Shedding light on the ART laboratory.

This commentary examines the impact of light conditions in the assisted reproductive technology (ART) laboratory, specifically considering gametes and embryo culture. While these processes traditionally occur in the absence of light within the female reproductive tract, laboratory conditions often involve exposure to varying wavelengths, intensities and light sources. Although literature reports describe potential detrimental effects of certain wavelengths of light on biological material, these findings are often based on experiments that might not reflect actual laboratory conditions. Current ART laboratory practices aim to minimize light exposure; however, some procedures necessitate light exposure, typically involving microscopy. Results from the authors' cross-sectional survey on light-intensity practices in ART laboratories revealed the frequent use of inadequate lighting, leading to errors and impacting staff well-being. A failure mode and effects analysis was used to identify potential failure modes and their impacts due to poor lighting. Overall, this manuscript stresses the importance of maintaining proper ambient lighting in the ART laboratory, balancing the potentially detrimental effects of light on gametes and embryos against the need for proper lighting for accurate procedures and staff well-being. Adequate lighting not only ensures the safety of reproductive cells, but also improves process management and the operators' psychological conditions.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

A technical note on the assessment of human sperm vitality using eosin-nigrosin staining.

RESEARCH QUESTION: How many spermatozoa and slides need to be counted to make a reliable assessment of sperm vitality? Currently, various authorities recommend assessing human sperm vitality on counts of at least 200 cells, but on one or two slides. DESIGN: This was an observational study on duplicate eosin-nigrosin stained sperm vitality slides made from 58 ejaculates. Assessments were made using counts of 2 â€¯×  100 and 1 â€¯×  200 cells per slide, all performed by the same trained expert observer. RESULTS: Although assessments tend to show fewer and smaller outlier values when based on counts of 200 spermatozoa than 100, and on 2 â€¯×  200 than 1 â€¯×  200, counting 200 spermatozoa from one slide provides a result with sufficient accuracy for the clinical purpose of ascertaining whether the immotile spermatozoa in an ejaculate showing low sperm motility are alive or dead. CONCLUSIONS: While the increased accuracy of results derived from counts of 2 â€¯×  200 cells might be important in research studies where sperm vitality is the specific end-point of interest, counting at least 200 spermatozoa from one smear is sufficiently accurate for the clinical purpose of establishing whether the immotile spermatozoa seen in ejaculates with low sperm motility are alive or not. Consequently, the extra workload of performing replicate counts and the associated calculations does not increase the clinical value of the result, and hence is unnecessary in routine semen analysis. Careful laboratory technique as well as proper staff training and competence are essential. The conclusions might not be applicable to staining methods other than the recommended one-step eosin-nigrosin technique.

Male reproductive health statement (XIIIth international symposium on Spermatology, may 9th-12th 2018, Stockholm, Sweden.

On the occasion of the XIIIth International Symposium on Spermatology held from 9 to 13 May 2018 in Stockholm (Sweden), participants (guest speakers and audience) collectively felt the need to make a public statement on the general issue of male reproductive health. Our intention is to raise awareness of what we believe is a neglected area of research despite alarming situations around the world. The disclosure strategy desired by the co-authors is to bring it to the attention of the greatest number partly by considering co-publication in the various periodicals dealing with Reproductive Biology and Andrology. BaCA's editorial office accepted this mission and found it natural that our periodical, the official journal of the French Andrology Society (SALF), should carry this message.

A l'occasion du XIII eme Symposium international sur la Spermatologie qui s'est. tenu du 9 au 13 Mai 2018 à Stockholm (Suède), les participants (orateurs invités et l'auditoire) ont ressenti collectivement le besoin de faire une déclaration publique sur la question générale de la santé reproductive masculine. Notre intention est. de mieux faire connaître ce que nous pensons être un domaine de recherche négligé malgré des situations alarmantes dans le monde entier. La stratégie de divulgation souhaitée par les co-auteurs est. de le porter à l'attention du plus grand nombre en envisageant pour partie une co-publication dans les différents périodiques traitant de Reproduction et d'Andrologie. Le bureau éditorial de BaCA, a accepté cette mission et a trouvé naturel que notre périodique, journal officiel de la Société d'Andrologie en Langue Française (SALF) porte ce message.

The functional anatomy of the human spermatozoon: relating ultrastructure and function.

The Internet, magazine articles, and even biomedical journal articles, are full of cartoons of spermatozoa that bear minimal resemblance to real spermatozoa, especially human spermatozoa, and this had led to many misconceptions about what spermatozoa look like and how they are constituted. This review summarizes the historical and current state of knowledge of mammalian sperm ultrastructure, with particular emphasis on and relevance to human spermatozoa, combining information obtained from a variety of electron microscopic (EM) techniques. Available information on the composition and configuration of the various ultrastructural components of the spermatozoon has been related to their mechanistic purpose and roles in the primary aspects of sperm function and fertilization: motility, hyperactivation, capacitation, the acrosome reaction and sperm-oocyte fusion.

UK dietary exposure to PCDD/Fs, PCBs, PBDD/Fs, PBBs and PBDEs: comparison of results from 24-h duplicate diets and total diet studies.

Chemicals in food are monitored to check for compliance with regulatory limits and to evaluate trends in dietary exposures, among other reasons. This study compared two different methods for estimating human dietary exposure to lipophilic persistent organic pollutants (POPs) during 2011/12: (1) the 2012 Total Diet Study (TDS) conducted by the UK Food Standards Agency (FSA) and (2) a 24-h duplicate diet (DD) study of 20 adults from the North East of England. The equivalence of the two approaches was assessed; anything less than an order of magnitude could be considered reasonable and within three-fold (equivalent to 0.5 log) as good. Adult dietary exposure estimates derived from the DD study for both average and high-level (97.5th percentile) consumers compared well with those from the TDS. Estimates from the DD study when compared with those from the TDS were within 10% for P97.5 for total PCDD/F/PCB with divergence increasing to a factor of 3.4 for average BDE-209. Most estimates derived from the TDS were slightly higher than those derived from the DD. Comparison with earlier UK TDS data over the last 30 years or so confirmed a gradual decline in levels of PCDD/F/PCBs in food. Such comparisons also indicated peaks in dietary exposure to ∑PBDE (excluding BDE-209) between 2000 and 2005. Exposure estimates for all measured compounds using both TDS and DD data were found to be within recommended tolerable daily intakes where available or within acceptable margins of exposure.

'How to count sperm properly': checklist for acceptability of studies based on human semen analysis.

STUDY QUESTION: Can a tool be developed for authors, reviewers and editors of the ESHRE Journals to improve the quality of published studies which rely on semen analysis data? SUMMARY ANSWER: A basic checklist for authors, reviewers and editors has been developed and is presented. WHAT IS KNOWN ALREADY: Laboratory work which includes semen analysis is burdened by a lack of standardization. This has significant negative effects on the quality of scientific and epidemiological studies, potential misclassification of patients and the potential to impair clinical treatments/diagnoses that rely on accurate semen quality information. Robust methods are available to reduce laboratory error in semen analysis, inducing adherence to World Health Organization techniques, participation in an external quality control scheme and appropriate training of laboratory personnel. However, journals have not had appropriate systems to assess if these methods have been used. STUDY DESIGN, SIZE, DURATION: After discussion at a series of Associate Editor Meetings of the ESHRE Journals the authors of the present text were asked to propose a tool for authors, reviewers and editors of the ESHRE Journals to ensure a high quality assessment of submitted manuscripts which rely on semen analysis data, including a detailed verification of the relevance and the quality of the methods used. PARTICIPANTS/MATERIALS, SETTING, METHODS: N/A. MAIN RESULTS AND THE ROLE OF CHANCE: A basic checklist for authors, reviewers and editors is presented. The checklist contains key points which should be considered by authors when designing studies and which provides essential information for when the submitted manuscript is evaluated. For published articles the answers in the checklist are suitable to be available as supplementary data, which will also reduce the space necessary for technical details in the printed article. LIMITATIONS, REASONS FOR CAUTION: Guidelines such as these should not be used uncritically. It is therefore important that submitting authors, in situations where their study does not comply with the basic requirements for semen analysis, not only explain all methodological deviations but also declare the level of uncertainty in their analyses and how it complies with, or might confound, the aims of the study. WIDER IMPLICATIONS OF THE FINDINGS: The fundamental importance of appropriate and robust methodology to facilitate advances in scientific understanding and patient management and treatment, is now accepted as being paramount. Use of the semen analysis checklist should be part of this process, and when completed and signed by the corresponding author at the time of submitting a manuscript should result in greater transparency, and ultimately uniformity. It is hoped that this initiative will pave the way for wider adoption of the methodology/reporting by other biomedical, epidemiological and scientific journals, and ultimately become the standard of practice for papers reporting semen analysis results obtained in laboratory and clinical andrology. Systems to assist referees, authors and editors to present high quality findings should have a significant impact on the field of reproductive medicine. STUDY FUNDING/COMPETING INTERESTS: No funding was obtained for this work. The authors have no competing interests in relation to the present publication and checklist. TRIAL REGISTRATION NUMBER: N/A.

The future of computer-aided sperm analysis.

Computer-aided sperm analysis (CASA) technology was developed in the late 1980s for analyzing sperm movement characteristics or kinematics and has been highly successful in enabling this field of research. CASA has also been used with great success for measuring semen characteristics such as sperm concentration and proportions of progressive motility in many animal species, including wide application in domesticated animal production laboratories and reproductive toxicology. However, attempts to use CASA for human clinical semen analysis have largely met with poor success due to the inherent difficulties presented by many human semen samples caused by sperm clumping and heavy background debris that, until now, have precluded accurate digital image analysis. The authors review the improved capabilities of two modern CASA platforms (Hamilton Thorne CASA-II and Microptic SCA6) and consider their current and future applications with particular reference to directing our focus towards using this technology to assess functional rather than simple descriptive characteristics of spermatozoa. Specific requirements for validating CASA technology as a semi-automated system for human semen analysis are also provided, with particular reference to the accuracy and uncertainty of measurement expected of a robust medical laboratory test for implementation in clinical laboratories operating according to modern accreditation standards.

The effects of flooding on dioxin and PCB levels in food produced on industrial river catchments.

This research examined the effect of regular flooding upon PCDD/F and PCB levels in milk, beef and lamb, produced on the floodplains of industrial river catchments. Our unique dataset included more than 200 samples analysed for PCDD/Fs and PCBs over two data collection phases (1998-1999 & 2008-2010) from working farms. A robust paired study design was adopted with samples taken from flood-prone farms and nearby control farms not subject to flooding. On industrial river catchments regular flooding is associated with higher PCDD/F and PCB levels in soils and grass. This contamination may be transferred to food but the impact varied by food type. These contrasts may be due to physiological differences between animals, the ages at which they are sent to market and differences in animal husbandry. To minimise the risks of producing food on flood-prone land in industrial river catchments, as well as on any land with elevated PCDD/F and PCB levels, this research suggests a number of options. The choice of livestock may be important and as an example in our study beef cattle accumulated PCDD/Fs to a higher degree than sheep. Land management may also play a role and could include minimising the time that livestock spend on such land or feeding commercial feed, low in PCDD/Fs and PCBs, where appropriate.

Investigation into the formation of PAHs in foods prepared in the home to determine the effects of frying, grilling, barbecuing, toasting and roasting.

The effects of frying, grilling, barbecuing, toasting and roasting on the formation of 27 different PAHs in foods were investigated. A total of 256 samples from in-house cooking experiments were produced. There was little evidence of PAH formation during the grilling, frying, roasting and toasting experiments. Comparison with the raw materials used in the experiments showed little or no increase in PAH concentrations for all of the sample types, regardless of distances from the heat source, cooking mediums and intensity of cooking conditions. Barbecuing with charcoal plus wood chips however resulted in the formation of benzo[a]pyrene in most foods; for beef burgers only, barbecuing over charcoal (without the use of wood chips) gave the highest levels. In general PAH levels increased when the food was barbequed closer to the heat source. For sausages cooked over briquettes, and for beef burgers, beef and salmon cooked over charcoal, the concentration of PAHs was lower when the food was closer to the heat source. Cooking time may result in a moderate increase of PAHs in some foods, although concentrations in beef burgers appeared to fall when cooking time was extended by 50-100%.

Contamination of fish in UK fresh water systems: risk assessment for human consumption.

There is growing evidence that more people in the UK are consuming fish taken from inland waterways. This may be partly due to the increased numbers of migrants from Eastern Europe where this is part of traditional culture and partly because of a desire to try new foods encouraged by celebrity chefs. Fish can bioaccumulate environmental contaminants and so could contribute a significant amount to dietary exposure to these chemicals. This study examined the changing habits of anglers and consumers and characterised a range of existing and emerging contaminants in freshwater fish species with a view to determining current levels of occurrence and possible risk from consumption. The project was conducted in two stages. The first stage included (a) a study that identified freshwater systems that are contaminated either by anthropogenic activity or as a result of the geology of the area; and (b) socioeconomic research to assess the consumption habits of the public, particularly anglers, with respect to fish and shellfish from unmanaged inland waterways. Based on the outcome from the first stage, specific rivers and other inland waterways were chosen for investigation, along with the range of contaminants to be included in the analytical programme. Predicted contamination levels and prevalence of anglers were among the factors taken into consideration. The second stage of the project involved sampling and analysis of fish taken from selected locations on the chosen waterways. A range of fish species from a variety of inland water habitats were obtained. These were analysed for the following contaminants: heavy metals, chlorinated dioxins (PCDD/Fs), polybrominated biphenyls (PBBs), polychlorinated biphenyls (PCBs), brominated dioxins (PBDD/Fs), polychlorinated naphthalenes (PCNs), polybrominated diphenylethers (PBDEs), OC pesticides, organotin compounds and organo-fluorine compounds. Legal limits for contaminants apply only to food traded commercially, but some samples were in excess of the regulatory limits for PCDD/Fs and PCBs in such fish. The maximum detected WHO-TEQ (1998) for PCDD/Fs plus PCBs was over 32ngkg(-1) on a whole weight basis for a sample of barbel from the River Don, and 6 other samples were also above the 8ngkg(-1) limit.

Mixed halogenated dioxins/furans (PXDD/Fs) and biphenyls (PXBs) in food: occurrence and toxic equivalent exposure using specific relative potencies.

The occurrence of nineteen mixed halogenated (bromo-chloro) dibenzo-p-dioxins, dibenzofurans (PXDD/Fs) and biphenyls (PXBs) in a range of foods (n>100) was investigated. The analytical methodology used dual activated carbon column fractionation with high resolution mass spectrometric measurement (13,500-15,000 res). Occurrence was observed in most commonly consumed foods but the most frequent detections of these environmental contaminants were made in shellfish and offal. The concentrations of the individual compounds were condensed into toxic equivalents (TEQs) using recently reported relative potency values. Although representing only a small subset of the full range of toxic PXDD/Fs and PXBs, the TEQs estimated for these compounds ranged from 0.2% to approximately 15% (depending on the food matrix) of the corresponding TEQ for the fully chlorinated analogues. This finding is of great toxicological importance as it implies that a potentially greater magnitude of TEQ could be associated with the full range of toxic PXDD/Fs and PXBs, thus making a significant contribution to dioxin-like toxicity from the diet, to human exposure.

Investigation into the occurrence in food of veterinary medicines, pharmaceuticals, and chemicals used in personal care products.

Human exposure to emerging contaminants by indirect routes is of increasing interest. This study assessed the contamination of food by chemicals used in human pharmaceuticals (HPs), veterinary medicines (VMs), and personal care products (PCPs). A prioritization study was undertaken to identify the chemicals and food-producing scenarios most likely to result in contamination of food. Around 400 samples of mushrooms, vegetables, aquaculture products, and animal tissues were collected from sites in the United Kingdom, along with aquaculture products imported from Southeast Asia. A number of multianalyte methods were developed and validated for the analysis of the prioritized compounds in these samples. The analysis of all sample-method combinations required approximately 18000 determinations. Around 325 individual residues, including parabens, musk compounds, and antibiotics, were detected in 118 individual samples, but mostly at low nanograms per gram concentrations. Results suggest that the limited contamination of target chemicals occurred in the realistic food-producing scenarios investigated.

The effects of river flooding on dioxin and PCBs in beef.

In 2008-2010, samples of meat from 40 beef cattle, along with grass, soil and commercial feed, taken from ten matched pairs of flood-prone and control farms, were analysed for PCDD/Fs and PCBs. Concentrations were higher in soil and grass from flood-prone farms. The beef samples from flood-prone farms had total TEQ levels about 20% higher than on control farms. A majority of flood-prone farms (7/10) had higher median levels in beef than on the corresponding control farm. This first controlled investigation into PCDD/F and PCB contamination in beef produced on flood-prone land, presents robust evidence that flooding is a contaminant transfer mechanism to cattle raised on river catchments with a history of urbanisation and industrialisation. PCDD/F and PCB sources in these river systems are likely to be a result of the legacy of contamination from previous industrialisation, as well as more recent combustion activity or pollution events.

Exposure to phthalic acid, phthalate diesters and phthalate monoesters from foodstuffs: UK total diet study results.

Phthalates are ubiquitous in the environment and thus exposure to these compounds can occur in various forms. Foods are one source of such exposure. There are only a limited number of studies that describe the levels of phthalates (diesters, monoesters and phthalic acid) in foods and assess the exposure from this source. In this study the levels of selected phthalate diesters, phthalate monoesters and phthalic acid in total diet study (TDS) samples are determined and the resulting exposure estimated. The methodology for the determination of phthalic acid and nine phthalate monoesters (mono-isopropyl phthalate, mono-n-butyl phthalate, mono-isobutyl phthalate, mono-benzyl phthalate, mono-cyclohexyl phthalate, mono-n-pentyl phthalate, mono-(2-ethylhexyl) phthalate, mono-n-octyl phthalate and mono-isononyl phthalate) in foods is described. In this method phthalate monoesters and phthalic acid are extracted from the foodstuffs with a mixture of acidified acetonitrile and dichloromethane. The method uses isotope-labelled phthalic acid and phthalate monoester internal standards and is appropriate for quantitative determination in the concentration range of 5-100 µg kg⁻¹. The method was validated in-house and its broad applicability demonstrated by the analysis of high-fat, high-carbohydrate and high-protein foodstuffs as well as combinations of all three major food constituents. The methodology used for 15 major phthalate diesters has been reported elsewhere. Phthalic acid was the most prevalent phthalate, being detected in 17 food groups. The highest concentration measured was di-(2-ethylhexyl) phthalate in fish (789 µg kg⁻¹). Low levels of mono-n-butyl phthalate and mono-(2-ethylhexyl) phthalate were detected in several of the TDS animal-based food groups and the highest concentrations measured corresponded with the most abundant diesters (di-n-butyl phthalate and di-(2-ethylhexyl) phthalate). The UK Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) considered the levels found and concluded that they did not indicate a risk to human health from dietary exposure alone.

What should it take to describe a substance or product as 'sperm-safe'.

BACKGROUND: Male reproductive potential continues to be adversely affected by many environmental, industrial and pharmaceutical toxins. Pre-emptive testing for reproductive toxicological (side-)effects remains limited, or even non-existent. Many products that come into direct contact with spermatozoa lack adequate testing for the absence of adverse effects, and numerous products that are intended for exposure to spermatozoa have only a general assumption of safety based on the absence of evidence of actual harm. Such assumptions can have unfortunate adverse impacts on at-risk individuals (e.g. couples who are trying to conceive), illustrating a clear need for appropriate up-front testing to establish actual 'sperm safety'. METHODS: After compiling a list of general areas within the review's scope, relevant literature and other information was obtained from the authors' personal professional libraries and archives, and supplemented as necessary using PubMed and Google searches. Review by co-authors identified and eliminated errors of omission or bias. RESULTS: This review provides an overview of the broad range of substances, materials and products that can affect male fertility, especially through sperm fertilizing ability, along with a discussion of practical methods and bioassays for their evaluation. It is concluded that products can only be claimed to be 'sperm-safe' after performing objective, properly designed experimental studies; extrapolation from supposed predicate products or other assumptions cannot be trusted. CONCLUSIONS: We call for adopting the precautionary principle, especially when exposure to a product might affect not only a couple's fertility potential but also the health of resulting offspring and perhaps future generations.

Determination of phthalate diesters in foods.

Methodology for the determination of 15 phthalate diesters (dimethyl phthalate, diethyl phthalate, diisopropyl phthalate, diallyl phthalate, diisobutyl phthalate, di-n-butyl phthalate, di-n-pentyl phthalate, di-n-hexyl phthalate, benzyl butyl phthalate, dicyclohexyl phthalate, di-(2-ethylhexyl) phthalate, di-n-octyl phthalate, diisononyl phthalate, diisodecyl phthalate, and di-n-decyl phthalate) is described. The method was validated in-house and its broad applicability demonstrated by the analysis of high-fat, high-carbohydrate and high-protein foodstuffs as well as combinations of all three major food constituents. Following on from the analysis of the 20 UK Total Diet Study samples, 261 foodstuffs were purchased and tested for their phthalate levels. Phthalate diesters were confirmed to be present in 77 samples. Di-(2-ethylhexyl) phthalate was the most frequently detected (66 samples), although the highest levels found were for the isomeric mixture diisononyl phthalate. Additional studies confirmed that, for some foodstuffs, packaging materials did contribute to the phthalate diester concentration in the foodstuff and one example is presented.

Manual methods for sperm motility assessment.

Progressive motility is a vital functional characteristic of ejaculated human spermatozoa that governs their ability to penetrate into, and migrate through, both cervical mucus and the oocyte vestments, and ultimately fertilize the oocyte. A detailed protocol, based on traditional manual/visual methods, is provided for performing an accurate four-category differential count including the reliable identification of rapid progressive (grade "a") spermatozoa-the most biologically, and hence clinically, important subpopulation. Thorough prior training and the use of a microscope fitted with a heated stage are both essential requirements for achieving accuracy and an acceptable uncertainty of measurement of no more than ±10%.

Computer-Aided Sperm Analysis (CASA) of sperm motility and hyperactivation.

Progressive motility is a vital functional characteristic of ejaculated human spermatozoa that governs their ability to penetrate into, and migrate through, both cervical mucus and the oocyte vestments, and ultimately fertilize the oocyte. A detailed protocol, based on the most common computer-aided sperm analysis (CASA) system with phase contrast microscope optics, is provided for performing reliable assessments of sperm movement pattern characteristics ("kinematics") in semen. The protocol can also be used with washed sperm suspensions where, in addition, the percentages of motile and progressively motile spermatozoa can also be derived. Using CASA technology it is also possible to identify biologically, and hence clinically, important subpopulations of spermatozoa (e.g., those in semen with good mucus-penetrating characteristics, or those showing hyperactivation when incubated under capacitating conditions) by applying multi-parametric definitions on a cell-by-cell basis.