■ Abstract Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process. The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among the death proteases. Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage via upstream proteases in an intracellular cascade. Regulation of caspase activation and activity occurs at several different levels: ( a) Zymogen gene transcription is regulated; ( b) antiapoptotic members of the Bcl-2 family and other cellular polypeptides block proximity-induced activation of certain procaspases; and ( c) certain cellular inhibitor of apoptosis proteins (cIAPs) can bind to and inhibit active caspases. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases. Collectively, these scissions disrupt survival pathways and disassemble important architectural components of the cell, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death.

Mammalian caspases: structure ,a ctivation ,s ubstrates, and functions during apoptosis

WHO laboratory manual for the examination and processing of human semen

Reactive oxygen species

In a healthy body, ROS (reactive oxygen species) and antioxidants remain in balance When the balance is disrupted towards an overabundance of ROS, oxidative stress (OS) occurs OS influences the entire reproductive lifespan of a woman and even thereafter (ie menopause) OS results from an imbalance between prooxidants (free radical species) and the body's scavenging ability (antioxidants) ROS are a double-edged sword – they serve as key signal molecules in physiological processes but also have a role in pathological processes involving the female reproductive tract ROS affect multiple physiological processes from oocyte maturation to fertilization, embryo development and pregnancy It has been suggested that OS modulates the age-related decline in fertility It plays a role during pregnancy and normal parturition and in initiation of preterm labor Most ovarian cancers appear in the surface epithelium, and repetitive ovulation has been thought to be a causative factor Ovulation-induced oxidative base damage and damage to DNA of the ovarian epithelium can be prevented by antioxidants There is growing literature on the effects of OS in female reproduction with involvement in the pathophsiology of preeclampsia, hydatidiform mole, free radical-induced birth defects and other situations such as abortions Numerous studies have shown that OS plays a role in the pathoysiology of infertility and assisted fertility There is some evidence of its role in endometriosis, tubal and peritoneal factor infertility and unexplained infertility This article reviews the role OS plays in normal cycling ovaries, follicular development and cyclical endometrial changes It also discusses OS-related female infertility and how it influences the outcomes of assisted reproductive techniques The review comprehensively explores the literature for evidence of the role of oxidative stress in conditions such as abortions, preeclampsia, hydatidiform mole, fetal embryopathies, preterm labour and preeclampsia and gestational diabetes The review also addresses the growing literature on the role of nitric oxide species in female reproduction The involvement of nitric oxide species in regulation of endometrial and ovarian function, etiopathogenesis of endometriosis, and maintenance of uterine quiescence, initiation of labour and ripening of cervix at parturition is discussed Complex interplay between cytokines and oxidative stress in the etiology of female reproductive disorders is discussed Oxidant status of the cell modulates angiogenesis, which is critical for follicular growth, corpus luteum formation endometrial differentiation and embryonic growth is also highlighted in the review Strategies to overcome oxidative stress and enhance fertility, both natural and assisted are delineated Early interventions being investigated for prevention of preeclampsia are enumerated Trials investigating combination intervention strategy of vitamin E and vitamin C supplementation in preventing preeclampsia are highlighted Antioxidants are powerful and there are few trials investigating antioxidant supplementation in female reproduction However, before clinicians recommend antioxidants, randomized controlled trials with sufficient power are necessary to prove the efficacy of antioxidant supplementation in disorders of female reproduction Serial measurement of oxidative stress biomarkers in longitudinal studies may help delineate the etiology of some of the diosorders in female reproduction such as preeclampsia

/pdf/role-of-oxidative-stress-in-female-reproduction-2fcw0uqz6p.pdf

Role of oxidative stress in female 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.

/pdf/oxidative-stress-and-male-infertility-a-clinical-perspective-2nu3kdi0jn.pdf

Oxidative stress and male infertility—a clinical perspective

The selection of vital, non-apoptotic spermatozoa is a prerequisite for achieving optimal conception rates in assisted reproductive techniques. Magnetic cell sorting using annexin-V microbeads can effectively separate apoptotic and non-apoptotic spermatozoa. The objective of the present study was to optimize the integration of magnetic cell sorting in standard sperm preparations and to correlate the effect of different sperm preparation procedures on apoptotic markers. Semen specimens collected from 15 healthy donors were prepared by either density gradient centrifugation or by one-step sperm wash technique separately and in combination with magnetic cell sorting. The preparation methods were evaluated by assessment of semen parameters (motility, viability and morphology) as well as markers of apoptosis (levels of active caspase-3, integrity of membrane mitochondrial potential and externalization of phosphatidylserine). The apoptotic markers were measured using fluorochrome dyes coupled with flow cytometry. The results showed that the combination of density gradient centrifugation and annexin-V magnetic cell sorting was superior to all other sperm preparation methods in terms of providing motile, viable and non-apoptotic spermatozoa. This study clearly shows the advantage of integrating magnetic cell sorting as a part of sperm preparation, which in turn may positively affect the success rates of assisted reproductive techniques.

https://www.rbmojournal.com/article/S1472-6483(10)61118-2/pdf

Advantage of combining magnetic cell separation with sperm preparation techniques

http://www.ccf.org/ReproductiveResearchCenter/docs/agradoc179.pdf

Infliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human spermatozoa: an in vitro model

https://www.clevelandclinic.org/reproductiveresearchcenter/docs/agradoc153.pdf

Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility.

Accurate assessment of reactive oxygen species (ROS) concentrations may help in the diagnosis of infertility. The chemiluminescence technique, which uses a luminometer to measure ROS, is a common method of assessment. A better understanding of the chemiluminescence technique will allow its proper application in reproductive medicine. A wide range of luminometers are available in the market, and laboratories should select the instruments that suit their individual needs.

https://clevelandclinic.org/reproductiveresearchcenter/docs/agradoc152.pdf

Chemiluminescence technique for measuring reactive oxygen species

Although the enhanced chemiluminescence assay is commonly used to measure the nonenzymatic total antioxidant capacity (TAC) of the human seminal plasma, it is cumbersome, expensive, and time-consuming. We describe herein an alternate method to measure TAC that is based on the ability of antioxidants in seminal plasma to interfere with a reaction between 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate] and metmyoglobin with H(2)O(2). This reaction produces a relatively stable blue-green color with absorbance maxima at 600 nm. We compared this colorimetric assay with our established chemiluminescence method and assessed quality control parameters (ie, intra-assay and interassay variabilities) in addition to intraobserver and interobserver differences. Our results show that the colorimetric assay was fairly predictive of antioxidant capacity similar to the chemiluminescence assay (P <.001). Furthermore, there was a high level of agreement between the duplicate measures by the same observer (intraobserver) and intra-assay variability, with a concordance correlation coefficient of 0.99. The interassay coefficient of variation was 4.7% (overall). The mean +/- SD of the difference between the 2 observers was 2.98% +/- 4.1%. In conclusion, we found that the colorimetric assay is a reliable and accurate method to evaluate seminal TAC, and it could be used as a simpler, rapid, and cheaper alternative to the chemiluminescence assay.

/pdf/enhanced-chemiluminescence-assay-vs-colorimetric-assay-for-35w870nvsm.pdf

Tamer M. Said

Papers

Advantage of combining magnetic cell separation with sperm preparation techniques

Infliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human spermatozoa: an in vitro model

Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility.

Chemiluminescence technique for measuring reactive oxygen species

Enhanced chemiluminescence assay vs colorimetric assay for measurement of the total antioxidant capacity of human seminal plasma