Effect of sperm selection techniques in frozen/thawed cat spermatozoa on sperm motility analyzed by CASA system / Efecto de técnicas de selección espermática en espermatozoides congelados/descongelados de gato sobre la motilidad espermática analizada mediante sistema CASA

SUMMARY: Freeze/thawing process reduces sperm survival and fertilizing ability of cat spermatozoa, with sperm motility being the most sensitive sperm parameter altered, due to cryo-damage. In this context, swim-up and density gradient processing methods can help to recover high motile and normal spermatozoa. Maximizing the use of frozen semen sample is essential, especially in endangered felids or high value cats in which sample size, number of samples or access to semen collection is reduced. To our knowledge, there is no previous report describing an in depth analysis of sperm motility improvement, after sperm selection techniques in frozen cat semen. Accordingly, we evaluated the effect of percoll gradient (PG) and swim up (SU) sperm selection techniques on sperm motility parameters and sperm recovery rate in frozen/thawed spermatozoa of domestic cat. Next, we evaluated the individual effect of the cat over sperm motility after PG sperm selection of frozen/thawed spermatozoa. SU and PG improved significantly all sperm motility parameters of frozen/thawed cat spermatozoa compared to simple washing. However, PG allows better sperm recovery from the original frozen sample and works mostly homogeneously among individual cats. This new information could help to maximize the use of frozen semen in endangered felids or high value domestic cats for its subsequent application on in vitro fertilization and artificial insemination.

RESUMEN: El proceso de congelación/descongelación reduce la sobrevivencia espermática y la habilidad para fertilizar en los espermatozoides de gato, siendo la motilidad espermática el parámetro más sensiblemente alterado debido al daño por frío. En este contexto, los métodos de procesamiento de swim-up y gradiente de densidad pueden ayudar a recuperar los espermatozoides normales y de alta motilidad. Maximizar el uso de una muestra de semen congelado es esencial, especialmente en felinos amenazados o en gatos de alto valor en los cuales el tamaño de muestra, número de muestras o el acceso a la colecta de semen son reducidos. Para nuestro conocimiento, no hay reportes previos que describan un análisis profundo del mejoramiento de la motilidad luego de técnicas de selección espermática en semen congelado de gato. De acuerdo a esto, evaluamos el efecto de las técnicas de selección espermática gradiente de percoll (PG) y swim up (SU) sobre los parámetros de motilidad y porcentaje de recuperación de espermatozoides congelados/descongelados de gato doméstico. Luego, evaluamos el efecto individual del gato sobre la motilidad espermática luego de la selección espermática con PG en espermatozoides congelados/descongelados. SU y PG mejoraron significativamente todos los parámetros de motilidad espermática de los espermatozoides congelados/descongelados comparado con el lavado simple. Sin embargo, PG permitió una mejor recuperación de espermatozoides desde la muestra congelada original y funcionó en su mayoría de manera homogénea entre los gatos individualmente. Esta nueva información puede ayudar a maximizar el uso del semen congelado en felinos amenazados o en gatos de alto valor para su posterior aplicación en fecundación in vitro e inseminación artificial.

Sperm DNA fragmentation and sex chromosome aneuploidy after swim-up versus density gradient centrifugation / 대한생식의학회지

OBJECTIVE: The aim of this study was to compare the efficacy of swim-up and density gradient centrifugation (DGC) for reducing the amount of sperm with fragmented DNA, sex chromosome aneuploidy, and abnormal chromatin structure. METHODS: Semen samples were obtained from 18 healthy male partners who attended infertility clinics for infertility investigations and were processed with swim-up and DGC. The percentages of sperm cells with fragmented DNA measured by the sperm chromatin dispersion test, normal sex chromosomes assessed by fluorescence in situ hybridization, and abnormal chromatin structure identified by toluidine blue staining were examined. RESULTS: The percentage of sperm cells with fragmented DNA was significantly lower in the swim-up fraction (9.7%, p=0.001) than in the unprocessed fraction (27.0%), but not in the DGC fraction (27.8%, p=0.098). The percentage of sperm cells with normal X or Y chromosomes was comparable in the three fractions. The percentage of sperm cells with abnormal chromatin structure significantly decreased after DGC (from 15.7% to 10.3%, p=0.002). The swim-up method also tended to reduce the percentage of sperm cells with abnormal chromatin structure, but the difference was not significant (from 15.7% to 11.6%, p=0.316). CONCLUSION: The swim-up method is superior for enriching genetically competent sperm.

The effect of semen optimization on the rate of sperm DNA fragmentation and its effect on pregnancy out-come of IVF-ET / 实用医学杂志

Objective To investigate the method of effectively density gradient centrifugation combined with swim-up improves sperm nuclear integrity and determine whether the sperm chromatin dispersion test of sperm DNA fragmentation in raw or DGC-swim-up treated semen can influence the outcome of IVF. Method The DNA integrity of spermatozoa from 120 patients underwent IVF were analyzed by SCD before and after DGC and swim-up. The predictive value of the SDFI for IVF outcomes were assessed in a cohort of 100 patients who were underwent new embryo transfer. Result In male infertility group,DGC combined with swim-up decreased the SDFI from 22.75(14.44,30.25)to 11.50(5.60,22.79),while the control group decreased the SDFI from 20.86(15.00,26.81) to 7.50(3.63,15.44),respectively(P<0.05);SDFI after optimization in clinical pregnancy group was significantly lower than that of non-pregnant group. The area under the receiver operating characteristic curve was 0.667. The patients with low sperm DFI had a higher implantation rate and pregnancy rate compared with patients with high sperm DFI. Conclusions DGC and swim-up treated Sperm DNA fragmentation can predict the outcome of IVF. The effect of semen optimization on the rate of sperm DNA fragmentation is limited,once exceed,pregnancy rate and birth rate are decreased although fertilization is normal.

The effect of semen optimization on the rate of sperm DNA fragmentation and its effect on pregnancy out-come of IVF-ET / 实用医学杂志

Objective To investigate the method of effectively density gradient centrifugation combined with swim-up improves sperm nuclear integrity and determine whether the sperm chromatin dispersion test of sperm DNA fragmentation in raw or DGC-swim-up treated semen can influence the outcome of IVF. Method The DNA integrity of spermatozoa from 120 patients underwent IVF were analyzed by SCD before and after DGC and swim-up. The predictive value of the SDFI for IVF outcomes were assessed in a cohort of 100 patients who were underwent new embryo transfer. Result In male infertility group,DGC combined with swim-up decreased the SDFI from 22.75(14.44,30.25)to 11.50(5.60,22.79),while the control group decreased the SDFI from 20.86(15.00,26.81) to 7.50(3.63,15.44),respectively(P<0.05);SDFI after optimization in clinical pregnancy group was significantly lower than that of non-pregnant group. The area under the receiver operating characteristic curve was 0.667. The patients with low sperm DFI had a higher implantation rate and pregnancy rate compared with patients with high sperm DFI. Conclusions DGC and swim-up treated Sperm DNA fragmentation can predict the outcome of IVF. The effect of semen optimization on the rate of sperm DNA fragmentation is limited,once exceed,pregnancy rate and birth rate are decreased although fertilization is normal.

The gradient technique improves success rates in intrauterine insemination cycles / 实用医学杂志

Objective To compare the efficacy of gradient and swim-up semen preparation techniques on pregnancy rates in couples undergoing intrauterine insemination (IUI) cycles with human menopausal gonadotropin (HMG) stimulation. Methods Five hundred and seventy one cycles were devided into the swim up or the gradient technique groups for sperm preperation. The clinical pregnancy rates per cycle were evaluated. Results The clinical pregnancy rates per cycle were significantly higher in the gradient group (17.8%) than that in the swim up group (11.4%)(P < 0.05). In the subgroup of unexplained subfertile couples, the gradient group also revealed significantly higher clinical pregnancy rates per cycle (33.7%) than that in the swim up group (20.3%)(P<0.05). In couples with mild male factor subfertility, the gradient group also revealed significantly higher clinical pregnancy rates per cycle (11.6%) than that in the swim up group (7.6%)(P < 0.05). Conclusion The gradient technique significantly improves clinical outcome in IUI cycles of unexplained subfertile and male subfertile couples.

Comparison of the effect of different media on the clinical outcomes of the density-gradient centrifugation/swim-up and swim-up methods / 대한생식의학회지

OBJECTIVE: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. METHODS: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). RESULTS: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. CONCLUSION: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.

Selección espermática en semen congelado/descongelado de equino: evaluación de las membranas plasmática, acrosomal y potencial de membrana mitocondrial / Sperm selection in frozen/thawed semen of equine: evaluation of plasma, acrosome membranes and mitochondrial membrane potential

Los procedimientos de criopreservación inducen cambios morfofuncionales en los espermatozoides. Es importante post descongelación espermática utilizar procedimientos de selección que permitan recuperar espermatozoides altamente funcionales. El objetivo del presente estudio fue comparar la eficiencia del Swim-up y Equipure® en la selección de espermatozoides funcionales en semen descongelado de equino. Semen de 4 potros reproductores Criollos Chilenos (A, B, C y D), fueron descongelados separadamente y procesados (n=15) por: I.- Swim-up (SU) y II.- Equipure® (EQ). Post descongelación se determinó por citometría de flujo la viabilidad e integridad de membrana plasmática (SYBR-14/PI), potencial de membrana mitocondrial (YDm; JC-1), integridad de la membrana acrosomal (FITC-PSA/PI). La motilidad progresiva (%) en dos animales fue más alta (P<0,05) por SU comparado con EQ: A (55,7±5,8% v/s 38,17±3,7%) y C (37,5±7% vs. 32±2,1%, respectivamente). La integridad de la membrana plasmática (%), tres animales presentaron diferencias (P<0,05), siendo más alta por SU en dos animales comparado con EQ (A: 54,3±1,7 vs. 36,7±1,9, C: 36,1±5,7 vs. 29,4±4,8 y D: 34,4±9,4 vs. 52,7±5,2; respectivamente), solamente un animal fue superior EQ. En el YDm (%), diferencias significativas (P<0,05) fueron detectadas en los cuatro animales, siendo más altos en SU comparado con EQ (A: 69,1±8,6% vs. 47,4±3,3%, B: 59,34±12,3% vs. 24,8±1,5%, C: 54,9±12,3% vs. 43,2±3,1% y D: 53,1±17,6% vs. 37,5±5,7%; respectivamente). Los resultados obtenidos en el presente estudio demostraron que los métodos de selección espermática Swim-up y Equipure® permiten recuperar espermatozoides de diferente calidad funcional en semen congelado-descongelado de equino, presentándose diferencias individuales entre los animales con respecto a los métodos. Se observó una tendencia del método Swim-up en seleccionar espermatozoides de equino descongelados con mayor calidad funcional comparado con Equipure®.

Freeze-thaw procedures induce structural and functional changes in sperm. It is important to use post thaw sperm selection procedures that can retrieve highly functional sperm. The aim of this study was to compare the efficiency of the Swim-up and Equipure® in the selection of functional sperm of thawed equine semen. Semen of four Chilean Criollo reproductive stallions (A, B , C and D) were frozen and thawed using a standard protocol and processed separately (n = 15) : I. Swim-up (SU) and II. Equipure® (EQ). Post sperm selection,was determined by flow cytometry. Viability and plasma membrane integrity (SYRB-14/PI), mitochondrial membrane potential (YDm, JC -1), acrosome membrane integrity (FITC-PSA/PI). Progressive motility (%) was higher (P<0.05) in two animals per SU compared with EQ, A (55.7±5.8% vs. 38.17±3.7%) and C (37.5±7.0% vs. 32±2.1%, respectively). The viability and integrity of the plasma membrane (%), three animals showed differences (P<0.05), being higher for SU in two animals compared with EQ (A: 54.3±1.7 vs. 36.7±1.9, C: 36.1±5.7 vs. 29.4±4.8 and D: 34.4±9.4 vs. 52.7±5.2, respectively), only one animal was higher EQ. In YDm (%), significant differences were detected (P<0.05) in all four animals, being higher in SU compared with EQ (A: 69.1±8.6% vs. 47.4±3.3% B: 59.34±12.3% vs. 24.8±1.5%, C: 54.9±12.3% vs. 43.2±3.1% and D: 53.1±17.6% vs. 37.5±5.7%, respectively). The results obtained in this study showed that sperm selection methods Swim-up and Equipure® can retrieve different functional sperm quality in frozen-thawed equine semen, and that individual differences were registered among animals with respect to methods. In the Swim-up method a tendency for selecting higher functional quality in thawed equine sperm was observed when compared to Equipure®.

Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production / Avaliação do swim-up e do gradiente descontinuo de densidade na pré-seleção do sexo de espermatozoides na produção de embriões bovinos

The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y- specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05) in deviation of sex ratio when comparing the control group (45.2% females) with the other spermatozoa selection procedures (60.6% females) (P<0.05). The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively) and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.

O objetivo do presente trabalho foi associar o método de swim-up modificado à centrifugação em gradiente de densidade para a separação de espermatozoides portadores do cromossomo X. A viabilidade e a integridade espermática foram avaliadas pelo método de coloração Azul de Tripan e Giemsa. O controle de qualidade dos espermatozoides centrifugados foi realizado por meio da produção in vitro de embriões bovinos. Os resultados foram validados pela técnica de PCR para verificar a proporção sexual dos embriões produzidos in vitro, com o uso de sequências Y especificas presente no DNA genômico de machos bovinos. Após determinar o sexo genético dos embriões produzidos in vitro, os resultados não mostraram diferença (P<0,05) no desvio da proporção do sexo quando comparou o grupo controle (45,2% de fêmeas) com os outros processos de seleção de espermatozoides (60,6% de fêmeas) (P<0,05). Os métodos de seleção de espermatozoides são capazes de selecionar espermatozoides portadores do cromossomo X sem comprometer a fertilidade, medida pelas taxas de clivagem e blastocisto de 70% e 26%, respectivamente, e foram considerados métodos de relevância para serem introduzidos nos programas de produção in vitro de embriões bovinos.

Evaluación de las técnicas de capacitación espermática y su efecto en la fragmentación del ADN / Assesment of sperm capacitation techniques and their effect on DNA fragmentation

Objetivo: evaluar el efecto de las técnicas de capacitación espermática en la fragmentación del ADN. Materiales y métodos: estudio experimental en el cual se describió el efecto de las técnicas de capacitación espermática swim up y gradientes de densidad de Isolate en la fragmentación del ADN. Se utilizaron las muestras de 41 pacientes remitidos a espermograma a la Unidad de Fertilidad del Tolima (Unifertil). Requisitos: 1) abstinencia sexual de 3 a 5 días, 2) no haber consumido medicamentos como antibióticos o antiparasitarios, 3) obtención de la muestra por masturbación, y 4) no presentar procesos virales o febriles. Después de realizar la primera evaluación de las muestras dentro de una hora posterior a su obtención se incluyeron aquellas que habían sido recolectadas completamente, que tenían una concentración mínima de 2 millones de espermatozoides/ml, y que no tenían contaminación con sangre. Se evaluaron la concentración, la movilidad, la morfología y la fragmentación del ADN de los espermatozoides, midiendo la longitud de los cometas resultantes del ensayo. Para el análisis estadístico se utilizaron análisis de varianza (ANOVA) y el coeficiente de correlación de Pearson, teniendo un valor de p < 0,05 como significativo estadísticamente. Resultados: la media de edad de los pacientes fue 35,1 ± 7,9 años. Se encontró que las técnicas de capacitación espermática, tanto swim up como gradientes de densidad de Isolate, disminuyen la fragmentación del ADN de los espermatozoides (p < 0,0001). No se encontraron diferencias significativas en cuanto a la fragmentación del ADN entre las dos técnicas de capacitación estudiadas. No se observaron correlaciones significativas entre los niveles de fragmentación del ADN espermático y los diferentes parámetros del espermograma (concentración, movilidad y morfología) (p = 0,9061). El ensayo cometa detectó diferencias en el grado de la fragmentación del ADN de los espermatozoides entre las muestras frescas y capacitadas. Conclusión: la capacitación espermática tanto por swim up como por gradientes de densidad de isolate tiene un efecto positivo al disminuir la fragmentación del ADN de los espermatozoides.

Objective: This study was aimed at evaluating the effect of sperm capacitation techniques on DNA fragmentation. Materials and methods: This was an experimental study which observed and described the effect of two sperm capacitation techniques on DNA fragmentation (i.e. swim up and isolate density gradients). Samples from 41 patients were used; these were sent for spermogram analysis at the Tolima Fertility Unit (Unifertil), taking the following requirements into account: sexual abstinence for 3 to 5 days, not having taken drugs such as antibiotics or antiparasitics, samples obtained by masturbation and not presenting viral or febrile processes. The first evaluation of the samples was made one hour after they had been obtained; those which had been collected completely, which had a minimum 2 million spermatozoid/ml concentration and which were not contaminated by blood were included. Concentration, mobility, morphology and DNA spermatic fragmentation were evaluated by measuring comet length in the comet assay in fresh samples. Analysis of variance (ANOVA) and Pearson’s correlation coefficient were used for statistical analysis (p < 0.05 being statistically significant). Results: It was found that sperm capacitation techniques reduced spermatozoid DNA fragmentation (p < 0.0001). No significant differences were found between the two different capacitation techniques studied regarding DNA fragmentation. No significant correlation was observed between sperm DNA fragmentation levels and spermogram parameters (concentrations, mobility and morphology) (p = 0.9061). The comet assay detected differences regarding the degree of spermatozoid DNA fragmentation between fresh and capacitated samples. Conclusion: Both swim up and isolate density gradient sperm capacitation had a positive effect by reducing spermatozoid DNA fragmentation.

Correlation of Post Swim-Up Acrosome Index with in-vitro Fertilization Outcomes

PURPOSE: A prospective study was planned to determine the relationship between post swim-up acrosome index (AI) evaluation and fertilization outcomes in an in vitro fertilization (IVF) program. MATERIALS AND METHODS: Infertile couples who have applied to IVF were admitted into this study when the male partner's sperm concentration was > 20x106/mL and motility > 30%. Pre- and post swim-up semen quality parameters including concentration, motility, sperm morphology and AI were evaluated in a prospective, randomized and blinded fashion. The couples were divided prospectively into 2 groups. In group I (25 couples) 50 000 sperm per oocyte were used for insemination considering post swim-up acrosome index, and in group II (25 couples) 50 000 sperm per oocyte were used for insemination without considering post swim-up acrosome index. RESULTS: Pre- and post swim-up AI were 30.8 +/- 3.4 and 17.8 +/- 4.5 in group I, and 31.4 +/- 3.6 and 16.3 +/- 4.7 in group II (p > 0.05) respectively. The significant improvement in morphology and motility after double wash swim-up procedure has been observed. However, double wash swim-up procedure could not eliminate head and especially acrosomal defects which would directly effect fertilization capacity in conventional IVF program. In group I, 85.3% of oocytes were fertilized, with a 48% pregnancy rate; in group II, 71.0% of oocytes were fertilized, with a pregnancy rate of 20%. Fertilization and pregnancy rates were significantly different (p < 0.05) between the two groups. CONCLUSION: We have concluded that it could be useful to consider post swim-up AI of sperm inseminated in conventional IVF cycles, which correlates with high fertilization and pregnancy rates.

Assessment of the two preparation techniques of sperm: swim \ufffd?up and discontinuous density gradient

Background: In Vietnam, the proportion infertile couples was about 7-10% of reproductive couples. Assisted reproductive technologies appeared and expanded rapidly. In 1970, sperm preparation techniques were found out, artificial insemination became widespread. Sperm preparation stage is very important in assisted reproductive techniques and increasingly improved. Objectives: To compare the efficiency of two sperm preparation techniques: swim \ufffd?up and discontinuous density gradient. Subjects and method: This was a cross-sectional described study included 30 normal semen samples were chosen based on WHO 1999 criteria. We examined sperm vitality, concentration, motility, morphology before and after applied these techniques. Results: Both procedures resulted in a significant higher percentage of vitality, motility, morphology compared with the original semen sample (p<0.01), but the concentration reduced approximately 5 times from whole semen to sediment when swim \ufffd?up was used and 3 times with density gradient, the difference was statically significant (p< 0.01). Conclusion: Normal semen should be prepared by these method. But in oligozoospermic man, to obtain a higher concentration of sperm, semen should be prepared by density gradient method.

Comparison of sperm motility between the swim-up and swim-down methods.

The objective of this study was to compare two methods for the extraction of high motility sperm; the swim-up and swim-down techniques. The result showed that both methods produced greater sperm motility than the original semen. The motility of the sperm was lower in the swim-down method compared with the swim-up method (65.5+11.2% vs 76.5+10.1%; p<0.01).

A comparison of swim-up and simple swim-up methods for the extraction of high motility sperm.

The objective of this study was to compare two methods for the extraction of high motility sperm; swim-up and simple swim-up technique. The results were comparable in concentration, motility and morphology.

Comparison between Isolate(R) gradient and Swim-up Procedures for Sperm Preparation: Effects on Freeze-thawing in Normal Semen Sample / 대한불임학회지

No abstract available in English.

Hypo-osmotic swelling test of washing and swim-up sperm treated with pentoxifylline.

An experimental study was carried out in order to evaluate the effect to of pentoxifylline on the membrane function of washing and swim-up sperm in the process of sperm preparation for assisted reproductive techniques. Normal semen samples were taken according to World Health Organization criterias, from 30 male partners who attended the infertility clinic from November 1996 to January 1997, and divided into two aliquots. In the process of sperm preparation by the washing and swim-up method, pentoxifylline was added in only one aliquot. The hypo-osmotic swelling test was used to evaluate the outcome of both aliquots. From the study, the percentage of sperm that showed swollen tails in the hypo-osmotic solution prepared by the washing and swim-up method and treated by pentoxifylline was higher than that in the group which did not have pentoxifylline added in the process of sperm preparation. However, the difference was not atatistically significant (54.9 and 49.2 percent; P>0.01). It was therefore concluded that pentoxifylline added to washing and swim-up sperm in the process of sperm preparation dose not improve membrane function of spermatozoa. Thus, it may not be necessary to use pentoxifylline in the treatment of male infertility.