WHO laboratory manual for the examination and processing of human semen

The Endocrine Society's first Scientific Statement in 2009 provided a wake-up call to the scientific community about how environmental endocrine-disrupting chemicals (EDCs) affect health and disease. Five years later, a substantially larger body of literature has solidified our understanding of plausible mechanisms underlying EDC actions and how exposures in animals and humans-especially during development-may lay the foundations for disease later in life. At this point in history, we have much stronger knowledge about how EDCs alter gene-environment interactions via physiological, cellular, molecular, and epigenetic changes, thereby producing effects in exposed individuals as well as their descendants. Causal links between exposure and manifestation of disease are substantiated by experimental animal models and are consistent with correlative epidemiological data in humans. There are several caveats because differences in how experimental animal work is conducted can lead to difficulties in drawing broad conclusions, and we must continue to be cautious about inferring causality in humans. In this second Scientific Statement, we reviewed the literature on a subset of topics for which the translational evidence is strongest: 1) obesity and diabetes; 2) female reproduction; 3) male reproduction; 4) hormone-sensitive cancers in females; 5) prostate; 6) thyroid; and 7) neurodevelopment and neuroendocrine systems. Our inclusion criteria for studies were those conducted predominantly in the past 5 years deemed to be of high quality based on appropriate negative and positive control groups or populations, adequate sample size and experimental design, and mammalian animal studies with exposure levels in a range that was relevant to humans. We also focused on studies using the developmental origins of health and disease model. No report was excluded based on a positive or negative effect of the EDC exposure. The bulk of the results across the board strengthen the evidence for endocrine health-related actions of EDCs. Based on this much more complete understanding of the endocrine principles by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability, these findings can be much better translated to human health. Armed with this information, researchers, physicians, and other healthcare providers can guide regulators and policymakers as they make responsible decisions.

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EDC-2: The Endocrine Society's Second Scientific Statement on Endocrine-Disrupting Chemicals

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

Role of reactive oxygen species in the pathophysiology of human reproduction

Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems.

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Genome Sequence of the Pea Aphid Acyrthosiphon pisum

Oxidative stress (OS), a state characterized by an imbalance between pro-oxidant molecules including reactive oxygen and nitrogen species, and antioxidant defenses, has been identified to play a key role in the pathogenesis of subfertility in both males and females. The adverse effects of OS on sperm quality and functions have been well documented. In females, on the other hand, the impact of OS on oocytes and reproductive functions remains unclear. This imbalance between pro-oxidants and antioxidants can lead to a number of reproductive diseases such as endometriosis, polycystic ovary syndrome (PCOS), and unexplained infertility. Pregnancy complications such as spontaneous abortion, recurrent pregnancy loss, and preeclampsia, can also develop in response to OS. Studies have shown that extremes of body weight and lifestyle factors such as cigarette smoking, alcohol use, and recreational drug use can promote excess free radical production, which could affect fertility. Exposures to environmental pollutants are of increasing concern, as they too have been found to trigger oxidative states, possibly contributing to female infertility. This article will review the currently available literature on the roles of reactive species and OS in both normal and abnormal reproductive physiological processes. Antioxidant supplementation may be effective in controlling the production of ROS and continues to be explored as a potential strategy to overcome reproductive disorders associated with infertility. However, investigations conducted to date have been through animal or in vitro studies, which have produced largely conflicting results. The impact of OS on assisted reproductive techniques (ART) will be addressed, in addition to the possible benefits of antioxidant supplementation of ART culture media to increase the likelihood for ART success. Future randomized controlled clinical trials on humans are necessary to elucidate the precise mechanisms through which OS affects female reproductive abilities, and will facilitate further explorations of the possible benefits of antioxidants to treat infertility.

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The effects of oxidative stress on female reproduction: a review

The molecular basis of many forms of male infertility is poorly defined. One area of research that has been studied intensely is the integrity of the DNA in the nucleus of mature ejaculated spermatozoa. It has been shown that, in men with abnormal sperm parameters, the DNA is more likely to possess strand breaks. However, how and why this DNA damage originates in certain males and how it may influence the genetic project of a mature spermatozoon is unknown. Two theories have been proposed to describe the origin of this DNA damage in mature spermatozoa. The first arises from studies performed in animal models and is linked to the unique manner in which mammalian sperm chromatin is packaged, while the second attributes the nuclear DNA damage in mature spermatozoa to apoptosis. One of the factors implicated in sperm apoptosis is the cell surface protein, Fas. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa, how these spermatozoa arrive in the ejaculate of some men, and what consequences they may have if they succeed in their genetic project.

Origin of dna damage in ejaculated human spermatozoa

Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.

Nature of DNA Damage in Ejaculated Human Spermatozoa and the Possible Involvement of Apoptosis

BACKGROUND: The relationship between early embryo post-implantation development in couples undergoing assisted reproductive techniques (ARTs) and sperm chromatin alterations has not been satisfactorily explained. The aim of this study was to assess the relationship between sperm DNA fragmentation in IVF/ICSI patients, sperm parameters (concentration, motility and morphology) and ART outcome, especially with regard to clinical pregnancy and pregnancy loss (spontaneous miscarriage or biochemical pregnancy). METHODS: DNA fragmentation was evaluated by TUNEL assay, performed on sperm suspensions after density gradient separation, in 132 men undergoing an ART cycle (82 IVF and 50 ICSI) and correlated with sperm parameters and ART outcome. RESULTS: A highly significant negative correlation was found between DNA fragmentation and sperm parameters. There was a close relationship between DNA fragmentation and post-implantation development in ICSI patients: the clinical pregnancy and pregnancy loss rates significantly differed between patients with high and low sperm DNA fragmentation (P = 0.007 and P = 0.009, respectively). CONCLUSIONS: Sperm DNA fragmentation seems to affect embryo post-implantation development in ICSI procedures: high sperm DNA fragmentation can compromise 'embryo viability', resulting in pregnancy loss.

Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART

In this study we investigated whether morphology and chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We examined unfertilized oocytes, using the fluorochrome Hoechst 33342, to determine whether a relationship exists between failure of fertilization and sperm chromatin quality. Sperm chromatin packaging quality was assessed using the chromomycin A3 (CMA3) fluorochrome, and the presence of DNA damage in spermatozoa, using in-situ nick translation, Normal males present sperm parameters with a normal morphology of > 20%, CMA3 fluorescence of < 30% and exhibit endogenous nicks in < 10% of their spermatozoa. When patients were separated according to these values no difference was observed in their fertilization rates after ICSI. When the unfertilized ICSI oocytes were examined, we found that patients with CMA3 fluorescence of <30% and nicks in < 10% of their spermatozoa had only 17.5 and 21.6% respectively of their unfertilized oocytes containing spermatozoa that remained condensed. In contrast, patients with higher CMA3 and nick values had a significantly higher number, 41.2 and 48.9%, of their unfertilized oocytes containing condensed spermatozoa. sperm morphology did not show any such pattern. The percentage of spermatozoa which had initiated decondensation in unfertilized oocytes was not influenced by morphology, CMA3 fluorescence or nicks. In light of these results we postulate that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation after ICSI and result in failure of fertilization.

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Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection

During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.

Davide Bizzaro

Papers

Origin of dna damage in ejaculated human spermatozoa

Nature of DNA Damage in Ejaculated Human Spermatozoa and the Possible Involvement of Apoptosis

Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART

Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection

Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility.