World Health Organization Manual Research Articles

Leukocytospermia and/or Bacteriospermia: Impact on Male Infertility.

Infertility is a globally underestimated public health concern affecting almost 190 million people, i.e., about 17.5% of people during their lifetime, while the prevalence of male factor infertility is about 7%. Among numerous other causes, the prevalence of male genital tract infections reportedly ranges between 10% and 35%. Leukocytospermia is found in 30% of infertile men and up to 20% in fertile men. Bacterial infections cause an inflammatory response attracting leukocytes, which produce reactive oxygen species (ROS) and release cytokines, both of which can cause damage to sperm, rendering them dysfunctional. Although leukocytospermia and bacteriospermia are both clinical conditions that can negatively affect male fertility, there is still debate about their impact on assisted reproduction outcomes and management. According to World Health Organization (WHO) guidelines, leukocytes should be determined by means of the Endtz test or with monoclonal antibodies against CD15, CD68 or CD22. The cut-off value proposed by the WHO is 1 × 106 peroxidase-positive cells/mL. For bacteria, Gram staining and semen culture are regarded as the "gold standard", while modern techniques such as PCR and next-generation sequencing (NGS) are allowing clinicians to detect a wider range of pathogens. Whereas the WHO manual does not specify a specific value as a cut-off for bacterial contamination, several studies consider semen samples with more than 103 colony-forming units (cfu)/mL as bacteriospermic. The pathogenic mechanisms leading to sperm dysfunction include direct interaction of bacteria with the male germ cells, bacterial release of spermatotoxic substances, induction of pro-inflammatory cytokines and ROS, all of which lead to oxidative stress. Clinically, bacterial infections, including "silent" infections, are treatable, with antibiotics being the treatment of choice. Yet, non-steroidal antiphlogistics or antioxidants should also be considered to alleviate inflammatory lesions and improve semen quality. In an assisted reproduction set up, sperm separation techniques significantly reduce the bacterial load in the semen. Nonetheless, contamination of the semen sample with skin commensals should be prevented by applying relevant hygiene techniques. In patients where leukocytospermia is detected, the causes (e.g. infection, inflammation, varicocele, smoking, etc.) of the leukocyte infiltration have to be identified and addressed with antibiotics, anti-inflammatories or antioxidants in cases where high oxidative stress levels are detected. However, no specific strategy is available for the management of leukocytospermia. Therefore, the relationship between bacteriospermia and leukocytospermia as well as their specific impact on functional sperm parameters and reproductive outcome variables such as fertilization or clinical pregnancy must be further investigated. The aim of this narrative review is to provide an update on the current knowledge on leukocytospermia and bacteriospermia and their impact on male fertility.

Incidence of influenza virus-associated Severe Acute Respiratory Infections in three districts in 2018 at Gharbia governorate, Egypt.

Influenza remains a contributor to substantial global morbidity and mortality. There is very limited data on disease burden in Egypt. The purpose of this study was to estimate the incidence of influenza-associated Severe Acute Respiratory Illness (SARI) in three districts in Gharbia governorate in 2018. This study Followed the World Health Organization (WHO) manual for estimating disease burden associated with seasonal influenza. The hospital admission database was screened for SARI patients in three districts at Gharbia governorate in 2018. A hospital admission survey (HAS) was used to define the catchment population. The incidence rate estimation was computed as the number of influenza-positive SARI cases per 100,000 population. A total of 180 SARI cases were identified in the catchment area. The median age was 23 years [IQR: 2-53], and 45% were males. Out of the total SARI cases, 33.3% influenza was confirmed by the laboratory test of RP-PCR. Influenza A(H3N2) virus predominated representing 55.0% of patients, thanA(H1N1) 26.7% and Flu-B virus 18.3%. Influenza prevailed in winter and spring; no deaths from influenza were reported. The annual incidence of influenza-associated SARIs found higher in <2 years (282 /100,000) and ≥65 years patients (215/100,000) at significant level p<0.001. The WHO Manual for estimating disease burden associated with seasonal influenza was successfully operationalized in the three districts of Gharbia governorate. It can be used in other districts. A considerable burden was associated with influenza viruses requiring hospitalization, especially among the older adult group.

Isolated teratozoospermia: revisiting its relevance in male infertility: a narrative review.

Basic semen analysis is the first step in the evaluation of male infertility. It includes an assessment of sperm morphology which is believed to reflect on overall spermatogenesis and sperm function. Teratozoospermia, defined as abnormal sperm morphology, is frequently present in association with severe oligoasthenozoospermia, but isolated teratozoospermia (in the presence of normal counts and motility) is a poorly understood clinical entity with conflicting implications in terms of fertility potential and treatment strategies. The following paper aims to: (I) discuss the classification of sperm morphology, causes, and molecular mechanism of teratozoospermia; (II) analyze the clinical significance and potential treatment options of isolated teratozoospermia as a cause of male infertility and a predictor of fertility outcome; and (III) provide a SWOT (strengths, weaknesses, opportunities, and threats) analysis based on the existing literature on this topic. A comprehensive search from database inception to 25 April 2023 was conducted in PubMed for relevant papers relating to isolated teratozoospermia in male infertility. Finally, seven systematic reviews/reviews/meta-analyses and 81 original articles were synthesized into the current narrative review. Classification of sperm morphology has evolved significantly since the first edition of the World Health Organization (WHO) Manual of Human Semen Analysis. Kruger's strict criteria are the most used classification and have been shown to correlate with fertility outcomes. There are many causes of teratozoospermia including genetic and environmental factors and physical conditions like varicocele. Teratozoospermia correlates with sperm DNA damage, elevated oxidative stress, low antioxidant function, and apoptotic alterations, which can result in impaired spermatozoa function and lower pregnancy rates. However, the clinical correlation between teratozoospermia and assisted reproductive technology (ART) outcome shows conflicting data with recent meta-analyses suggesting that isolated teratozoospermia was not associated with poor fertility outcomes from ART and that intrauterine insemination (IUI) can be an effective option even in the presence of teratozoospermia. There is very limited data on effective therapeutic options to treat idiopathic isolated teratozoospermia. The opportunity for future research is huge to fill the gap in the medical literature on this topic. Contemporary literature on isolated teratozoospermia shows conflicting results in terms of its actual clinical implication in male infertility and the utility of available treatment options. Further research is warranted on this clinical entity to improve sperm function and future paternity.

Seminal Plasma Examination Test Result Agreements Using the WHO Manual Recommendation: With Semi-Automatic and Automatic Methods

Objective: To analyze the suitability of the measurement results of neutral alpha-glucosidase and fructose in seminal plasma, between the manual method recommended by the World Health Organization (WHO) Sixth Edition and the semi-automatic method, using Biosystems BTS 350 and the automatic method using Biosystems A15.Material and Methods: This study involved 47 subjects at the Andrology Outpatient Department of Dr. Soetomo General Academic Hospital; from October 2022 and February 2023. The measurement of neutral alpha-glucosidase and fructose levels in seminal plasma was conducted, via three different approaches: the manual method, as recommended by the WHO Sixth Edition, the semi-automatic Biosystems BTS 350, and the automatic Biosystems A15. The manual method recommended by the WHO Sixth Edition was employed as the reference system.Results: The average disparity in neutral alpha-glucosidase levels among the manual method, as recommended by the WHO Sixth Edition, compared to the semi-automatic and automatic methods were recorded as: -1.0215 IU/l and -2.3428 IU/l, respectively. Similarly, the fructose levels exhibited differences of -2.1234 mmol/l and 2.2834 mmol/l. Notably, within the method comparisons, 44 values of neutral alpha-glucosidase levels fell within the limit of agreement, while fructose levels showed 44 and 45 values within the agreement limit.Conclusion: The results obtained from the manual method, as recommended by the WHO Sixth Edition, demonstrated agreement with the measurements of neutral alpha-glucosidase and fructose obtained when using both the semi-automatic and automatic methods.

Seminal Plasma Examination Test Result Agreements Using the WHO Manual Recommendation: With Semi-Automatic and Automatic Methods

Objective: To analyze the suitability of the measurement results of neutral alpha-glucosidase and fructose in seminal plasma, between the manual method recommended by the World Health Organization (WHO) Sixth Edition and the semi-automatic method, using Biosystems BTS 350 and the automatic method using Biosystems A15.Material and Methods: This study involved 47 subjects at the Andrology Outpatient Department of Dr. Soetomo General Academic Hospital; from October 2022 and February 2023. The measurement of neutral alpha-glucosidase and fructose levels in seminal plasma was conducted, via three different approaches: the manual method, as recommended by the WHO Sixth Edition, the semi-automatic Biosystems BTS 350, and the automatic Biosystems A15. The manual method recommended by the WHO Sixth Edition was employed as the reference system.Results: The average disparity in neutral alpha-glucosidase levels among the manual method, as recommended by the WHO Sixth Edition, compared to the semi-automatic and automatic methods were recorded as: -1.0215 IU/l and -2.3428 IU/l, respectively. Similarly, the fructose levels exhibited differences of -2.1234 mmol/l and 2.2834 mmol/l. Notably, within the method comparisons, 44 values of neutral alpha-glucosidase levels fell within the limit of agreement, while fructose levels showed 44 and 45 values within the agreement limit.Conclusion: The results obtained from the manual method, as recommended by the WHO Sixth Edition, demonstrated agreement with the measurements of neutral alpha-glucosidase and fructose obtained when using both the semi-automatic and automatic methods.

Sixth edition of the World Health Organization laboratory manual of semen analysis: Updates and essential take away for busy clinicians

ABSTRACT Background Semen analysis is the cornerstone for male fertility evaluation. In 2021, the World Health Organization (WHO) released its 6th edition of semen analysis manual. Methods We highlight the main changes in the latest 6th edition of the WHO manual of semen analysis and their possible interpretations. Results The manual is highly comprehensive, offering detailed information, and is widely regarded as an excellent technical reference for laboratory staff. Nevertheless, several aspects of the manual require further elucidation for infertility practitioners. Conclusion The recently published 6th edition of the WHO manual provides a strong framework for the assessment and processing of human semen. Grasping the modifications introduced in this updated edition and their clinical significance can enhance the quality of patient care.

Effect of the technical variability of counting chambers upon the interpretation of sperm concentration results

Research questionDoes the choice of sperm-counting chamber affect the proportion of samples generating results with an erroneous interpretation? DesignLaboratories in an external quality assurance programme were sent 141 semen samples over a 12-year period and asked to return the sperm concentration and whether or not the result was abnormal. Only those using 5th edition of the World Health Organization manual (WHO5) interpretation criteria were included. Submissions from specialist fertility laboratories were used to calculate assigned values for each sample. Laboratory50 values determined the sperm concentration at which the laboratories reported a majority transition from abnormal to normal interpretations, i.e. the tipping point, which should coincide with the lower reference limit. ResultsThe median and range of bias from the assigned values of each sample were determined for the Makler (–3.3%; –88.6% to +332.8%), haemocytometer (10.6%; –93.3% to +645.5%), Kova (+65.3%; –71.7% to +581.8%) and Vetriplast (+72.4%; –100.0% to +709.1) chambers. Laboratory50 values for the Makler (17.3 × 106/ml), haemocytometer (13.6 × 106/ml), Kova (10.0 × 106/ml) and Vetriplast chambers (8.8 × 106/ml) reflected the under- and overestimation of the chambers and confirmed a shift in the adjusted lower reference limit then used. The proportion of laboratories reporting erroneous interpretations of the four chambers for oligozoospermic samples were 10.9%, 15.1.%, 40.1% and 44.0%, respectively, and rose as the adjusted lower reference limit decreased. ConclusionsThe between-laboratory and within-sample variation for all the chambers was high and remains a concern. The main impact of an increasing bias of the chambers was a lowering of the laboratory50 tipping point, resulting in an under-reporting of abnormal semen samples.

Aberrant protamination in sperm correlates to anomalous nuclear and cytoplasmic architectures in infertile males with sperm dysmorphology.

Aberrant sperm protamination is linked to sperm dysmorphology and nuclear chromatin condensation. Yet, its effects on sperm cytoplasmic maturation remain largely unexplored. The relationships of protamines, sperm morphology, DNA damage, and cytoplasmic remodeling were illustrated in this study to provide fresh perspectives on the mechanisms of male infertility. A total of 205 infertile males were allocated into 5 groups according to the percentage of spermatozoa exhibiting abnormal morphology within their samples. Sperm concentration, motility, abnormal sperm morphology, cytoplasmic droplets (CDs), and excess residual cytoplasm (ERC) were analyzed according to the World Health Organization manual (2010). Sperm nuclear vacuoles (NVs) were determined by propidium iodide (PI) staining. Sperm protamine expressions (P1 and P2) were detected by western blot. DNA damage was measured by acridine orange test (AOT) to calculate the proportion of sperm with single-strand DNA breaks (SSBs). Our data showed that sperm concentration and motility in infertile males significantly decreased with the severity of abnormal sperm morphology (both P < 0.01). P1 level, P1/P2 ratio, and SSB rate increased with the severity of sperm dysmorphology, whilst the P2 level decreased (all P < 0.01). NVs, CDs, and ERC were more common in males with sperm dysmorphology and positively correlated with the SSB rate (all P < 0.01). The relationships between the SSB rate and the P1/P2 ratio were also significant (P < 0.01). Aberrant protamination may cause sperm dysmorphology and compromise male fertility by impairing sperm's nucleus and cytoplasm maturation, with the P1/P2 ratio potentially serving as a valuable indicator of sperm quality and male fertility.

A national survey on registered products, availability, prices, and affordability of 100 essential medicines in community pharmacies across Sri Lanka

IntroductionAvailability of essential medicines that meet the expected quality standards, in appropriate dosage forms at affordable prices is a fundamental prerequisite to fulfill healthcare needs of given a population. This study assessed available products, prices and affordability of essential medicines (EM) in community pharmacies in Sri Lanka with comparison of registration status from the National Medicines Regulatory Authority(NMRA).MethodsA cross-sectional island-wide survey of 80 pharmacies was conducted according to World Health Organization and Health Action International Manual (WHO/HAI). Hundred medicines were selected from the global core list(n = 14), regional core list(n = 16) and the Sri Lanka Essential Medicine List (SL-EML) (n = 70) based on healthcare needs. Number of registered products in 2015 and 2021 were compared.FindingsAverage availability was 85.4%(± 12.31) and availability was lowest in the Northern province (69.38 ± 21.18%)(p = 0.008). Availability between the state owned, franchise and privately owned pharmacies was not significantly different (p > 0.05). 89.4% medicines were affordable except for amiodarone, hydroxychloroquine, sitagliptin, soluble insulin, isophane insulin, losartan, levodopa carbidopa combination, clonazepam and ceftriaxone. The median price ratio (MPR) of 33.7% of medicines was less than 1 and MPR of 37.1% originator brands (OB) was over 3. Median number of generic brands in the market was 8(range 2–44), 9% of medicines had 20 or more products in the market and 72.7% medicines had more products available than the number registered in 2015. The average number of registered products were similar in 2015 (8.27) and 2021(7.59) (p = 0.15).ConclusionThe overall availability of EMs in Sri Lanka was high in all categories of community pharmacies. Medicines were largely affordable and reasonably priced in 2015, although OBs were generally more expensive. Majority of medicines had more products in the market than the number of registered products.

Evaluación del recolector de orina en la toma de muestra de líquido seminal y tinción diferencial de fluorescencia en espermatozoides

Objective: To verify that urine collectors are not toxic to sperm. Methods: The toxicity of glass (control) and plastic (urine collector) containers with semen samples of good concentration and sperm motility was evaluated by studying sperm motility at intervals of 1 hour, at room temperature, for 4 hours, following guidelines of the fifth edition of the World Health Organization Manual for the processing of semen samples. The progressive motility between both types of containers (glass and plastic) was compared and the morphotintorial characteristics of the spermatozoa were evaluated with the modified fluorescence differential stain. Results: No statistically significant differences (p = 0.334) were found between the groups. Spermatozoa that emitted a green fluorescence were categorized as normal and presented a strong correlation (r = 1,000; p = 0.000); Whereas, yellow (r = -0.838, p = 0.009) and orange (r = 0.940, p = 0.000) stains were highly correlated with sperm abnormalities, highlighting that orange fluorescence had a high correlation (r = 0.940, p = 0.000) with head abnormalities. Conclusion: The plastic collector has no toxic effects on sperm, it satisfies the needs of collection of the semen sample. With modified fluorescence differential staining, green staining is associated with morphologically normal spermatozoa and yellow and orange stains are associated with abnormalities of the head, middle piece, and main piece of sperm.

O-285 The 6th edition of the WHO manual for the examination and processing of human semen: How should it affect our practice?

Abstract The WHO laboratory manual (World Health Organization 2021) has widened the scope of semen examination from primarily a prognostic tool for Medically Assisted Reproduction (MAR) by indicating the importance of evaluating men with possible disorders in male reproductive functions. Practical instructions for basic examination have been clarified and focused to measures of sperm production and motility as judged from sperm number, motility (vitality if poor motility) and sperm morphology. In addition to the recommendations by the WHO, an international standard based on the same laboratory science as the WHO manual has been published (International Organization for Standardization 2021) to facilitate for laboratories to provide reliable results of semen examination. Each ejaculate must be handled with great care before analysis begins. Time and temperature between sample collection and start of assessments is crucial. To reduce assessment variability, it is essential to ascertain that the examined aliquots are representative for the entire ejaculate and that the number of observations is sufficient to reduce the risk for significant influence of random factors. The former is handled both by aliquot volume (at least 50 µL for sperm concentration) and replicate assessment and comparison that the two assessments don’t differ too much. Regarding number of observations, 400 sperm counted in sperm concentration and in sperm motility assessments is required to reduce this source of errors to ± 10%. For assessment of sperm morphology and vitality a minimum number of observations is 200 spermatozoa. To achieve acceptable performance, thorough in-house training is necessary. There are protocols how to set up and run such schemes (Mortimer 1994, 1994). Basic medical laboratory standards also require that the laboratory regularly performs internal quality control to monitor that inter- and intra-personal variability is under control. Each laboratory should also participate in an external scheme for quality assessment (EQA) (International Organization for Standardization 2012). For the proper interpretation of semen examination results reference limits are important. However, the reference limits suggested by the WHO (World Health Organization 2021) come from a very mixed group of men and should therefore not be mistaken for true limits between fertility and infertility. The new WHO manual therefore argues for development of decision limits (when is it reasonable to act?) that are much more important than limits from a mixed population. It has been argued that semen examination must be better standardized (Björndahl et al. 2016), but the compliance has been almost non-existent (Vasconcelos et al. 2022) leading to even stronger appeals for stronger improvement of basic laboratory investigations of male reproductive functions (Björndahl et al. 2022) References Björndahl L, Barratt CL, Mortimer D, and Jouannet P. ‘How to count sperm properly': checklist for acceptability of studies based on human semen analysis. Hum Reprod 2016: 31; 227-232. Björndahl L, Barratt CLR, Mortimer D, Agarwal A, Aitken RJ, Alvarez JG, Aneck-Hahn N, Arver S, Baldi E, Bassas L, et al. Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology. Hum Reprod 2022. International Organization for Standardization. ISO 15189:2012 Medical Laboratories – Requirements for Quality and Competence 2012. Geneva. International Organization for Standardization. ISO 23162:2021 Basic semen examination — Specification and test methods 2021. ISO, Geneva. Mortimer D. Laboratory standards in routine clinical andrology. Reproductive Medicine Review 1994: 3; 97-111. Mortimer D. Practical Laboratory Andrology 1994. Oxford University Press, Oxford. Vasconcelos AL, Campbell MJ, Barratt CLR, and Gellatly SA. Do studies published in two leading reproduction journals between 2011 and 2020 demonstrate that they followed WHO5 recommendations for basic semen analysis? Hum Reprod 2022. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 6th edn, 2021. World Health Organization, Geneva.

Does an increase in adipose tissue 'weight' affect male fertility? A systematic review and meta-analysis based on semen analysis performed using the WHO 2010 criteria.

Obesity negatively impact on the metabolism of sex hormones, leading to reduced testosterone serum levels. However, how the obesity could negatively impact on the overall gonadal function, particularly on male fertility, remained unclear so far. To systematically review evidences regarding the influence of body weight excess on the sperm production. A meta-analysis was conducted, searching all prospective and retrospective observational studies reporting male subjects older than 18 years old, with body weight excess from overweight to severe obesity were considered. Only studies using the V edition of the World Health Organization (WHO) manual for semen analysis interpretation were considered. No specific interventions were considered. Search was focused on studies comparing overweight/obese to normal weight subjects. Twenty-eight studies were considered. Total sperm count and sperm progressive motility were significantly lower in overweight compared to normal weight subjects. Meta-regression analyses demonstrated that patients' age impacted on sperm parameters. Similarly, obese men showed lower sperm concentration, total sperm number, progressive and total motilities, and normal morphology lower than normal weight subjects. Reduced sperm concentration in obese men was influenced by age, smoking habit, varicocele, and total testosterone serum levels at meta-regression analyses. The male potential fertility is reduced in subjects with increased body weight, compared to normal weight men. The higher was the increased body weight, the worst was the sperm quantity/quality. This result comprehensively included obesity among non-communicable risk factor for male infertility, shedding new lights on the negative impact of increased body weight on overall gonadal function.

Analytical variability and interpretation of results of a 3-category sperm motility assessment: 5 years’ of an Australian external quality assurance programme

Research questionHow do laboratories perform when assessing sperm motility with a 3-category system and interpreting results as per the fifth edition of the World Health Organization manual (WHO5), and will the use of a 4-category system as per the sixth edition of the WHO manual improve their performance? DesignEighty video recordings of sperm samples were sent to over 200 laboratories spanning a 5-year period for the assessment of progressive motility. The results were reviewed relative to the all-laboratory trimmed mean (ALTM) in terms of the minimum and maximum values reported, the coefficient of variation and the proportion of laboratories indicating an abnormal result. A further 20 video recordings were sent over 1 year, with 6–11 laboratories per distribution adjusting to reporting rapid progressive motility using the 4-category system. ResultsFor the 3-category system, the videos covered a mean assessed progressive motility range of 12.0–81.1%. The mean difference between the minimum and maximum values per sample was 50.3% and the coefficients of variation were negatively correlated with the ALTM (r = –0.87, P < 0.00001). Progressive motility abnormality reporting formed a sigmoid curve, and the inflection point (50% of laboratories identifying an abnormality) gave an ALTM value of 32.01%. Preliminary results for laboratories using the 4-category system showed no performance improvement but the number of laboratories was small. ConclusionsAnalytical variation can result in laboratories crossing the clinical cut-off of the lower reference limit for samples whose motility is close to the WHO5 lower reference limit, but is less important for samples with extreme values. The benefits of a 4-category motility system are yet to be shown.

Do studies published in two leading reproduction journals between 2011 and 2020 demonstrate that they followed WHO5 recommendations for basic semen analysis?

STUDY QUESTIONDo publications that involve the interpretation of the results of a basic semen analysis, published in Human Reproduction and Fertility & Sterility between 2011 and 2020, give sufficient evidence in their methodology to demonstrate that they followed the technical methods recommended in the fifth edition of the World Health Organization (WHO) laboratory manual, entitled WHO Laboratory Manual for the Examination and Processing of Human Semen (WHO5)?SUMMARY ANSWEREvidence of methodological agreement of studies with the WHO5 recommendations was low, despite 70% of papers stating that they followed WHO5 recommendations.WHAT IS KNOWN ALREADYA basic semen analysis is currently an integral part of infertility investigations of the male, but method standardization in laboratories remains an issue. The different editions of the WHO manual for the basic semen analysis (WHO1–6) have attempted to address this by providing increasingly rigorous methodological protocols to reduce experimental error. However, to what extent these methods are followed by studies that involve the interpretation of the results of basic semen analysis remains unknown.STUDY DESIGN, SIZE, DURATIONA survey of the technical methods used to perform a basic semen analysis was conducted on studies published in two leading reproduction journals (Human Reproduction and Fertility & Sterility) between 2011 and 2020.PARTICIPANTS/MATERIALS, SETTING, METHODSThe literature search was performed on the electronic databases PUBMED and MEDLINE Ovid between January 2021 and March 2021. The MeSH terms included in the search were ‘sperm concentration’ OR ‘sperm motility’ OR ‘sperm morphology’ OR ‘sperm vitality’ OR ‘male fertility’ AND ‘human spermatozoa’ NOT ‘animals’. A total of 122 studies were available for analysis.MAIN RESULTS AND THE ROLE OF CHANCEIn total, 70% of the studies cited WHO5 in their methods section. Of the remaining studies, 10% cited the fourth edition of the WHO laboratory manual (WHO4), 7% cited both WHO4 and WHO5, 1% cited the third edition of the WHO laboratory manual (WHO3), and 12% did not cite the WHO at all. Overall methodological agreement with WHO5 recommendations was poor, with the main reason for this lack of agreement being that the research studies did not disclose specific details of the technical methods and equipment used.LIMITATIONS, REASONS FOR CAUTIONIn the case of studies that did not disclose any specific technical methods that they used, we did not attempt to contact these authors and so were unable to confirm the agreement between their technical methods and WHO5 recommendations.WIDER IMPLICATIONS OF THE FINDINGSOur findings suggest there is an urgent need to develop strategies to address standardization in reporting the results of a semen analysis for publication. This is particularly timely given the recent publication of WHO6 and ISO standard 23162 for the basic examination of human semen.STUDY FUNDING/COMPETING INTEREST(S)There was no funding for this project. C.L.R.B., as an employee of the University of Dundee, serves on the Scientific Advisory board of ExSeed Health (from October 2021, financial compensation to the University of Dundee) and is a scientific consultant for Exscientia (from September 2021, financial compensation to the University of Dundee). C.L.R.B. has previously received a fee from Cooper Surgical for lectures on scientific research methods outside the submitted work (2020) and Ferring for a lecture on male reproductive health (2021). C.L.R.B. is Editor for RBMO.TRIAL REGISTRATION NUMBERN/A.

The statistical foundation of the reference population for semen analysis included in the sixth edition of the WHO manual: a critical reappraisal of the evidence.

In the most recent version of the ‘WHO Laboratory Manual For The Examination And Processing Of Human Semen’, the updated target population used to infer reference values included 3589 fertile subjects, representative of 12 countries and 5 continents, and 10 studies. We have critically evaluated the newly proposed distribution of semen examination results using an approach borrowed from clinical chemistry laboratories and based on the recommendations of the International Federation of Clinical Chemistry for estimation of reference intervals. Surprisingly, most prerequisites to produce common reference intervals through multicentric data were not met. Moreover, when we assessed with the bootstrap method the descriptive reference values obtained from raw data of the 10 individual studies for sperm concentration, sperm number, motility and normal forms, we found that none of the populations was completely correctly described by the reference centiles. We concluded that aggregated data used to build the reference distribution cannot be considered to originate from the same population, and this can result from real differences among individuals or different methodological approaches used in the various studies. Transferability conditions across studies did not seem to have been met. Our findings strengthen the relevance of concerns regarding the use of reference populations in the World Health Organization manual to discriminate between fertile and infertile men.

Survey of semen analysis practices in India and need for standardization and improvement.

Infertility is a sensitive subject carrying with it economic, social, and psychological implications. Work up of male infertility is often hampered by a lack of infrastructure and facilities, as well as inadequate training of pathology residents. The purpose of this research survey was to evaluate the current status of semen analysis practices and compare them to the standards laid down by the World Health Organization (WHO). A web-based questionnaire was designed consisting of questions related to semen analysis practices and procedures being followed currently by pathologists in India. A total of 194 pathologists responded. Questions regarding the procedures followed for semen collection, sperm count, volume, normal range, lower normal limit of sperm count, morphology, etc., were included in the survey. These data were recorded. The differences and gaps in the practice with respect to the WHO standards were analyzed. The survey revealed that the printed instructions for semen analysis were available with 38.7% of the respondents; 58.8% of the respondents had a separate room for semen collection; 95.9% performed the analysis manually, and; only 4.1% used automated analyzers. Only 53.6 and 52.6% of the respondents were correctly reporting the normal range of semen volume and sperm counts, respectively. Only 19.6% stated as having read the WHO manual and were also practicing its guidelines, while 14.4% had not read the WHO manual even once. The present study showed a large gap between the practice of semen analysis by respondents from various parts of India and standard procedures as laid down by the WHO. Many laboratories do not follow the standard instructions. There is a need to improve the quality of practice related to semen analysis in this country through appropriate teaching and training in medical institutions as well as through Continuing Medical Education (CMEs) or regular update programs.

An evaluation of changes over time in the semen parameters data used for the World Health Organization semen analysis reference ranges.

Previous meta-analyses concluded that there is a decline in sperm parameters over time. This conclusion might be incorrect due to inherent biases or focusing only on a single parameter - sperm concentration. To study trends in sperm parameters over the past 20 years using data from the trials that defined the reference ranges of the World Health Organization manual. Retrospective evaluation of the data used to define the World Health Organization reference ranges. The data from 11 studies, including 3589 participants between 1996 and 2016, were divided into three period groups based on the decade of study. Differences in semen parameters' distribution were presented in boxplot. p-values were calculated by the Kruskal-Wallis rank-sum test followed by Dunn post hoc test. Analyses were conducted using the R programming language. A small decrease was noted in mean sperm concentrations (88.1 million/ml, 87.6 million/ml, and 77.2 million/ml for the first, second, and third decades, respectively) (p<0.01). However, the 5th percentile of sperm concentration for the third decade was higher than the first or second decades (18 million/ml versus 14.9 million/ml and 15 million/ml, respectively). No significant differences were noted in progressive motility over the years (p=0.32). The percent of morphologically normal sperm decreased between the first (24.2%) and the second (12.6%) periods of the study (p<0.001) and then increased in the third decade (14.2%) (p<0.01). Total motile sperm count (TMC) declined between the second and third decades (189 million and 153.9 million, respectively, p<0.001), at levels unlikely to decrease fertility. However, the 5th percentile of the TMC remained stable at 24.9, 20.8, and 20.6 million, for the first, second, and third decades respectively (p=0.36). Trends in sperm parameters over the last three decades do not seem to be clinically significant.

O-040 The evolution of WHO Manual. Where are we now?

Abstract text Male factor infertility is associated with impaired overall health, decreased life expectancy, lower quality of life and may affect reproductive outcomes even under assisted reproductive technology (ART) settings. Male factors, alone or combined with female factors, contribute to at least 50% of reported infertility cases. Despite this, the male partner is often overlooked in the evaluation and treatment of infertility. A routine semen analysis is frequently the only test carried out to assess a man’s fertility potential. The state-of-art on how the human semen should be assessed is provided by the World Health Organization (WHO), which periodically releases manuals that include specific protocols and reference standards. These manuals include detailed laboratory methods for semen examination, protocols for sperm preparation and cryopreservation, quality assurance and quality control, results’ interpretation, and reference ranges. Unlike the previous four versions, the latest 2010 WHO reference values relied on clinical chemistry principles to generate 95% intervals for sperm volume, count, motility, vitality, and morphology from recent fathers. The fifth centile was deemed suitable for representing semen characteristics at lower limits. The reference values ultimately obtained were markedly lower than those previously reported, raising concerns about its clinical utility and generalizability. Criticisms included the limited geographical area of patients analyzed, the methods used for semen evaluation, and the potential impact of the new reference range on patient referral, diagnosis, and treatment guidance. An updated new WHO manual (6th edition) is about to be released with much expectation. Although semen analysis remains one of the cornerstones of the infertility evaluation, a male infertility workup primarily based on routine semen analysis does not provide men with an optimal fertility pathway for many reasons. First, reference intervals do not reliably distinguish fertile from subfertile subjects. Second, an individual patient’s results have limited prognostic value for both natural and assisted conception unless at extreme lower limits. Third, there is a wide variation in how laboratories perform a semen analysis. Lastly, routine semen analysis does not detect sperm DNA defects that might adversely impact embryo development, implantation, and offspring’s health. Guidelines issued by professional societies recommend that a full andrological assessment be performed in all men with couple infertility. Well-trained reproductive urologists or clinical andrologists should perform the male evaluation, including a detailed history, physical examination, semen analysis, endocrine assessment, and other tests as needed. Therefore, the importance of WHO manuals remains critical. However, the goals of a comprehensive male infertility workup go beyond the laboratory assessment of human semen. It comprises i. Diagnosis, i.e., detection of any underlying relevant medical or lifestyle conditions potentially impairing the (reproductive) health of the male or his offspring; ii. Counselling, particularly regarding the impact of infertility, genetic factors, age, and lifestyle on pregnancy prospects, reproductive and overall health, and offspring’s well-being; and iii. Management Guidance, i.e., identifying optimal treatment options to improve the likelihood of achieving natural pregnancy or ART success. The prevention and management of male infertility are integral components of comprehensive sexual and reproductive health services needed to attain a sustainable development goal.

Short Communication: Phytochemical diversity and bioactivity of Malaysian Premna cordifolia (Lamiaceae)

Abstract. Daud D, Dewa MSAM, Mahbob ENM, Razak WRWA. 2021. Short Communication: Phytochemical diversity and bioactivity of Malaysian Premna cordifolia (Lamiaceae). Biodiversitas 22: 3245-3248. The aims of the present study are to determine the chelation potential and protective effect of Premna cordifolia leaves ethanolic extract in lead intoxicated mice. Twenty-four male mice were divided into four groups with six animals each. Group 1 was healthy mice and received 2 ml/kg bwt distilled water. Group 2 was healthy mice and supplemented with 200 mg/kg bwt P. cordifolia leaves ethanolic extract. Group 3 was lead intoxicated mice and received 2 ml/kg bwt distilled water. Group 4 was lead intoxicated mice and supplemented with 200 mg/kg bwt P. cordifolia leaves ethanolic extract. All treatments were given for 14 days. Chelation activity was determined by measuring the concentrations of lead in the liver, kidney and testis by using atomic absorption spectroscopy. Sperm quality was tested according to World Health Organization manual and testosterone was estimated by using ELISA kit. Spectroscopic data showed that P. cordifolia leaves ethanolic extract was able to lower lead concentrations in liver, kidney and testis of lead intoxicated mice. Following a decrease in lead concentrations, sperm quality and testosterone levels were restored in lead intoxicated mice supplemented with P. cordifolia leaves ethanolic extract. In conclusion, P. cordifolia leaves ethanolic extract has the potential to be developed as an alternative remedy for treating lead poisoning effects.

Medication selection for the treatment of acute infective diarrhea in Thai pharmacies: a qualitative study.

World Health Organization (WHO) released the treatment manual of diarrhea in 2005. We aimed to investigate the rationale for selecting medications for acute infective diarrhea in Thai community pharmacies and to see if the selection complied with the WHO manual. A theoretical 18-year-old patient with acute infective diarrhea was used for interviews. The protocol and materials for the research were approved by Institutional Review Board. A total of 30 drugstore personnel were selected by convenience sampling and included. The first author manually coded, extracted for themes, and translated the transcript. Participants did not dispense oral rehydration salt because of the feeling that diarrhea was not severe. Absorbents were dispensed because they were perceived as the first-line medication for noninfective or mild diarrhea. Antibiotics were dispensed because of the concerns for the prognosis and the expected patient pressure. None provided zinc to the patient because of the lack of knowledge of the indication of zinc. We found that dispensing for acute infective diarrhea in Thai drugstores deviated from the WHO treatment guideline. The reasons were that the pharmacy personnel were not practicing evidence-based medicine, the lack of knowledge, the patient pressure, the unavailability of products, and the perceived availability of information in local guidelines.