WHO laboratory manual for the examination and processing of human semen

Stem cells are defined as cells able to both extensively self-renew and differentiate into progenitors. Research data show that more resistant stem cells than common cancer cells exist in cancer patients.To identify unrecognized differences between cancer stem cells and cancer cells might be able to develope effective classification,diagnose and treat ment for cancer.

Stem Cells,Cancer and Cancer Stem Cells

https://www.clevelandclinic.org/ReproductiveResearchCenter/docs/agradoc114.pdf

Role of reactive oxygen species in the pathophysiology of human reproduction

Oxidative stress occurs when the production of potentially destructive reactive oxygen species (ROS) exceeds the bodies own natural antioxidant defenses, resulting in cellular damage. Oxidative stress is a common pathology seen in approximately half of all infertile men. ROS, defined as including oxygen ions, free radicals and peroxides are generated by sperm and seminal leukocytes within semen and produce infertility by two key mechanisms. First, they damage the sperm membrane, decreasing sperm motility and its ability to fuse with the oocyte. Second, ROS can alter the sperm DNA, resulting in the passage of defective paternal DNA on to the conceptus. This review will provide an overview of oxidative biochemistry related to sperm health and will identify which men are most at risk of oxidative infertility. Finally, the review will outline methods available for diagnosing oxidative stress and the various treatments available.

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Oxidative stress and male infertility—a clinical perspective

Oxidative stress is involved in the aetiology of defective embryo development. Reactive oxygen species (ROS) may originate from embryo metabolism and/or embryo surroundings. Embryo metabolism generates ROS via several enzymatic mechanisms. The relative contribution of each source seems different depending on the species, the stage of development, and the culture conditions. Several exogenous factors and culture conditions can enhance the production of ROS by embryos. ROS can alter most types of cellular molecules, and also induce development block and retardation. Multiple mechanisms of embryo protection against ROS exist, and these have complementary actions. External protection, present in follicular and tubal fluids, mainly comprises non-enzymatic antioxidants such as hypotaurine, taurine and ascorbic acid. Internal protection mainly comprises antioxidant enzymes: superoxide dismutase, glutathione peroxidase and gamma-glutamylcysteine synthetase. Transcripts encoding for these enzymes are present in the oocyte, embryo and oviduct. It may be important that these transcripts are stored during oocyte maturation in order to allow the embryo to acquire the aptitude to develop. It is now common to add antioxidant compounds to culture media. Nevertheless, maintaining the pro-oxidant-antioxidant equilibrium in embryos through such supplementation is a complex problem. Further studies are necessary to limit oxidative stress during embryo culture.

Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings

Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men Semen samples were obtained from 17 fertile and 40 infertile men All samples were prepared by discontinuous Percoll density centrifugation (950:475) Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen Thawing was carried out slowly at room temperature Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men

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Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

The aim of this study was to determine the effects of supplementation with ascorbate and a-tocopherol, both singly and in combination, during sperm preparation on subsequent sperm DNA integrity, induced DNA damage and reactive oxygen species (ROS) generation. Semen samples with normozoospermic and asthenozoospermic profiles (n J 15 for each control and antioxidant group) were prepared by Percoll density centrifugation (95.0‐ 47.5%) where the medium had been supplemented with these antioxidants to a number of different concentrations, all within physiological levels. Controls were included which had no ascorbate or a-tocopherol added. DNA damage was induced using hydrogen peroxide (H 2O2) and DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay, while ROS generation was measured using chemiluminescence. Addition of ascorbate to sperm preparation medium did not affect baseline DNA integrity but did provide sperm with complete protection against H2O2-induced DNA damage. Generation of H2O2induced ROS was also significantly reduced after treatment with ascorbate, although baseline levels were unaffected by this antioxidant. Supplementation of sperm preparation medium with a-tocopherol did not influence baseline DNA integrity but provided sperm with dose-dependent protection against H2O2-induced DNA damage. Generation of H2O2-induced ROS was significantly reduced after treatment with a-tocopherol, although baseline ROS levels were unaffected by this antioxidant. Addition of both ascorbate and a-tocopherol in combination to sperm preparation medium actually induced DNA damage and intensified the damage induced by H2O2, however, H2O2-induced ROS production was significantly reduced in a dose-dependent manner by supplementation with both vitamins.

The effect of ascorbate and α-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa

https://www.fertstert.org/article/S0015-0282(98)00146-0/pdf

Eilish T. Donnelly

Papers

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa

Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

The effect of ascorbate and α-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa

In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity