Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

The primary objective of the studies described in this thesis was to improve the cryopreservation success of koala spermatozoa for the purpose of establishing a genome resource bank for this species. A defining feature of the studies in this thesis was the implementation of an organelle-specific approach to better understand the causes of koala sperm cryo-injury. The functional attributes of spermatozoa, such as mitochondrial function, plasma membrane fluidity, membrane lipid asymmetry and DNA integrity were assessed as an indication of cryo-injury. Sperm mitochondrial function and plasma membrane integrity were examined by cryomicroscopy using the fluorescent probes JC-1 and propidium iodide (PI) respectively in a dual staining technique. Cooling and re-warming koala spermatozoa were more detrimental to mitochondrial function than to plasma membrane integrity. Mitochondrial membrane potential (MMP) was suppressed by freezing and thawing treatments; after thawing, MMP declined significantly during rewarming (from 5ºC to 35ºC). The distribution of GM1 ganglioside was examined using fluorescent-labelled cholera toxin B. No significant redistribution of GM1 was observed after chilling or cryotreatment. The externalisation of phosphatidylserine (PS) was examined using fluorescent-labelled annexin V. There was no significant increase in translocation of PS after chilling or cryopreservation. These observations imply that cryotreatment had little effect on plasma membrane lipid asymmetry. Koala spermatozoa were incubated in a range of anisotonic media to investigate whether nuclear swelling was caused by osmotic flux during the cryopreservation process. Although the most hypotonic solution tested (64 mOsm/kg) induced the highest incidence of nuclear relaxation (mean ± SEM; 12 ± 3%), this was not as severe as that previously documented following cryopreservation. Chromatin relaxation is a phenomenon observed in koala spermatozoa, where the sperm nucleus expands due to the result of structural changes in the natural conformation of the sperm DNA/protamine complex. DNA fragmentation was not a primary cause of cryopreservation-induced sperm chromatin relaxation, although in situ nick translation of putative DNA breaks indicated that these increased as the sperm head became progressively more relaxed. Using a Sperm Chromatin Dispersion test (SCDt) specifically developed and validated for koala spermatozoa, a continuum of nuclear morphotypes was observed, ranging from no apparent DNA fragmentation to spermatozoa with highly dispersed and degraded chromatin. A double comet assay was also developed to investigate DNA fragmentation in the koala spermatozoa. Conducted under neutral followed by alkaline conditions, this assay was able to differentiate between single- (SSB) and double-stranded (DSB) DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa; the majority of the koala spermatozoa had nuclei with DNA abasic-like residues. The ubiquity of these residues suggested that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with “true” DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA, to nuclei that exhibited both SSB and DSB. Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of inter-molecular disulphide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulphide bonds within their chromatin, but nevertheless, have been successfully cryopreserved. To examine the hypothesis that the cryoprotectants used for fish sperm cryopreservation will confer a similar degree of protection on koala spermatozoa, various concentrations of five cryoprotectants (dimethyl sulphoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Dimethylacetamide at a concentration of 12.5% (v/v) was found to be comparable to glycerol in the successful cryopreservation of koala spermatozoa although high inter-male variability was observed. However, when the new protocol was subsequently validated for a larger population of captive koalas (n = 22), glycerol emerged the better cryoprotectant with respect to all sperm viability parameters assessed except for that of the incidence of chromatin relaxation, which was not affected by the cryoprotectant. Significant difference was also observed in the post-thaw survival of spermatozoa from different animals, which was independent of pre-freeze semen quality. Based on post-thaw semen viability parameters, the koalas could be divided into two distinct groups, where one group had significantly higher sperm viability compared to the other group, regardless of cryoprotectant used. Positive correlation between motility and MMP was observed before and after cryopreservation. However, cryopreservation significantly reduced the dependency between these variables (P < 0.001), suggesting that cryopreservation reduced the dependency between mitochondrial function and motility.

The primary objective of the studies described in this thesis was to improve the cryopreservation success of koala spermatozoa for the purpose of establishing a genome resource bank for this species. A defining feature of the studies in this thesis was the implementation of an organelle-specific approach to better understand the causes of koala sperm cryo-injury. The functional attributes of spermatozoa, such as mitochondrial function, plasma membrane fluidity, membrane lipid asymmetry and DNA integrity were assessed as an indication of cryo-injury. Sperm mitochondrial function and plasma membrane integrity were examined by cryomicroscopy using the fluorescent probes JC-1 and propidium iodide (PI) respectively in a dual staining technique. Cooling and re-warming koala spermatozoa were more detrimental to mitochondrial function than to plasma membrane integrity. Mitochondrial membrane potential (MMP) was suppressed by freezing and thawing treatments; after thawing, MMP declined significantly during rewarming (from 5ºC to 35ºC). The distribution of GM1 ganglioside was examined using fluorescent-labelled cholera toxin B. No significant redistribution of GM1 was observed after chilling or cryotreatment. The externalisation of phosphatidylserine (PS) was examined using fluorescent-labelled annexin V. There was no significant increase in translocation of PS after chilling or cryopreservation. These observations imply that cryotreatment had little effect on plasma membrane lipid asymmetry. Koala spermatozoa were incubated in a range of anisotonic media to investigate whether nuclear swelling was caused by osmotic flux during the cryopreservation process. Although the most hypotonic solution tested (64 mOsm/kg) induced the highest incidence of nuclear relaxation (mean ± SEM; 12 ± 3%), this was not as severe as that previously documented following cryopreservation. Chromatin relaxation is a phenomenon observed in koala spermatozoa, where the sperm nucleus expands due to the result of structural changes in the natural conformation of the sperm DNA/protamine complex. DNA fragmentation was not a primary cause of cryopreservation-induced sperm chromatin relaxation, although in situ nick translation of putative DNA breaks indicated that these increased as the sperm head became progressively more relaxed. Using a Sperm Chromatin Dispersion test (SCDt) specifically developed and validated for koala spermatozoa, a continuum of nuclear morphotypes was observed, ranging from no apparent DNA fragmentation to spermatozoa with highly dispersed and degraded chromatin. A double comet assay was also developed to investigate DNA fragmentation in the koala spermatozoa. Conducted under neutral followed by alkaline conditions, this assay was able to differentiate between single- (SSB) and double-stranded (DSB) DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa; the majority of the koala spermatozoa had nuclei with DNA abasic-like residues. The ubiquity of these residues suggested that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with “true” DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA, to nuclei that exhibited both SSB and DSB. Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of inter-molecular disulphide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulphide bonds within their chromatin, but nevertheless, have been successfully cryopreserved. To examine the hypothesis that the cryoprotectants used for fish sperm cryopreservation will confer a similar degree of protection on koala spermatozoa, various concentrations of five cryoprotectants (dimethyl sulphoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Dimethylacetamide at a concentration of 12.5% (v/v) was found to be comparable to glycerol in the successful cryopreservation of koala spermatozoa although high inter-male variability was observed. However, when the new protocol was subsequently validated for a larger population of captive koalas (n = 22), glycerol emerged the better cryoprotectant with respect to all sperm viability parameters assessed except for that of the incidence of chromatin relaxation, which was not affected by the cryoprotectant. Significant difference was also observed in the post-thaw survival of spermatozoa from different animals, which was independent of pre-freeze semen quality. Based on post-thaw semen viability parameters, the koalas could be divided into two distinct groups, where one group had significantly higher sperm viability compared to the other group, regardless of cryoprotectant used. Positive correlation between motility and MMP was observed before and after cryopreservation. However, cryopreservation significantly reduced the dependency between these variables (P < 0.001), suggesting that cryopreservation reduced the dependency between mitochondrial function and motility.

The primary objective of the studies described in this thesis was to improve the cryopreservation success of koala spermatozoa for the purpose of establishing a genome resource bank for this species. A defining feature of the studies in this thesis was the implementation of an organelle-specific approach to better understand the causes of koala sperm cryo-injury. The functional attributes of spermatozoa, such as mitochondrial function, plasma membrane fluidity, membrane lipid asymmetry and DNA integrity were assessed as an indication of cryo-injury. Sperm mitochondrial function and plasma membrane integrity were examined by cryomicroscopy using the fluorescent probes JC-1 and propidium iodide (PI) respectively in a dual staining technique. Cooling and re-warming koala spermatozoa were more detrimental to mitochondrial function than to plasma membrane integrity. Mitochondrial membrane potential (MMP) was suppressed by freezing and thawing treatments; after thawing, MMP declined significantly during rewarming (from 5ºC to 35ºC). The distribution of GM1 ganglioside was examined using fluorescent-labelled cholera toxin B. No significant redistribution of GM1 was observed after chilling or cryotreatment. The externalisation of phosphatidylserine (PS) was examined using fluorescent-labelled annexin V. There was no significant increase in translocation of PS after chilling or cryopreservation. These observations imply that cryotreatment had little effect on plasma membrane lipid asymmetry. Koala spermatozoa were incubated in a range of anisotonic media to investigate whether nuclear swelling was caused by osmotic flux during the cryopreservation process. Although the most hypotonic solution tested (64 mOsm/kg) induced the highest incidence of nuclear relaxation (mean ± SEM; 12 ± 3%), this was not as severe as that previously documented following cryopreservation. Chromatin relaxation is a phenomenon observed in koala spermatozoa, where the sperm nucleus expands due to the result of structural changes in the natural conformation of the sperm DNA/protamine complex. DNA fragmentation was not a primary cause of cryopreservation-induced sperm chromatin relaxation, although in situ nick translation of putative DNA breaks indicated that these increased as the sperm head became progressively more relaxed. Using a Sperm Chromatin Dispersion test (SCDt) specifically developed and validated for koala spermatozoa, a continuum of nuclear morphotypes was observed, ranging from no apparent DNA fragmentation to spermatozoa with highly dispersed and degraded chromatin. A double comet assay was also developed to investigate DNA fragmentation in the koala spermatozoa. Conducted under neutral followed by alkaline conditions, this assay was able to differentiate between single- (SSB) and double-stranded (DSB) DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa; the majority of the koala spermatozoa had nuclei with DNA abasic-like residues. The ubiquity of these residues suggested that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with “true” DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA, to nuclei that exhibited both SSB and DSB. Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of inter-molecular disulphide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulphide bonds within their chromatin, but nevertheless, have been successfully cryopreserved. To examine the hypothesis that the cryoprotectants used for fish sperm cryopreservation will confer a similar degree of protection on koala spermatozoa, various concentrations of five cryoprotectants (dimethyl sulphoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Dimethylacetamide at a concentration of 12.5% (v/v) was found to be comparable to glycerol in the successful cryopreservation of koala spermatozoa although high inter-male variability was observed. However, when the new protocol was subsequently validated for a larger population of captive koalas (n = 22), glycerol emerged the better cryoprotectant with respect to all sperm viability parameters assessed except for that of the incidence of chromatin relaxation, which was not affected by the cryoprotectant. Significant difference was also observed in the post-thaw survival of spermatozoa from different animals, which was independent of pre-freeze semen quality. Based on post-thaw semen viability parameters, the koalas could be divided into two distinct groups, where one group had significantly higher sperm viability compared to the other group, regardless of cryoprotectant used. Positive correlation between motility and MMP was observed before and after cryopreservation. However, cryopreservation significantly reduced the dependency between these variables (P < 0.001), suggesting that cryopreservation reduced the dependency between mitochondrial function and motility.

Introdução: Doze a 29 % dos ejaculados tem viscosidade seminal aumentada. Uma das técnicas recomendadas para diminuir a viscosidade do sêmen consiste num processo físico em que se passa o sêmen, 4 vezes, por uma seringa de 10 mL com agulha 18G. Ao fim desse processo, ter-se-ia modificado o comportamento molecular das fibrilas protéicas do esperma, tornando-o mais liquefeito, sem dano aparente a morfologia ou motilidade dos espermatozoides. Atualmente, discute-se sobre a total inocuidade desta técnica, uma vez que ela poderia provocar aumento da fragmentação do DNA dos espermatozoides. A hiperviscosidade seminal, além de reduzir a concentração e a motilidade dos espermatozoides, pode afetar a capacitação e a reação acrossômica, podendo ter implicações na integridade do DNA e no desenvolvimento embrionário normal. Objetivo: Comparar a fragmentação do DNA de espermatozoides humanos em amostras com viscosidade diminuída, fisiológica e aumentada, e avaliar se o processo de expulsão do sêmen através da agulha e seringa altera significativamente as taxas de fragmentação do DNA dos espermatozoides. Método: Após a coleta e avaliação dos parâmetros seminais das amostras de sêmen, aquelas que estiverem com viscosidade aumentada passarão pelo processo de expulsão através da seringa de 10 mL com agulha 18G por 4 vezes, para diminuir a viscosidade seminal. Posteriormente, serão feitas análises da fragmentação do DNA dos espermatozoides através da técnica de TUNEL em todas as amostras. Resultados: A fragmentação do DNA entre amostras com viscosidade fisiológica, diminuída e aumentada não apresentou diferença estatisticamente significativa (P=0,857). A fragmentação do DNA aumentou significativamente (P=0,035) nas amostras com viscosidade aumentada após passar na seringa e agulha quando comparadas com as mesmas amostras antes de passar pela mesma. Conclusão: O processo de expulsão do sêmen através de seringa e agulha visando redução da viscosidade seminal aumenta a fragmentação do DNA no espermatozoide, devendo este processo ser substituído por um processo inócuo. / Background: Twelve to 29 % of the ejaculated have the semen viscosity increased. One of the techniques recommended to decrease the viscosity of semen is a physical process in which the semen passes four times through a 10mL syringe with a 18G needle. At the end of this process would have modified the molecular behavior of the protein fibrils of the sperm making it more liquefied, without apparent damage to the morphology or motility of spermatozoa. Currently, the debate is about the complete safety of this technique since it could result in increased DNA fragmentation of spermatozoa. The seminal viscosity moreover reduces the concentration and motility of sperm, can affect the capacitation and acrosome reaction which may have implications for DNA integrity and normal embryonic development. Objective: To compare the DNA fragmentation in human semen samples with reduced, physiological and increased viscosity and evaluate if the process of expulsion of semen through the needle and syringe significantly alter rates of sperm DNA fragmentation. Methods: After collecting and evaluating semen parameters of semen samples, those that are with increased viscosity will pass through the process of expulsion through the 10 mL syringe with a 18G needle four times to reduce the viscosity of semen. After that, analysis of spermatozoa DNA fragmentation through the TUNEL technique will be made in all samples. Results: The fragmentation of DNA between samples with physiological, reduced and increased viscosity is not statistically significant (P=0.857). The DNA fragmentation increased significantly (P=0.035) in samples with increased viscosity after passing the syringe and needle when compared with the same samples before going through the same. Conclusion: The process of expulsion of semen through a syringe and needle in order to reduce the semen viscosity increases the DNA fragmentation in sperm, and this shall be replaced by a harmless process.

Introdução: Doze a 29 % dos ejaculados tem viscosidade seminal aumentada. Uma das técnicas recomendadas para diminuir a viscosidade do sêmen consiste num processo físico em que se passa o sêmen, 4 vezes, por uma seringa de 10 mL com agulha 18G. Ao fim desse processo, ter-se-ia modificado o comportamento molecular das fibrilas protéicas do esperma, tornando-o mais liquefeito, sem dano aparente a morfologia ou motilidade dos espermatozoides. Atualmente, discute-se sobre a total inocuidade desta técnica, uma vez que ela poderia provocar aumento da fragmentação do DNA dos espermatozoides. A hiperviscosidade seminal, além de reduzir a concentração e a motilidade dos espermatozoides, pode afetar a capacitação e a reação acrossômica, podendo ter implicações na integridade do DNA e no desenvolvimento embrionário normal. Objetivo: Comparar a fragmentação do DNA de espermatozoides humanos em amostras com viscosidade diminuída, fisiológica e aumentada, e avaliar se o processo de expulsão do sêmen através da agulha e seringa altera significativamente as taxas de fragmentação do DNA dos espermatozoides. Método: Após a coleta e avaliação dos parâmetros seminais das amostras de sêmen, aquelas que estiverem com viscosidade aumentada passarão pelo processo de expulsão através da seringa de 10 mL com agulha 18G por 4 vezes, para diminuir a viscosidade seminal. Posteriormente, serão feitas análises da fragmentação do DNA dos espermatozoides através da técnica de TUNEL em todas as amostras. Resultados: A fragmentação do DNA entre amostras com viscosidade fisiológica, diminuída e aumentada não apresentou diferença estatisticamente significativa (P=0,857). A fragmentação do DNA aumentou significativamente (P=0,035) nas amostras com viscosidade aumentada após passar na seringa e agulha quando comparadas com as mesmas amostras antes de passar pela mesma. Conclusão: O processo de expulsão do sêmen através de seringa e agulha visando redução da viscosidade seminal aumenta a fragmentação do DNA no espermatozoide, devendo este processo ser substituído por um processo inócuo. / Background: Twelve to 29 % of the ejaculated have the semen viscosity increased. One of the techniques recommended to decrease the viscosity of semen is a physical process in which the semen passes four times through a 10mL syringe with a 18G needle. At the end of this process would have modified the molecular behavior of the protein fibrils of the sperm making it more liquefied, without apparent damage to the morphology or motility of spermatozoa. Currently, the debate is about the complete safety of this technique since it could result in increased DNA fragmentation of spermatozoa. The seminal viscosity moreover reduces the concentration and motility of sperm, can affect the capacitation and acrosome reaction which may have implications for DNA integrity and normal embryonic development. Objective: To compare the DNA fragmentation in human semen samples with reduced, physiological and increased viscosity and evaluate if the process of expulsion of semen through the needle and syringe significantly alter rates of sperm DNA fragmentation. Methods: After collecting and evaluating semen parameters of semen samples, those that are with increased viscosity will pass through the process of expulsion through the 10 mL syringe with a 18G needle four times to reduce the viscosity of semen. After that, analysis of spermatozoa DNA fragmentation through the TUNEL technique will be made in all samples. Results: The fragmentation of DNA between samples with physiological, reduced and increased viscosity is not statistically significant (P=0.857). The DNA fragmentation increased significantly (P=0.035) in samples with increased viscosity after passing the syringe and needle when compared with the same samples before going through the same. Conclusion: The process of expulsion of semen through a syringe and needle in order to reduce the semen viscosity increases the DNA fragmentation in sperm, and this shall be replaced by a harmless process.

Introdução: Doze a 29 % dos ejaculados tem viscosidade seminal aumentada. Uma das técnicas recomendadas para diminuir a viscosidade do sêmen consiste num processo físico em que se passa o sêmen, 4 vezes, por uma seringa de 10 mL com agulha 18G. Ao fim desse processo, ter-se-ia modificado o comportamento molecular das fibrilas protéicas do esperma, tornando-o mais liquefeito, sem dano aparente a morfologia ou motilidade dos espermatozoides. Atualmente, discute-se sobre a total inocuidade desta técnica, uma vez que ela poderia provocar aumento da fragmentação do DNA dos espermatozoides. A hiperviscosidade seminal, além de reduzir a concentração e a motilidade dos espermatozoides, pode afetar a capacitação e a reação acrossômica, podendo ter implicações na integridade do DNA e no desenvolvimento embrionário normal. Objetivo: Comparar a fragmentação do DNA de espermatozoides humanos em amostras com viscosidade diminuída, fisiológica e aumentada, e avaliar se o processo de expulsão do sêmen através da agulha e seringa altera significativamente as taxas de fragmentação do DNA dos espermatozoides. Método: Após a coleta e avaliação dos parâmetros seminais das amostras de sêmen, aquelas que estiverem com viscosidade aumentada passarão pelo processo de expulsão através da seringa de 10 mL com agulha 18G por 4 vezes, para diminuir a viscosidade seminal. Posteriormente, serão feitas análises da fragmentação do DNA dos espermatozoides através da técnica de TUNEL em todas as amostras. Resultados: A fragmentação do DNA entre amostras com viscosidade fisiológica, diminuída e aumentada não apresentou diferença estatisticamente significativa (P=0,857). A fragmentação do DNA aumentou significativamente (P=0,035) nas amostras com viscosidade aumentada após passar na seringa e agulha quando comparadas com as mesmas amostras antes de passar pela mesma. Conclusão: O processo de expulsão do sêmen através de seringa e agulha visando redução da viscosidade seminal aumenta a fragmentação do DNA no espermatozoide, devendo este processo ser substituído por um processo inócuo. / Background: Twelve to 29 % of the ejaculated have the semen viscosity increased. One of the techniques recommended to decrease the viscosity of semen is a physical process in which the semen passes four times through a 10mL syringe with a 18G needle. At the end of this process would have modified the molecular behavior of the protein fibrils of the sperm making it more liquefied, without apparent damage to the morphology or motility of spermatozoa. Currently, the debate is about the complete safety of this technique since it could result in increased DNA fragmentation of spermatozoa. The seminal viscosity moreover reduces the concentration and motility of sperm, can affect the capacitation and acrosome reaction which may have implications for DNA integrity and normal embryonic development. Objective: To compare the DNA fragmentation in human semen samples with reduced, physiological and increased viscosity and evaluate if the process of expulsion of semen through the needle and syringe significantly alter rates of sperm DNA fragmentation. Methods: After collecting and evaluating semen parameters of semen samples, those that are with increased viscosity will pass through the process of expulsion through the 10 mL syringe with a 18G needle four times to reduce the viscosity of semen. After that, analysis of spermatozoa DNA fragmentation through the TUNEL technique will be made in all samples. Results: The fragmentation of DNA between samples with physiological, reduced and increased viscosity is not statistically significant (P=0.857). The DNA fragmentation increased significantly (P=0.035) in samples with increased viscosity after passing the syringe and needle when compared with the same samples before going through the same. Conclusion: The process of expulsion of semen through a syringe and needle in order to reduce the semen viscosity increases the DNA fragmentation in sperm, and this shall be replaced by a harmless process.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Introduction: Mondia whitei commonly known as "White Ginger" is a highly acclaimed medicinal plant that is extensively used across Africa. M. whitei is used as treatment for sexual dysfunction and is considered to be an aphrodisiac by traditional medicine practitioners. Yet, scientific evidence to support these claims are minimal and those that are published possess ambiguity. To date, only one study reporting the in vitro effect of the aqueous rhizome extract of M. whitei on human sperm motility is available. Therefore, the aim of the study was to determine the in vitro effects of M. whitei in human sperm functions. Materials and Methods: Roots of Mondia whitei obtained from the tropical Kakamega rain forest, located in the Western Province of Kenya, were cleaned and chopped into smaller segments. These pieces were ovendried at 25℃ for 3 days and milled to form a powdery substance which was infused with hot (about 70℃) distilled water for 1 hour. After cooling and filtration, the extract was frozen at -20℃ and subsequently freeze-dried. The dried extract was then stored at 4℃ in a closed container until experimentation. A total of 60 semen samples were collected: 28 of them represented healthy sperm donors and 32 infertile patients. Among these subjects, oligozoospermic and asthenozoospermic semen samples were identified and analysed separately. Sperm were washed using human tubular fluid medium supplemented with bovine serum albumin (HTF-BSA) and incubated for 1 hour at 37℃ with different concentrations of M. whitei (0.0185, 0.185, 1.85, 18.5 and 185 μg/ml). A sample without M. whitei served as control. Sperm cell motility, vitality, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), capacitation, acrosome reaction and DNA fragmentation were assessed. Results: Total motility and the percentage of sperm with intact MMP showed significant dose-dependent increases in both groups (patient and donor), while, the percentages of progressively motile sperm only revealed significant increases in the patient group. Besides, the percentage of ROS-positive spermatozoa showed significant trend towards higher concentrations in the patient group only. Conversely, a trend towards reduced sperm DNA-fragmentation could be observed in the patient, but not the donor group. Similar tendencies were noted in oligozoospermic and asthenozoospermic, but not for normozoospermic subjects. Yet, sperm vitality, capacitation, acrosome reaction and kinematic parameters were not affected. Conclusions: Phytochemicals present in M. whitei root extract maintains spermatozoa total motility, progressive motility and intact-MMP and DNA integrity. However, at therapeutic concentration (<1.85 μg/ml) it does not trigger sperm intrinsic superoxide production nor increase ROS by causing oxidative stress, that leads to DNA fragmentation. / National Research Foundation (NRF)