Evaluation of male fertility potential by Toluidine Blue test for sperm chromatin structure assessment

Tsarev, Igor; Bungum, Mona; Giwercman, Aleksander; Erenpreisa, J.; Ebessen, T.; Ernst, Emil Hagen; Ērenpreiss, Juris

doi:10.1093/humrep/dep068

Cited by 52 publications

(34 citation statements)

References 21 publications

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“…27 In TB staining, we also found a significant difference between control and case groups (P < 0.05). The percentage of men with SCI who had samples with more than 45% TB-reacted spermatozoa, 28 was notably higher than controls (P < 0.05) (Fig. 1).…”

Section: Resultsmentioning

confidence: 88%

Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury

Talebi

Khalili

Vahidi

et al. 2013

The Journal of Spinal Cord Medicine

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Objectives: To evaluate the effect of cord injury on (1) sperm parameters and (2) DNA chromatin status. Design: Case-control study. Setting: Data were collected from men referred to Research and Clinical Center for Infertility, Yazd, Iran. Participants: Thirty infertile men with the presence of any level of spinal cord injury (SCI) were compared with 30 healthy donors with definite fertility and normal sperm parameters. Interventions: Not applicable. Outcome measures: Sperm chromatin integrity was assessed using aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) assays. The rate of apoptotic spermatozoa was evaluated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. Results: Sperm concentration, motility, and morphology in men with SCI were significantly decreased compared with control group (P < 0.05). In addition, with regard to cytochemical staining and TUNEL test, the rate of reacted spermatozoa was increased significantly in SCI group when compared with the controls (P < 0.05). The majority of AB, TB, AO, and CMA3-reacted spermatozoa were higher than the "cut-off" value in men with SCI, as were the number of apoptotic spermatozoa stained with TUNEL. Conclusion: Results showed that SCI disturbs sperm parameters, nuclear maturity, and DNA integrity of spermatozoa. Therefore, the production of spermatozoa with less condensed chromatin and more apoptotic rate increases after cord injury and this may be one possible cause of infertility following SCI.

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Section: Resultsmentioning

confidence: 88%

Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury

Talebi

Khalili

Vahidi

et al. 2013

The Journal of Spinal Cord Medicine

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“…Esto permite, utilizar al DTT o al ME como controles positivos de la tinción con AT. Esta tinción se ha utilizado en varias especies para detectar defectos en la condensación de la cromatina (humano: Barrera et al, 1993;Erenpreiss et al, 2001;Erenpreisa et al, 2003;Tsarev et al, 2009;toro: Beletti y Mello, 1996;Beletti et al, 2005;conejo: Beletti y Mello, 2004;padrillo equino: Naves et al, 2004;Sardoy et al, 2008;Carretero et al, 2012a) y actualmente se ha empleado para evaluar la cromatina en caninos, felinos y porcinos (Monachesi y Carretero, 2014;Allera et al, 2016;Arraztoa et al, 2016).…”

Section: Revisión Del Temaunclassified

“…En nuestro laboratorio, se probó simplificar la técnica descripta por otros autores (Erenpreiss et al, 2001(Erenpreiss et al, , 2004Erenpreisa et al, 2003;Beletti y Mello, 2004;Beletti et al, 2005;Tsarev et al, 2009), quienes utilizaron una hidrólisis ácida previa a la tinción con la finalidad de aumentar la sensibilidad de la técnica. Estos autores reportaron que los espermatozoides que tienen una cromatina altamente compacta (normales) son apenas afectados por la hidrólisis ácida y en consecuencia se tiñen levemente celestes, mientras que, en los espermatozoides con una cromatina alterada, las protaminas pueden ser parcialmente extraídas por la hidrólisis permitiendo que las moléculas de AT se unan a los grupos fosfato, aumentando la sensibilidad de la técnica.…”

Section: Revisión Del Temaunclassified

“…Por otra parte, distintos autores han utilizado diferentes soluciones para fijar los extendidos: metanol (Barrera et al, 1993), etanol-ácido acético (3:1) más etanol 70% (Beletti y Mello, 2004;Beletti et al, 2005), etanolacetona (1:1) (Gagnon 1999;Erenpreiss et al, 2001;Erenpreisa et al, 2003;Tsarev et al, 2009) pero muchas de estas sustancias pueden ser altamente tóxicas si son inhaladas durante su manipulación (Macedo y Nava Muñoz, 2000). En CSA, equino, felino, canino y porcino, la fijación simple con etanol 96° durante 2 minutos fue efectiva (Sardoy et al, 2008;Carretero et al, 2009;2010a, 2010b2012a;Monachesi y Carretero, 2014;Allera et al, 2016;Arraztoa et al, 2016), dado que se encontraron patrones de coloración similares a los observados en otras especies (Barrera et al, 1993;Gagnon 1999;Erenpreiss et al, 2001;Erenpreisa et al, 2003;Beletti y Mello, 2004;Beletti et al, 2005;Tsarev et al, 2009). Todas las características mencionadas hacen que el AT sea una técnica sencilla de realizar, económica y rápida para determinar el grado de condensación de la cromatina de los espermatozoides.…”

Section: Revisión Del Temaunclassified

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Evaluación de la calidad del ADN espermático en camélidos sudamericanos y otras especies domésticas

Carretero¹,

Giuliano²,

Neild³

2017

SPERMOVA

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RESUMENLas anormalidades en el material genético pueden expresarse por defectos en la maduración o condensación nuclear, rupturas del ADN y anormalidades cromosómicas. Los parámetros de rutina utilizados para evaluar el semen (movilidad espermática, concentración, viabilidad, funcionalidad de membranas y morfología) no reflejan la calidad del ADN espermático. Esto cobra importancia para las técnicas de reproducción asistida, particularmente en la inyección intracitoplasmática de un espermatozoide en la que espermatozoides con genomas anormales pueden alcanzar el material genético del ovocito. Se postula que tres factores pueden estar involucrados en la etiología del daño del ADN: estrés oxidativo, aberraciones en el empaquetamiento de la cromatina y apoptosis. Varias técnicas han sido utilizadas para estudiar los defectos del ADN. A modo general, las técnicas de evaluación de ADN pueden ser divididas en 2 grupos: aquellas que evalúan el grado de condensación o compactación de la cromatina y aquellas que miden el grado de fragmentación del ADN. Muchas de estas técnicas son laboriosas, costosas y algunas dependen de enzimas cuya actividad y accesibilidad a las roturas de ADN pueden ser irregulares. En este artículo se comenta sobre el desarrollo y puesta a punto de varias de las técnicas de evaluación de la cromatina espermática en camélidos sudamericanos (CSA) y otras especies domésticas. Palabras clave: calidad de ADN, camélidos sudamericanos, espermatozoides ABSTRACTAbnormalities in sperm genetic material can be expressed as maturation or nuclear condensation defects, DNA damage or breakdown and chromosomal abnormalities. Routine semen evaluation (sperm motility, concentration, viability, membrane function and morphology) does not reflect sperm DNA quality. This becomes important when applying assisted reproductive techniques, especially intracytoplasmic sperm injection, in which sperm with abnormal genomes can reach the oocyte's genetic material. Three factors have been suggested to be involved in the etiology of DNA damage: oxidative stress, chromatin packaging anomalies and apoptosis. Various techniques have been used to study DNA defects. In general, DNA evaluation techniques can be divided into two groups: those that evaluate the degree of chromatin condensation or compactation and those that measure the degree of DNA fragmentation. Many of these techniques are laborious, costly and some depend on enzymes whose activity and accessibility to DNA fragments can be irregular. In this article, the development and setting up of various techniques to evaluate sperm chromatin in South American Camelids (SACs) and other domestic species is commented on.

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“…Generally, the quality of the sperm chromatin plays an important role in the success of ovum fertilization [de Lamirande et al 2012;Hofmann and Hilscher 1991]. Different simple methods based on different staining or fluorochromes have been proposed in recent years to assess sperm chromatin quality, using many staining methods for DNA integrity such as aniline blue (AB), toluidine blue (TB), and chromomycin A3 (CMA3) for protamination and chromatin integrity assay as well as fragmentation assays [de Lamirande et al 2012;Hekmatdoost et al 2009;Hofmann and Hilscher 1991;Kazerooni et al 2009;Kim et al 2013;Pizzol et al 2014;Sellami et al 2013;Tsarev et al 2009]. The usefulness of these methods in the evaluation of male infertility and as prognostic markers for successful assisted reproductive programs is still debatable, partly because standardization has not been reached for some of these methods [Pizzol et al 2014].…”

Section: Introductionmentioning

confidence: 99%

Sperm chromatin maturity and integrity correlated to zygote development in ICSI program

Asmarinah

Syauqy

Umar

et al. 2016

Systems Biology in Reproductive Medicine

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This study aimed to evaluate sperm chromatin maturity and integrity of that injected into goodquality oocytes in an in vitro fertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020); while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate; as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of <87% correlated significantly with the cleavage rate of the zygote (p=0.022; r=0.371); whereas poor sperm chromatin integrity at the level of <80% correlated with embryo formation (p=0.048; r=0,485). In conclusion, this study showed that poor maturity and integrity of sperm chromatin (AB<87% and TB<80%, respectively), could affect zygote development following ICSI.

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Evaluation of male fertility potential by Toluidine Blue test for sperm chromatin structure assessment

Abstract: The TB test can be suggested for clinical use as a complementary test for standard semen analysis to diagnose male infertility.

Cited by 52 publications

References 21 publications

Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury

Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury

Evaluación de la calidad del ADN espermático en camélidos sudamericanos y otras especies domésticas

Sperm chromatin maturity and integrity correlated to zygote development in ICSI program

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