/mL of CM resulted in a significant decrease of both parameters. No effect of CM on spontaneous or hFF-induced acrosome reaction or in sperm viability was observed. Conclusion(s): The results indicate that the incubation of spermatozoa in the presence of CM reduces sperm affinity for the zona pellucida. This effect can be partly explained by the decreased expression of d-mannose binding sites on the sperm surface.Fil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Serravalle, Analía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Caille, Adriana M.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Zumoffen, Carlos María. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Botti, Gustavo. Universidad Nacional de Rosario; ArgentinaFil: Cabada, Marcelo Oscar. 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: Ghersevich, Sergio Albino. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Study of the effect of ulipristal acetate on human sperm ability to interact with tubal tissue and cumulus-oocyte-complexes

Ulispristal acetate (UPA) is a selective progesterone receptor modulator widely used for emergency contraception (EC). The described main mechanism of action is by inhibiting or delaying ovulation; however, the postovulatory effects of the drug are still on debate. Therefore, the aim of this study was to determine whether UPA could interfere with human sperm fertilizing ability.Fil: Zumoffen, Carlos María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Gómez Elías, Matías Daniel. 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: Caille, Adriana M.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Bahamondes, Luis. Universidade Estadual de Campinas; BrasilFil: 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; 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: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Glucose-regulated protein 78 (Grp78/BiP) is secreted by human oviduct epithelial cells and the recombinant protein modulates sperm-zona pellucida binding

To determine the secretion of Grp78 by human oviduct epithelial cells, its association to spermatozoa, and its involvement in gamete interaction. Prospective study. Basic research laboratory. Semen samples obtained from normozoospermic volunteers. Tubal tissue provided by patients undergoing hysterectomies. Oocytes collected from women undergoing IVF-ET. Analysis of Grp78 expression and secretion by oviductal tissue. Gamete incubation with recombinant Grp78 (rec-Grp78).Fil: 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: González Echeverría, María F.. Centro Médico Fertilab; ArgentinaFil: Munuce, María José. Universidad Nacional de Rosario; ArgentinaFil: Ghersevich, Sergio Albino. Universidad Nacional de Rosario; ArgentinaFil: Caille, Adriana M.. Universidad Nacional de Rosario; ArgentinaFil: Hellman, Ulf. Uppsala Universitet; SueciaFil: Corrigall, Valerie M.. King´s College London; Reino UnidoFil: Vazquez, Monica Hebe. 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