Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction.

BACKGROUND: Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development. In some mammals, like pigs, ejaculates are emitted in three separate fractions: pre-sperm, sperm-rich (SRF) and post sperm-rich (PSRF). These fractions are known to vary in volume, sperm concentration and quality, as well as in the origin and composition of seminal plasma (SP), with differences being also observed within the SRF one. Yet, whether disparities in the DNA integrity and chromatin condensation and protamination of their sperm exist has not been interrogated. RESULTS: This study determined chromatin protamination (Chromomycin A3 test, CMA3), condensation (Dibromobimane test, DBB), and DNA integrity (Comet assay) in the pig sperm contained in the first 10 mL of the SRF (SRF-P1), the remaining portion of the sperm-rich fraction (SRF-P2), and the post sperm-rich fraction (PSRF). While chromatin protamination was found to be similar between the different ejaculate fractions (P > 0.05), chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF (P = 0.018 and P = 0.004, respectively). Regarding DNA integrity, no differences between fractions were observed (P > 0.05). As the SRF-P1 has the highest sperm concentration and ejaculate fractions are known to differ in antioxidant composition, the oxidative stress index (OSi) in SP, calculated as total oxidant activity divided by total antioxidant capacity, was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF (0.42 ± 0.06 vs. 0.23 ± 0.09 and 0.08 ± 0.00, respectively; P < 0.01); this index, in addition, was observed to be correlated to the sperm concentration of each fraction (Rs = 0.973; P < 0.001). CONCLUSION: While sperm DNA integrity was not found to differ between ejaculate fractions, SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF. This could be related to the OSi of each fraction.

Therapeutic potential of testosterone on sperm parameters and chromatin status in fresh and thawed normo and asthenozoospermic samples / Potencial terapéutico de la testosterona sobre los parámetros espermáticos y el estado de la cromatina en muestras frescas y descongeladas de normo y astenozoospermia

Background: Hormonal changes alter the physiological level of ROS and cause oxidative stress in the cell. As estimated, hormonal deficiencies, environmental and ideological factors make up about 25% of male infertility. Pathogenic reactive oxygen species (ROS) is a chief cause of unexplained infertility. Limited studies exist on the effects of testosterone on human sperm culture. Therefore, in the current study, the effect of different doses of testosterone on sperm parameters and chromatin quality was investigated. Materials and methods: Semen samples from 15 normospermic and 15 asthenospermic patients were prepared by swim up method, and then were divided into four groups by exposing to different concentrations of testosterone (1, 10, and 100nM) for 45min. Samples without any intervention were considered as control group. All samples were washed twice. Sperm parameters and chromatin protamination were assessed in each group and the remains were frozen. After two weeks, all tests were repeated for sperm thawed. Also, the MSOM technique was used to determine the sperm morphology of class 1. Results: Although sperm parameters were not show any significant differences in normospermic and asthenospermic samples exposed to different concentrations of testosterone before and after freezing, chromatin protamination was significantly decreased in the normospermic samples exposed to 10nM of testosterone before freezing (p<0.006), as well as 1 and 10nM of testosterone after freezing compared to control samples (p=0.001 and p=0.0009, respectively). (AU)

Antecedentes: Los cambios hormonales alteran el nivel fisiológico de las especies reactivas de oxígeno (reactive oxygen species [ROS]) patógenas y provocan estrés oxidativo en la célula. Según estimaciones, las deficiencias hormonales, los factores ambientales y los ideológicos constituyen alrededor del 25% de la infertilidad masculina. Las ROS son una causa principal de infertilidad inexplicable. Existen estudios limitados sobre los efectos de la testosterona en el cultivo de esperma humano. Por lo tanto, en el estudio actual se ha investigado el efecto de diferentes dosis de testosterona sobre los parámetros del esperma y la calidad de la cromatina. Materiales y métodos: Se prepararon muestras de semen de 15 pacientes normospérmicos y 15 astenospérmicos mediante el método swim up, y luego se dividieron en cuatro grupos exponiéndolos a diferentes concentraciones de testosterona (1, 10 y 100nM) durante 45min. Las muestras sin ninguna intervención se consideraron como grupo control. Todas las muestras se lavaron dos veces. En cada grupo se evaluaron los parámetros espermáticos y la protaminación de la cromatina, y los restos se congelaron. Dos semanas después se repitieron todas las pruebas de esperma descongelado. Asimismo, se utilizó la técnica MSOM para determinar la morfología espermática de clase 1. Resultados: Aunque los parámetros espermáticos no mostraron diferencias significativas en las muestras normospérmicas y astenospérmicas expuestas a diferentes concentraciones de testosterona antes y después de la congelación, la protaminación de la cromatina disminuyó significativamente en las muestras normospérmicas expuestas a 10nM de testosterona antes de la congelación (p<0,006), así como a 1 y 10nM de testosterona después de la congelación, en comparación con las muestras de control (p=0,001 y p=0,0009, respectivamente). (AU)

Therapeutic potential of testosterone on sperm parameters and chromatin status in fresh and thawed normo and asthenozoospermic samples.

BACKGROUND: Hormonal changes alter the physiological level of ROS and cause oxidative stress in the cell. As estimated, hormonal deficiencies, environmental and ideological factors make up about 25% of male infertility. Pathogenic reactive oxygen species (ROS) is a chief cause of unexplained infertility. Limited studies exist on the effects of testosterone on human sperm culture. Therefore, in the current study, the effect of different doses of testosterone on sperm parameters and chromatin quality was investigated. MATERIALS AND METHODS: Semen samples from 15 normospermic and 15 asthenospermic patients were prepared by swim up method, and then were divided into four groups by exposing to different concentrations of testosterone (1, 10, and 100nM) for 45min. Samples without any intervention were considered as control group. All samples were washed twice. Sperm parameters and chromatin protamination were assessed in each group and the remains were frozen. After two weeks, all tests were repeated for sperm thawed. Also, the MSOM technique was used to determine the sperm morphology of class 1. RESULTS: Although sperm parameters were not show any significant differences in normospermic and asthenospermic samples exposed to different concentrations of testosterone before and after freezing, chromatin protamination was significantly decreased in the normospermic samples exposed to 10nM of testosterone before freezing (p<0.006), as well as 1 and 10nM of testosterone after freezing compared to control samples (p=0.001 and p=0.0009, respectively). Similarly, chromatin protamination in the asthenospermic samples was significantly decreased at concentration of 1nM of testosterone before and after freezing (p=0.0014 and p=0.0004, respectively), and at concentration of 10nM of testosterone before and after freezing (p=0.0009, p=0.0007) compared to control samples. CONCLUSION: Using a low dose of testosterone in the sperm culture medium, has positive effects on chromatin quality.

The role of spermatozoa-zona pellucida interaction in selecting fertilization-competent spermatozoa in humans.

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The time-course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis.

Integridad Del ADN Espermático Y La Toxicidad Reproductiva Del Nitrato De Plomo En Ratones Machos Adultos

Introducción: La toxicidad del plomo se ha relacionado a diferentes patologías en humanos y varias evidencias sugieren una fuerte relación con el daño observado sobre la función reproductiva en humanos y roedores. Método: Se proporcionó a ratones una dosis única de nitrato de plomo (NP) (50mg/kg/pc), los cuales fueron eutanizados siete días postinyección con el objetivo de evaluar los espermatozoides que han salido de los túbulos seminíferos y están en tránsito por el epidídimo; además, se evaluó la fragmentación del ADN espermático mediante el ensayo tunel. Resultados: La disminución del peso corporal en ratones, tratados con NP (p 0,05); de igual manera, los valores fisiológicos como conteo y movilidad espermática no disminuyeron con el tratamiento (p > 0.05). El tránsito y maduración de los espermatozoides en el epidídimo no sería afectado por el NP, y al no observar aumento en la fragmentación del ADN espermático en el grupo tratado (p > 0,05), la protaminación espermática estaría cumpliendo su rol protector sobre el material genético murino, por lo que no hubo daños genotóxicos por el NP. Conclusión: La administración intraperitoneal de 50mg/kg/pc de NP, por siete días, no causa toxicidad sistémica ni efecto en la espermatogénesis en ratón.

Introduction: Lead toxicity has been linked to different diseases in humans and several evidences suggest a strong relationship with the observed damage on reproductive function in humans and rodents. Methods: Mice were given a single dose of lead nitrate (NP) (50mg/kg/bw), which were euthanized seven days post-injection with the aim of evaluating sperm to come out from the seminiferous tubules and are in transit through the epididymis. Also, the Tunel test was done to evaluate the sperm DNA fragmentation. Results: The decrease in body weight in mice treated with ln (p 0.05), in the same way physiological values such as sperm concentration and motility didn´t decrease with the treatment (p > 0.05). Transit and sperm maturation in the epididymis would not be affected by the ln, and because we did not observe increased sperm DNA fragmentation in the treated group (p > 0.05), sperm protamination would be fulfilling its protective role on murine genetic material avoiding genotoxic damage by ln. Conclusion: The intraperitoneal administration of 50mg/kg/pc of ln for seven days does not cause systemic toxicity or effect on spermatogenesis in mice.

Condensation and protamination of sperm chromatin affect ICSI outcomes when gametes from healthy individuals are used.

STUDY QUESTION: Do defects in sperm chromatin protamination and condensation have an impact on ICSI outcomes? SUMMARY ANSWER: Sperm protamination is related to fertilization rates in healthy donors, and the in vitro capacity of sperm to condense their chromatin is linked to blastocyst rates, both associations being more apparent in women <33 years of age. WHAT IS KNOWN ALREADY: Previous data on how sperm chromatin damage affects ICSI outcomes are inconsistent. Revealing which sperm factors influence embryo development is necessary to understand the male contribution to ICSI success and to develop novel sperm selection techniques or male-based treatments. Sperm chromatin is mainly condensed in protamines, which are cross-linked through disulphide bridges. This study aimed to determine whether sperm protamination and the integrity of disulphide bonds (condensation) are related to embryo development after ICSI. STUDY DESIGN, SIZE, DURATION: The design was a retrospective study with a blind analysis of sperm chromatin. Gametes were divided into two groups: double donation (DD) cohort and single donation (SD) cohort. Samples from 45 semen donors used in 55 ICSI cycles with oocyte donors (age range 19-33 years), generating 491 embryos, were included in the DD cohort. The SD cohort consisted of samples from 34 semen donors used in 41 ICSI cycles with oocytes from healthy females (single-parent families or lesbian couples, age range 20-44 years), generating a total of 378 embryos. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Donor sperm samples from DD and SD cohorts were used for standard ICSI, and embryo development was observed by time-lapse imaging. The incidence of thiol reduction (dibromobimane, DBB) and the degree of chromatin protamination (chromomycin A3, CMA3, indicating non-protaminated regions) in sperm were determined by flow cytometry at 0 and 4 h post-thawing. MAIN RESULTS AND THE ROLE OF CHANCE: Percentages ± standard deviation of CMA3 were 21.08 ± 9.09 and 35.01 ± 14.68 at 0 and 4 h post-thawing, respectively, in the DD cohort and 22.57 ± 9.48 and 35.79 ± 12.58, at 0 and 4 h post-thawing, respectively, in the SD cohort. Percentages of DBB+ were 16.57 ± 11.10 and 10.51 ± 8.40 at 0 and 4 h post-thawing (P < 0.0001), respectively, in the DD cohort and 17.98 ± 10.19 and 12.72 ± 8.76 at 0 and 4 h post-thawing (P < 0.0001), respectively, in the SD cohort. Female age correlated with fertilization rates, and the relation between sperm chromatin and embryo development was determined through multiple linear regression. While CMA3 was associated with fertilization rates, with no influence of female age, in the DD cohort (ß1 = -1.036, P < 0.001 for CMA3; ß2 = 0.667, P = 0.304 for female age), this was not observed in the SD cohort, where female age had a significant effect, masking the effects of CMA3 (ß1 = -0.066, P = 0.804 for CMA3; ß 2 = -1.451, P = 0.003 for female age). The in vitro capacity of sperm to condense their chromatin after 4 h of incubation was associated with blastocyst rates, independent of female age (DD cohort: ß1 = -0.238, P = 0.008 for %DBB+ variation; ß2 = 0.404, P = 0.638 for female age; SD cohort: ß1 = -0.278, P = 0.010 for %DBB+ variation; ß2 = -0.292, P = 0.594 for female age). The in vitro capacity of sperm to condense their chromatin was also related to the time required for the embryo to reach blastocyst stage in the DD cohort (P = 0.007). Finally, multiple logistic regression showed that both chromatin protamination and condensation, together with the age of the oocyte donors and the embryo recipients, had an impact on pregnancy achievement (P < 0.01) and on live birth rates (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The main limitation was the restrictive selection of couples, which led to a relatively small sample size and could influence the observed outcomes. For this reason, and to reduce Type I error, the level of significance was set at P ≤ 0.01. On the other hand, the use of cryopreserved samples could also be a limitation. WIDER IMPLICATIONS OF THE FINDINGS: This research demonstrated that protamination and condensation of sperm chromatin are related to embryo development after ICSI, but female age could be a confounding factor when oocytes from older females are used. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the European Union's Horizon 2020 Research and Innovation scheme under the Marie Sklodowska-Curie grant agreement No 801342 (Tecniospring INDUSTRY; TECSPR-19-1-0003); La Marató de TV3 Foundation (214/857-202039); the Ministry of Science and Innovation, Spain (IJC2019-039615-I); the Catalan Agency for Management of University and Research Grants, Regional Government of Catalonia, Spain (2017-SGR-1229); and the Catalan Institution for Research and Advanced Studies, Spain (ICREA). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Protamines and DNA integrity as a biomarkers of sperm quality and assisted conception outcome.

Present research aim was to identify functional tests in semen associated with DNA damage and chromatin maturity (protamination) which predict the outcome in assisted reproduction. Couples were grouped according to male partner semen parameters, into normozoospermia (NZs), severe male factor (SMF) and mild male factor (MMF). DNA fragmentation index (DFI) in spermatozoa was analysed by sperms chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and acridine orange testing (AOT). Chromomycin A3 (CMA3) and toluidine blue (TB) staining to measure sperm chromatin maturity (CM). DFI and chromatin decondensation were significantly lower in N compared to male factor categories (MMF and SMF). Aneuploidy embryos were significantly higher in couples with male factor infertility (MMF and SMF). A positive correlation was observed between fertilization rate (FR) and live birth rate (LBR) with sperm concentration, motility, vitality, normal sperm morphology and negative correlation between sperm DFI and sperm CM. No correlation was observed between embryo aneuploidy and sperm DFI or CM. Lower percentage of spermatozoa chromatin integrity are associated with low fertilization and live birth rate. Male factor infertility, due to impaired semen parameters and chromatin defects could be regarded in future as an indication of IVF/ICSI, and predictor of assisted reproductive techniques outcome.

A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs.

In order to accurately analyze the possible side effects of sperm cryopreservation, an in-depth screening of post-thaw sperm status is necessary. Thus, this study aimed to identify thorough effects of sperm cryopreservation, by evaluating the integrity of all specific structures of the canine spermatozoa. Thirteen (n = 13) mature dogs of different breeds were selected. Six dogs (n = 6) were subjected to sperm cryopreservation, whereas seven dogs (n = 7) were used as semen donors to validate a simultaneous assessment of sperm plasmatic, acrosomal, and mitochondrial membranes (triple stain) by fluorescent probes. Fresh and post-thaw semen samples were evaluated through a computer-assisted analysis of sperm motility, sperm morpho-functional evaluation, triple stain and sperm DNA integrity. Post-thaw semen samples had lower total and progressive motility, as well as higher percentage of minor and major defects. Moreover, post-thaw samples had higher percentage of sperm with plasma membrane and mitochondrial damage but intact acrosome, and also sperm with simultaneous damaged plasma, acrosomal and mitochondrial membranes. Furthermore, post-thaw sperm had higher protamination deficiency and DNA fragmentation. In conclusion, cryopreservation has a broad impact in sperm morphology and function, altering motility patterns, plasma, acrosome and mitochondrial membranes integrity, as well as sperm DNA.

Protective effects of zinc on rat sperm chromatin integrity involvement: DNA methylation, DNA fragmentation, ubiquitination and protamination after bleomycin etoposide and cis-platin treatment.

Testicular cancer is one of the most common malignancy in young men, chemotherapy induced damage in cancerous cells as well as healthy tissue, and we decided to investigate recovery effect of zinc (Zn) on chemotherapy-induced complications in rat chromatin integrity and testicular histomorphometry. The male rats (n = 40) were treated with BEP at appropriate dose levels of BEP (0.75, 7.5, and 1.5 mg/kg) for 9 weeks, with or without Zn; testicular histology, sperm DNA methylation, ubiquitination, DNA fragmentation and protamination were further assessed through immunofluorescence. BEP treatment significantly increased ubiquitination, and DNA fragmentation, considerably reducing global DNA methylation and protamination (P < 0.001), resulting in degenerative changes in testicular structure. Zn restored normal DNA methylation, protamination and structure of male gonads, maintained spermatogonial stem cells, and significantly reduced the mean percentage of ubiquitination and sperm DNA fragmentation as compared with BEP group (P < 0.001). We found that supplementation of Zn following chemotherapy can improve chromatin integrity, testicular organization and spermatogenesis.

Detection of protamine 2 in bovine spermatozoa and testicles.

BACKGROUND: Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. OBJECTIVES: The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. MATERIALS AND METHODS: For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. RESULTS: We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. CONCLUSION: Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Detection of protamine 2 in bovine spermatozoa and testicles

Background Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. Objectives The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. Materials and methods For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. Results We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. Conclusion Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Sperm telomere length as a parameter of sperm quality in normozoospermic men.

STUDY QUESTION: Could sperm telomere length (STL) represent a novel parameter and biomarker of sperm quality? SUMMARY ANSWER: STL is associated with standard semen quality parameters and, more importantly, it is significantly associated with levels of DNA fragmentation and sperm protamination. WHAT IS KNOWN ALREADY: Telomeres are fundamental for genome integrity. Recent studies have demonstrated that STL increases with age and men with oligozoospermia have shorter sperm telomeres than normozoospermic men. STUDY DESIGN, SIZE, DURATION: Cohort study conducted from September 2014 to June 2015 on 100 subjects with normal standard semen parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: STL was measured indirectly by quantitative polymerase chain reaction using telomere/single-copy gene ratio, sperm DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay and protamination by aniline blue staining. Data were analyzed for determining the relationships between STL, standard semen parameters and DNA fragmentation and protamination. MAIN RESULTS AND THE ROLE OF CHANCE: Among standard semen parameters, STL was positively associated with progressive motility (P = 0.004) and vitality (P = 0.007). STL was significantly and negatively associated with sperm DNA fragmentation (P = 0.001) and significantly and positively associated with protamination (P = 0.002). The role of chance was limited and the findings have biological relevance and a pathophysiological explanation. LIMITATIONS, REASONS FOR CAUTION: For the present study, we deliberately selected only men with normozoospermia to better analyze whether STL might represent a biomarker of sperm quality beyond traditional sperm parameters. Additional studies in proven fertile men with normal sperm parameters are needed. WIDER IMPLICATIONS OF THE FINDINGS: The measurement of STL is a simple and rapid method that offers further information about the quality of sperm. The results of this study demonstrate that STL could be considered as an additional sperm parameter and opens new perspectives in the evaluation of the infertile male. Additional studies will clarify the significance of this parameter also as a prognostic biomarker in assisted reproduction. STUDY FUNDING/COMPETING INTERESTS: No external funding was either sought or obtained for this study. There are no conflicts of interest to be declared.

Sperm nuclear DNA fragmentation and chromatin structure in one-day-old ejaculated sperm.

OBJECTIVE: To evaluate sperm nuclear DNA fragmentation and chromatin structure after 18 hours'incubation at room temperature. METHODS: Twenty-eight male partners who participating IVF treatment were prospectively included in this study. Ejaculated sperm count and motility were assessed. The sperm was then immediately processed by the conventional swim-up method. After utilization of some of the sample for routine clinical use, the remainder of each of the samples was divided into two aliquots. One aliquot was immediately assessed for sperm nuclear DNA fragmentation (TUNEL assay) and chromatin structure (toluidine blue [TB] staining). The other aliquot was incubated at room temperature for 18 hours and then assessed by two methods. Only dark-TB sperms were considered as having abnormal chromatin structure. Data before and after extended incubation were compared using a paired Student's t-test. RESULTS: Before and after extended culture, nuclear DNA fragmentation assessed by TUNEL was 4.9±4.7% and 7.0±6.4%, respectively (p=0.008). The proportion of abnormal chromatin structure (dark-TB sperm) was 8.2±5.6% and 10.3±6.5% (p<0.001), before and after incubation, respectively. CONCLUSION: After 18 hours' incubation at room temperature, sperm nuclear DNA and chromatin structure were significantly affected. The IVF practitioner should bear this information in mind when performing delayed insemination, especially for in vitro maturation cycles.

Sperm nuclear DNA fragmentation and chromatin structure in one-day-old ejaculated sperm / 대한생식의학회지

OBJECTIVE: To evaluate sperm nuclear DNA fragmentation and chromatin structure after 18 hours'incubation at room temperature. METHODS: Twenty-eight male partners who participating IVF treatment were prospectively included in this study. Ejaculated sperm count and motility were assessed. The sperm was then immediately processed by the conventional swim-up method. After utilization of some of the sample for routine clinical use, the remainder of each of the samples was divided into two aliquots. One aliquot was immediately assessed for sperm nuclear DNA fragmentation (TUNEL assay) and chromatin structure (toluidine blue [TB] staining). The other aliquot was incubated at room temperature for 18 hours and then assessed by two methods. Only dark-TB sperms were considered as having abnormal chromatin structure. Data before and after extended incubation were compared using a paired Student's t-test. RESULTS: Before and after extended culture, nuclear DNA fragmentation assessed by TUNEL was 4.9+/-4.7% and 7.0+/-6.4%, respectively (p=0.008). The proportion of abnormal chromatin structure (dark-TB sperm) was 8.2+/-5.6% and 10.3+/-6.5% (p<0.001), before and after incubation, respectively. CONCLUSION: After 18 hours' incubation at room temperature, sperm nuclear DNA and chromatin structure were significantly affected. The IVF practitioner should bear this information in mind when performing delayed insemination, especially for in vitro maturation cycles.