Human kinematic and morphometric sperm subpopulation analysis using CASA technology: A new approach to spermatozoa classification / Análisis cinemático y morfométrico de subpoblaciones de espermatozoides humanos utilizando tecnología CASA: un nuevo enfoque para la clasificación de los espermatozoides

Introduction: Semen analysis is a clinical method aimed at determining the fertility of a male individual. The traditional subjective method lacks the reliability that can be achieved by computer-assisted sperm analysis (CASA) technology. Unfortunately, this technology has only been used when taking into consideration individually different sperm characteristics. The aim of this work is to present an integrative mathematical approach that considers different seminal variables to establish human sperm subpopulations. Methods: Samples were obtained from thirteen volunteers via masturbation and were analyzed by the routine subjective method and two objective systems, CASA Motility (CASA-Mot) and CASA Morphology (CASA-Morph). Results: Seminogram variables were reduced to three principal components (PC) showing two subpopulations. Kinematics and morphometric variables each rendered three PCs for four subpopulations. Conclusions: These results lay the foundations for future studies including different geographical, social, ethnic and age range conditions with the aim of achieving a definitive view of the human semen picture. (AU)

Introducción: El análisis de semen es el método clínico para determinar la fertilidad masculina. El método subjetivo tradicional carece de la fiabilidad, que se puede obtener con el uso de la tecnología del análisis de semen asistido por ordenador (CASA). Desafortunadamente, esta tecnología se ha venido utilizando únicamente teniendo en cuenta de forma independiente las diversas características de los espermatozoides. El objetivo del presente estudio es presentar una aproximación matemática que incluye diversas variables seminales para definir las posibles subpoblaciones espermáticas. Métodos: Las muestras se obtuvieron por masturbación de 13 voluntarios, que se analizaron de forma subjetiva, así como con 2 sistemas objetivos, para el análisis de la movilidad (CASA-Mot) y la morfología (CASA-Morph). Resultados: Tanto las variables cinemáticas como las morfométricas rindieron 3 componentes principales y 4 subpoblaciones. Conclusión: Estos resultados sientan las bases para estudios futuros que incluyan diferencias geográficas, sociales, étnicas o de rango de edad con el ánimo de obtener una definición concluyente sobre las características seminales de la especie humana. (AU)

The benefit of Silybum marianum in ethanol-induced reprotoxicity of male Wistar rat

Abstract This study investigates the toxic effects of ethanol (Eth) on the reproductive system of male rats and the possible protective role of Silybum marianum seeds-infused solution (SMI) over six consecutive weeks of administration. Animals were divided into the following groups: control, SMI positive control (200 mg/kg/day), Eth1 (1 g/kg/day), Eth2 (2 g/kg/day), Eth1+SMI, and Eth2+SMI. Plasma testosterone concentration, epididymal spermatozoa biology, and testicular and epididymal MDA, GSH and GPx levels were evaluated. The results indicated a significant decrease in testis and epididymis weight, testosterone level, sperm concentration, sperm vitality and sperm motility (total motility, progressive motility, curvilinear velocity, straight-line velocity, velocity average path, beat cross frequency, and lateral head displacement) in both Eth1 and Eth2 compared to the control groups and the combined-treatment groups (Eth1+SMI and Eth2+SMI). Furthermore, results showed a significant elevation in MDA concentration with a significant decrease of testicular and epididymal GSH concentration and GPx activity in theEth1 and Eth2 groups compared to the combined-treatment groups. The administration of SMI succeeded in improving the parameters cited above in the combined-treatment groups compared to the Eth1 and Eth2 groups, and bring them to the levels seen in the control groups. To conclude, SMI has clearly protected reproductive indices against ethanol-induced reprotoxicity in male rats

Is there any relationship between human sperm parameters and protamine deficiency in different groups of infertile men? / ¿Existe alguna relación entre los parámetros del esperma humano y la deficiencia de protamina en varios grupos de varones infértiles?

OBJECTIVE: Abnormality in Histone-Protamine replacements has been indicated to cause sperm DNA damage and infertility. The aim of the present study was to investigate the relationships between sperm parameters in oligospermia, asthenospermia, and teratospermia with protamine deficiency in infertile men. MATERIAL AND METHOD: In this case-control study, we had three experimental groups including oligospermia (n=100), asthenospermia (n=100), and teratospermia (n=100) as well as normospermia (n=100) as controls. Sperm analyses were performed according to the recommendations of the World Health Organization (WHO, 2010) and sperm chromatin quality was assessed using Chromomycin A3 (CMA3) staining for each sample. RESULTS: The comparison of the data between groups indicated that the percentage of spermatozoa with protamine deficiency was significantly different in patients with oligospermia, asthenospermia, and teratospermia when compared with control ones. However, there was no significant correlation between sperm nuclear protamine deficiency and their parameters of the men with teratospermia using CMA3 test. Regarding the oligospermia and asthenospermia semen samples, the findings showed the negative correlations between the sperm nuclear protamine deficiency and progressive motility as well as immobility (p < 0.001). CONCLUSION: The higher proportion of spermatozoa with abnormal chromatin packaging was observed in asthenospermic samples than those from other experimental groups as well as controls. It seems that normal morphology cannot have a valuable predictive value for good chromatin quality of spermatozoa, as much as normal motility characteristics, since samples with high mobility rates often have lower protamine deficiencies. The findings may provide a supportable promoting the future wider clinical application of chromatin/DNA integrity testing along with the semen analysis in male infertility

OBJETIVO: Se ha indicado que la irregularidad en los reemplazos de histona-protamina provoca daño en el ADN del esperma e infertilidad. El objetivo del presente estudio fue investigar las relaciones entre los parámetros espermáticos en oligospermia, astenospermia y teratospermia con deficiencia de protamina en varones infértiles. MATERIAL Y MÉTODO: En este estudio de casos y controles, hubo 3 grupos experimentales que incluían oligospermia (n=100), astenospermia (n=100) y teratospermia (n=100), así como normospermia (n=100) como controles. Los análisis de esperma se realizaron de acuerdo con las recomendaciones de la Organización Mundial de la Salud (OMS, 2010), y se evaluó la calidad de la cromatina de los espermatozoides utilizando la tinción con Chromomycin A3 (CMA3) para cada muestra. RESULTADOS: La comparación de los datos entre los grupos indicó que el porcentaje de espermatozoides con deficiencia de protamina fue considerablemente diferente en pacientes con oligospermia, astenospermia y teratospermia en comparación con la de los controles. Sin embargo, no hubo una correlación importante entre la deficiencia de protamina nuclear de esperma y sus parámetros de los varones con teratospermia cuando se utilizaba la prueba de CMA3. En cuanto a las muestras de semen de oligospermia y astenospermia, los hallazgos mostraron las correlaciones negativas entre la deficiencia de protamina nuclear de esperma y la movilidad progresiva, así como la inmovilidad (p < 0,001). CONCLUSIÓN: La mayor proporción de espermatozoides con un empaquetado de cromatina anómalo se observó en las muestras astenospérmicas que en las de otros grupos experimentales, así como en los controles. Parece que la morfología normal no puede tener un valor diagnóstico valioso de la buena calidad de la cromatina de los espermatozoides, tanto como las características normales de movilidad, ya que las muestras con altas tasas de movilidad a menudo tienen menores deficiencias de protamina. Los hallazgos pueden ofrecer un soporte que promueva la futura aplicación clínica más amplia de las pruebas de integridad de la cromatina/ADN junto con el análisis del semen en la infertilidad masculina

The effect of modifiable lifestyle factors on semen quality / Efecto de los factores modificables asociados el estilo de vida en la calidad del semen

INTRODUCTION AND OBJECTIVES: To examine the association between lifestyle factors (body mass index, smoking, alcohol consumption, coffee intake, physical activity, sauna and cell phone usage, wearing tight-fitting underwear), and conventional semen parameters. MATERIALS AND METHODS: 1311 participants who attended the Andrology Clinic were included in the study. All participants were separated into two groups as men with normozoospermia and dysspermia. All participants answered a questionnaire which contains questions about the modifiable lifestyle factors. The total risk scores were calculated after all the positive lifestyle factors had been counted. RESULTS: Men with normozoospermia and dysspermia consisted of 852 (65.0%) and 459 (35.0%) participants respectively. A negative relationship between the wearing of tight underwear and having normal semen parameters was detected between the two groups (p = 0.004). While going to a sauna regularly was negatively related to semen concentration, wearing tight underwear was also related to both lower motility, normal morphology as well as semen concentration (p < 0.05). While the total score of all participants was 5.22±1.34 point, there were no statistical differences between the two groups (p = 0.332). It was found that having 3 more or fewer points was not related to any type of semen parameters and results of a spermiogram. CONCLUSION: The clinicians should give advice to infertile male patients about changing their risky lifestyle, for infertility, to a healthy lifestyle for fertility. Better designed studies, with larger sample sizes using conventional semen analysis with sperm DNA analysis methods, should be planned to identify the possible effects of lifestyle factors on semen quality

INTRODUCCIÓN Y OBJETIVOS: Examinar la asociación entre los factores asociados al estilo de vida (índice de masa corporal, tabaquismo, consumo de alcohol, ingesta de café, actividad física, sauna, uso del teléfono móvil y llevar ropa interior ajustada), y los parámetros seminales convencionales. MATERIALES Y MÉTODOS: Se incluyó en el estudio a 1.311 participantes que acudieron a la Clínica de Andrología. Se separó a los participantes en 2 grupos: varones con normozoospermia o dispermia. Todos los participantes respondieron a un cuestionario que contenía preguntas relativas a los factores modificables asociados al estilo de vida. Se calcularon las puntuaciones de riesgo total tras el recuento de todos los factores positivos del estilo de vida. RESULTADOS: De entre los participantes, el número de varones con normozoospermia ascendió a 852 (65%) y a 459 (35%) los varones con dispermia. Se detectó una relación negativa entre llevar ropa interior ajustada y tener parámetros normales de semen entre los 2 grupos (p = 0,004). Mientras el acudir regularmente a una sauna guardó una relación negativa con la concentración de semen, el llevar ropa interior ajustada se relacionó también con menor movilidad, morfología normal y concentración del semen (p < 0,05). A pesar de que la puntuación total de todos los participantes fue de 5,22±1,34 puntos, no se encontraron diferencias estadísticas entre los 2 grupos (p = 0,332). También se encontró que tener 3 puntos menos, o más, no guardaba relación con ningún tipo de parámetro seminal y los resultados del espermiograma. CONCLUSIÓN: Los clínicos deberían asesorar a los varones infértiles acerca de modificar su estilo de vida de riesgo de infertilidad, llevando una vida sana de cara a la fertilidad. Deberán planificarse y diseñarse estudios con mayores tamaños de muestra, utilizando análisis convencionales de semen con métodos analíticos de ADN espermático, para identificar los posibles efectos en la calidad del semen de los factores asociados al estilo de vida en DNA

A fully automated hybrid human sperm detection and classification system based on mobile-net and the performance comparison with conventional methods.

Sperm morphology, as an indicator of fertility, is a critical tool in semen analysis. In this study, a smartphone-based hybrid system that fully automates the sperm morphological analysis is introduced with the aim of eliminating unwanted human factors. Proposed hybrid system consists of two progressive steps: automatic segmentation of possible sperm shapes and classification of normal/ab-normal sperms. In the segmentation step, clustering techniques with/without group sparsity approach were tested to extract region of interests from the images. Subsequently, a novel publicly available morphological sperm image data set, whose labels were identified by experts as non-sperm, normal and abnormal sperm, was created as the ground truths of classification step. In the classification step, conventional and ensemble machine learning methods were applied to domain-specific features that were extracted by using wavelet transform and descriptors. Additionally, as an alternative to conventional features, three deep neural network architectures, which can extract high-level features from raw images after using statistical learning, were employed to increase the proposed method's performance. The results show that, for the conventional features, the highest classification accuracies were achieved as 80.5% and 83.8% by using the wavelet- and descriptor-based features that were fed to the Support Vector Machines respectively. On the other hand, the Mobile-Net, which is a very convenient network for smartphones, achieved 87% accuracy. In the light of obtained results, it is seen that a fully automatic hybrid system, which uses the group sparsity to enhance segmentation performance and the Mobile-Net to obtain high-level robust features, can be an effective mobile solution for the sperm morphology analysis problem. A fully automated hybrid human sperm detection and classification system based on mobile-net.

Motile sperm subpopulations in bull semen using different clustering approaches - Associations with flow cytometric sperm characteristics and fertility.

There are sperm subpopulations (SPs) with different kinematic characteristics in various species, however, biological relevance of these SPs is still uncertain. The objective of the present study was to investigate associations of motile sperm SPs with sperm characteristics determined by evaluations with flow cytometry and assessment of bull fertility, using multiple approaches for sperm clustering. Semen from 24 bulls was evaluated concomitantly using computer-assisted sperm analysis (CASA) and flow cytometry before freezing and after thawing. Motile SPs were determined utilizing two acknowledged clustering methods (TwoStep and K-Means) and one customized method. With the customized method, there was utilization of mean values of sperm velocity and linearity as thresholds for direct assignment of motile spermatozoa into four SPs. Regardless of approach for identifying SPs, sperm quality, as determined using flow cytometry, was correlated particularly with the subpopulation (SP) of fast and linear spermatozoa immediately after thawing and with the SP of fast and nonlinear spermatozoa before freezing and 3 h after thawing. Furthermore, there was a positive correlation between proportion of spermatozoa with fast and nonlinear movements before freezing and bull non-return to estrous rates. These results indicate that with different sperm SPs, there are different biological implications which can be evaluated to gain useful information concerning semen quality as determined using flow cytometry and fertility. Furthermore, determining SPs by assigning motile spermatozoa into clusters based on a combination of "below and "above" threshold values for sperm velocity and linearity might be considered a practical alternative to otherwise intricate clustering procedures.

Activation of Toll-like receptor 7/8 encoded by the X chromosome alters sperm motility and provides a novel simple technology for sexing sperm.

In most mammals, the male to female sex ratio of offspring is about 50% because half of the sperm contain either the Y chromosome or X chromosome. In mice, the Y chromosome encodes fewer than 700 genes, whereas the X chromosome encodes over 3,000 genes. Although overall gene expression is lower in sperm than in somatic cells, transcription is activated selectively in round spermatids. By regulating the expression of specific genes, we hypothesized that the X chromosome might exert functional differences in sperm that are usually masked during fertilization. In this study, we found that Toll-like receptors 7/8 (TLR7/8) coding the X chromosome were expressed by approximately 50% of the round spermatids in testis and in approximately 50% of the epididymal sperm. Especially, TLR7 was localized to the tail, and TLR8 was localized to the midpiece. Ligand activation of TLR7/8 selectively suppressed the mobility of the X chromosome-bearing sperm (X-sperm) but not the Y-sperm without altering sperm viability or acrosome formation. The difference in sperm motility allowed for the separation of Y-sperm from X-sperm. Following in vitro fertilization using the ligand-selected high-mobility sperm, 90% of the embryos were XY male. Likewise, 83% of the pups obtained following embryo transfer were XY males. Conversely, the TLR7/8-activated, slow mobility sperm produced embryos and pups that were 81% XX females. Therefore, the functional differences between Y-sperm and X-sperm motility were revealed and related to different gene expression patterns, specifically TLR7/8 on X-sperm.

Deep learning for the classification of human sperm.

BACKGROUND: Infertility is a global health concern, and couples are increasingly seeking medical assistance to achieve reproduction. Semen analysis is a primary assessment performed by a clinician, in which the morphology of the sperm population is evaluated. Machine learning algorithms that automate, standardize, and expedite sperm classification are the subject of ongoing research. METHOD: We demonstrate a deep learning method to classify sperm into one of several World Health Organization (WHO) shape-based categories. Our method uses VGG16, a deep convolutional neural network (CNN) initially trained on ImageNet, a collection of human-annotated everyday images, which we retrain for sperm classification using two freely-available sperm head datasets (HuSHeM and SCIAN). RESULTS: Our deep learning approach classifies sperm at high accuracy and performs well in head-to-head comparisons with earlier approaches using identical datasets. We demonstrate improvement in true positive rate over a classifier approach based on a cascade ensemble of support vector machines (CE-SVM) and show similar true positive rates as compared to an adaptive patch-based dictionary learning (APDL) method. Retraining an off-the-shelf VGG16 network avoids excessive neural network computation or having to learn and use the massive dictionaries required for sparse representation, both of which can be computationally expensive. CONCLUSIONS: We show that our deep learning approach to sperm head classification represents a viable method to automate, standardize, and accelerate semen analysis. Our approach highlights the potential of artificial intelligence technologies to eventually exceed human experts in terms of accuracy, reliability, and throughput.

The elimination of apoptotic sperm in IVF procedures and its effect on pregnancy rate.

OBJECTIVE: To identify the effect of apoptotic sperm elimination with MACS in patients that require IVF. METHODS: An experimental, cross-sectional, descriptive, prospective and non-blinded study of diagnostic tests performed in patients who required IVF and ICSI from July 2011 to July 2012. Ninety-two couples participated according to the treatment administered to the semen sample; in the control group: the samples were subjected only to density gradients before ICSI, in the study group: the same procedure was performed plus the addition of the MACS technique. Comparing the groups, we assessed the fertilization, division, viable embryos and clinical pregnancy rates in all cases. RESULTS: We found significant differences when using MACS technique in sperm parameters. We found no differences between the total samples of the control and study groups. When separating the own and donated eggs in each group, we found an improvement in the fertilization rates (p<0.001) of the own eggs. In both groups, the handling of donated eggs lead to a significant improvement in the immunological pregnancy test (IPT) and fetal heart rate (FHR) results. Only in the donated eggs group, where MACS was applied, could we see that all cases with positive IPT had a fetal heart rate, which shows a significant difference (p<0.002) when compared with the control group, where the percentage decreased abruptly. CONCLUSIONS: This study demonstrates the effectiveness of the use of annexins (MACS) in eliminating apoptotic sperm, and when the obtained sperm is applied to good-quality eggs.

Efficient GFP-labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry.

Spermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue-specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre-lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.

Ultrastructure of mature spermatozoa of three Bucephalidae (Prosorhynchus longisaccatus, Rhipidocotyle khalili and Bucephalus margaritae) and phylogenetic implications.

We describe here the mature spermatozoa of three species of bucephalids, namely Bucephalus margaritae, Rhipidocotyle khalili and Prosorhynchus longisaccatus. This study provides the first ultrastructural data on the genera Bucephalus and Rhipidocotyle and enabled us to confirm the model of the mature spermatozoon in the Bucephalinae. The spermatozoon exhibits two axonemes with the 9 + "1" pattern of the Trepaxonemata, one of which is very short, lateral expansion, external ornamentation of the plasma membrane located in the anterior extremity of the spermatozoon and associated with cortical microtubules, spine-like bodies, a mitochondrion, and a nucleus. The maximum number of cortical microtubules is located in the anterior part of the spermatozoon. However, more studies are needed to elucidate if spine-like bodies are present in all the Bucephalinae or not. In the Prosorhynchinae, the mature spermatozoon exhibits a similar ultrastructural pattern. Some differences are observed, particularly the axoneme lengths and the arrangement of the spine-like bodies. The posterior extremity of the spermatozoon in the Bucephalinae exhibits only the nucleus, but prosorhynchines have microtubules.

Relationship between motile sperm subpopulations identified in frozen-thawed dog semen samples and their ability to bind to the zona pellucida of canine oocytes.

Studies performed on ejaculates from several species have identified discrete subpopulations of motile sperm. In dogs, motile sperm subpopulations have also been described in fresh and frozen-thawed semen. The subpopulation of the most rapid and progressively motile sperm has been suggested to be the most likely source of fertilizing spermatozoa. However, the significance of subpopulation differences among dogs and ejaculates relative to fertility is not known. The aim of this study was to investigate the relationship between the relative proportion of motile sperm subpopulations in frozen-thawed dog semen samples and their ability to bind to the zona pellucida of canine oocytes. Multiple linear regression analysis indicated that the subpopulation of the most rapid and progressively motile sperm was significantly and positively correlated with zona pellucida-binding assays (ZBA) outcomes: each 10% increase in this subpopulation was associated with an increase of 1.5 sperm bound per oocyte. Subpopulations of hyperactivated-like or locally motile sperm were negatively correlated with the ZBA results. It was concluded that subpopulation differences among frozen-thawed dog semen samples determined differences in the number of sperm bound to the ZP of canine oocytes.

Can centrifugation force compromise the plasmatic membrane, acrosome and DNA integrity of goat spermatozoa? / A força de centrifugação pode comprometer a integridade de membrana plasmática, acrossomal e DNA de espermatozoides caprinos?

Protocols for cooling or freezing goat semen usually recommend centrifugation for seminal plasma removal. However, little is known about the effect of this process on goat sperm viability and functionality. The present study evaluated the effects of centrifugation force on the plasma membrane, acrosomes, and DNA integrity of goat semen. Four ejaculates from each of the four different Anglo Nubian male goats were used. Semen samples were obtained using artificial vagina, and immediately after collection, ejaculates were diluted using Ringer's sodium lactate solution and split into three groups: Control (CG, without centrifugation), G1 (centrifugation 600 x g/10 min), G2 (centrifugation 1200 x g/10 min). After centrifugation, seminal plasma was removed, the sperm pellets were resuspended using Tris-egg yolk extender (80 x 106 spermatozoa/mL) and the sperm morphology was analyzed. Samples were cooled at 5°C for 5, 24, 36, and 48 h and then sperm plasma membrane and acrosome integrity (PMAI, %) and sperm DNA fragmentation index (SDF, %) were evaluated at each time-point, using a flow cytometer. Additionally, sperm movement was determined using computer semen analysis (CASA) after 5, 24, and 48 h of refrigeration period. The semen centrifugation did not induce additional sperm morphology defect or reduction in sperm kinetics in the experimental groups. Differences were not observed (p > 0.05) in PMAI and SDF among different groups, in any of each time-point of the cooling process. In conclusion, centrifugation, even at high speeds, did not affect goat sperm integrity and functionality when submitted to refrigeration process. (AU)

A maior parte dos protocolos de refrigeração e criopreservação do sêmen caprino recomenda o uso de centrifugação para remoção do plasma seminal. No entanto, não existe consenso sobre o risco que esse tipo de processamento pode ocasionar à viabilidade espermática. Nesse contexto, o presente trabalho investigou os possíveis efeitos deletérios da centrifugação sobre a integridade estrutural e DNA de espermatozoides caprinos. Para a pesquisa foram selecionados quatro reprodutores para colheita de sêmen (n = 4 ejaculados/bode). Cada ejaculado foi fracionado em três alíquotas iguais, diluídas em ringer e divididas em três grupos: Controle (GC, não centrifugado), G1 (centrifugação a 600 g/10 minutos) e G2 (centrifugação a 1200 g/10 minutos). As amostras seminais por grupo foram diluídas em meio Tris gema respeitando-se a concentração final de 80 milhões de espermatozoides/mL e foram submetidas à avaliação de morfologia espermática. Todas as amostras foram acondicionadas a 5°C, sendo analisadas nos momentos 5, 24, 36 e 48 horas do processo de refrigeração por meio da avaliação da integridade de membrana plasmática e acrossomal (MPAI, %) e índice de fragmentação de DNA (IDF, %). Adicionalmente, a cinética espermática foi avaliada com o emprego de um sistema computadorizado de análise (CASA) nos momentos 5, 24 e 48 horas da refrigeração. A centrifugação não induziu a manifestação de defeitos morfológicos ou redução significativa da cinética de espermatozoides caprinos. Não foram observadas diferenças para a integridade de membrana plasmática e para o índice de fragmentação de DNA quando comparados, respectivamente, GC, G1 e G2 em cada um dos quatro momentos experimentais. Conclui-se que mesmo quando empregadas altas forças de rotação não ocorre lesão à ultraestrutura dos espermatozoides caprinos submetidos ao processo de refrigeração.(AU)

Sperm subpopulations in ejaculated sperm and spermatozoa recovered from ovine epididymides up to 48h after death.

The objectives of this study were threefold: to identify subpopulations of sperm based on the kinetics of frozen/thawed sheep epididymal spermatozoa or semen collected with an artificial vagina; to evaluate the effects on sperm subpopulations in the thawed samples of post mortem storage at room temperature and the addition of 20% of seminal plasma to the freezing extender and to correlate the percentage of subpopulations with gestation rate following artificial intrauterine insemination. The categorization of the subpopulations was based on sperm kinetic data from Computer Assisted Sperm Analysis (CASA). A hundred ewes were inseminated with thawed spermatozoa and gestation rate was correlated with the proportions of each subpopulation using Pearson correlation matrix and linear regression. Three distinct subpopulations were identified in the thawed samples of either ovine ejaculate collected in artificial vaginas (AV) or ovine spermatozoa retrieved from the cauda epididymis. Subpopulation 1 (SP1) was characterized by spermatozoa with slow and non-linear motion, subpopulation 2 (SP2) was classified as hyperactived spermatozoa and subpopulation 3 (SP3) was composed of spermatozoa with fast, linear motion. The largest subpopulation in all groups was SP1. The semen collected in an artificial vagina had a higher (P<0.05) percentage of SP2 and lower (P<0.05) percentage of SP1 when compared to spermatozoa recovered after death. Increasing time of storage after death had a detrimental effect on sperm samples, increasing (P<0.05) the percentage of SP1 and decreasing (P<0.05) SP2. Length of storage after death was the only variable that influenced, with an inversely proportional relationship, SP3. In samples stored for 48h after death no SP3 spermatozoa were present. The addition of seminal plasma to the cryopreservative decreased (P<0.05) the subpopulation of hyperactived spermatozoa (SP2). We conclude that, after thawing there are three sperm subpopulations in the spermatozoa obtained from the cauda epididymides and the semen collected in AVs and that the relative proportions of these subpopulations varies with the time of storage post mortem and the presence of 20% of seminal plasma in the extender. However, we conclude that these subpopulations do not correlate with fertility after intrauterine artificial insemination.

CASAnova: a multiclass support vector machine model for the classification of human sperm motility patterns.

The ability to accurately monitor alterations in sperm motility is paramount to understanding multiple genetic and biochemical perturbations impacting normal fertilization. Computer-aided sperm analysis (CASA) of human sperm typically reports motile percentage and kinematic parameters at the population level, and uses kinematic gating methods to identify subpopulations such as progressive or hyperactivated sperm. The goal of this study was to develop an automated method that classifies all patterns of human sperm motility during in vitro capacitation following the removal of seminal plasma. We visually classified CASA tracks of 2817 sperm from 18 individuals and used a support vector machine-based decision tree to compute four hyperplanes that separate five classes based on their kinematic parameters. We then developed a web-based program, CASAnova, which applies these equations sequentially to assign a single classification to each motile sperm. Vigorous sperm are classified as progressive, intermediate, or hyperactivated, and nonvigorous sperm as slow or weakly motile. This program correctly classifies sperm motility into one of five classes with an overall accuracy of 89.9%. Application of CASAnova to capacitating sperm populations showed a shift from predominantly linear patterns of motility at initial time points to more vigorous patterns, including hyperactivated motility, as capacitation proceeds. Both intermediate and hyperactivated motility patterns were largely eliminated when sperm were incubated in noncapacitating medium, demonstrating the sensitivity of this method. The five CASAnova classifications are distinctive and reflect kinetic parameters of washed human sperm, providing an accurate, quantitative, and high-throughput method for monitoring alterations in motility.

Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility.

Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as "hyperactivated" (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables.

Comparison of sperm subpopulation structures in first and second ejaculated semen from Japanese black bulls by a cluster analysis of sperm motility evaluated by a CASA system.

In the present study, bull sperm in the first and second ejaculates were divided into subpopulations based on their motility characteristics using a cluster analysis of data from computer-assisted sperm motility analysis (CASA). Semen samples were collected from 4 Japanese black bulls. Data from 9,228 motile sperm were classified into 4 clusters; 1) very rapid and progressively motile sperm, 2) rapid and circularly motile sperm with widely moving heads, 3) moderately motile sperm with heads moving frequently in a short length, and 4) poorly motile sperm. The percentage of cluster 1 varied between bulls. The first ejaculates had a higher proportion of cluster 2 and lower proportion of cluster 3 than the second ejaculates.

Post-thaw recovery of rare or very low concentrations of cryopreserved human sperm.

OBJECTIVE: To analyze cases in which no sperm could be identified after thawing among cryopreserved samples of rare or very low concentrations of sperm. DESIGN: Retrospective, single-institution, cross-sectional. SETTING: Male infertility clinic. PATIENT(S): We identified couples that underwent intracytoplasmic sperm injection (ICSI) with the use of either ejaculated or testicular cryopreserved-thawed sperm. Inclusion criteria were men with <100,000 total ejaculated sperm or men with azoospermia due to spermatogenic dysfunction who underwent microsurgical testicular sperm extraction with similarly low pre-cryopreservation sperm counts. Pre-cryopreservation specimens were categorized as "rare sperm only" (Group 1) or <100,000 total sperm (group 2). "Rare sperm only" applied to cases in which only one to three sperm were identified in a search of >20 high-power fields. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cases in which no sperm were able to be found post-thaw (i.e., complete cellular loss) for use at the time of a programmed IVF cycle. RESULT(S): We analyzed 55 men (83 ICSI cycles). There were five ICSI cycles (6.0%) among five different couples in which no sperm could be identified post-thaw. Of these, four cases were from group 1 (8.5%) and one from group 2 (2.8%). Complete cellular loss occurred in 5.8% of testicular sperm samples and 7.1% of ejaculated sperm samples. There were no statistical associations between the ability to locate sperm post-thaw and the pre-cryopreservation parameters or sperm source. CONCLUSION(S): Failure to retrieve any sperm after thawing of rare or very low concentrations of cryopreserved sperm is an infrequent event and largely limited to those patients with rare quantities of sperm.

Fish sperm subpopulations: Changes after cryopreservation process and relationship with fertilization success in tambaqui (Colossoma macropomum).

Fish tambaqui (Colossoma macropomum) is the native Brazilian fish with the highest agricultural production under intensive aquaculture in South America. However, the decrease in the genetic variability in fish farms has become necessary the improvement of cryopreservation process through new statistical studies of spermatozoa (like subpopulation studies). The evaluation of the kinetic data obtained with a computer-assisted sperm analysis system, applying a two-step cluster analysis, yielded in tambaqui three different subpopulations in fresh sperm: SP1, considered as a slow nonlinear subpopulation; SP2, considered as a fast nonlinear subpopulation, and finally; SP3, considered as a fast linear subpopulation. For cryopreserved sperm, the cluster analysis yielded only two sperm subpopulations: SP1', considered as a slow nonlinear subpopulation and SP2', which seemed to be an intermediate subpopulation (showing medium motility and velocity values) merged from SP2 and SP3 obtained from fresh sperm. Coefficients of correlation (r) and determination (r2) between the sperm subpopulations from fresh sperm and the fertilization rates were calculated, and SP2 and SP3 (the fast-spermatozoa subpopulations) showed a high-positive correlation with the fertilization rates (r = 0.93 and 0.79, respectively). In addition, the positive significant correlations found in curvilinear velocity (r = 0.78), straight line velocity (r = 0.57), and average velocity (r = 0.75) indicate that sperm kinetic features seem to be a key factor in the fertilization process in tambaqui, as occur in other fish species.

Variation in male reproductive system characters in Corydoradinae (Loricarioidei: Callichthyidae) reflects the occurrence of different lineages in this subfamily

Callichthyidae comprises a well-corroborated monophyletic group divided into two subfamilies: Corydoradinae and Callichthyinae. A recent proposal, based on molecular data, suggests that Corydoradinae is composed by nine monophyletic lineages, possibly genera. The species pertaining to those lineages have extensive modification in the size of genome, including diploid, tetraploid and octoploid species. Considering the occurrence of these monophyletic lineages and that the variations in DNA content may imply in significant alterations on the structure of spermatozoa, this study analyzed the morphology of the male reproductive system and the morphometry of the head of the spermatozoa of representatives of the nine lineages of Corydoradinae, seeking for particular characteristics of each lineage. Morphological data revealed a high intra-lineage variation, larger than that observed among species of different lineages. In contrast, morphometric data obtained for eight out of the nine lineages, revealed large congruency with the hypothesis that Corydoradinae is composed by different lineages. These results demonstrate that there is a correlation among variations in DNA content and the size of the spermatozoon head, thus providing additional subsides for the definition of the Corydoradinae lineages.(AU)

A família Callichthyidae compreende um grupo monofilético bem corroborado, dividida em duas subfamílias: Corydoradinae e Callichthyinae. Uma proposta recente, baseada em dados moleculares, sugeriu que a subfamília Corydoradinae é composta por nove linhagens, possivelmente gêneros. As espécies pertencentes a cada uma destas linhagens possuem extensivas modificações no tamanho do genoma, incluindo espécies diplóides, tetraplóides e octaplóides. Considerando a ocorrência dessas diferentes linhagens e que as extremas variações em conteúdo de DNA podem implicar em alterações significativas na estrutura dos espermatozoides, o presente estudo analisou a morfologia do sistema reprodutor masculino e a morfometria da cabeça dos espermatozoides de representantes das nove linhagens de Corydoradinae, procurando características particulares em cada uma. Os dados morfológicos revelaram a ocorrência de grande variação dentro das linhagens, maior que aquela observada entre espécies de diferentes linhagens. Diferentemente, os dados morfométricos obtidos para oito das nove linhagens revelaram grande congruência com a atual proposta para Corydoradinae. Estes resultados demonstram que há correlação entre as variações em conteúdo de DNA e o tamanho da cabeça dos espermatozoides, fornecendo, assim, subsídio adicional para a definição das nove linhagens de Corydoradinae.(AU)