Assessment Of Sperm Morphology Research Articles

P-034 Revolutionizing sperm morphology assessment: A novel artificial intelligence optic microscope (AIOM)-powered platform for consistent and reliable analysis in male infertility diagnosis

Abstract Study question Does AIOM-based live sperm morphology annotation provide consistent and accurate results for assessing sperm morphology? Summary answer AIOM offers precise and reproducible sperm morphology evaluations which can enhance diagnostic efficiency with a single and reliable test. What is known already Semen analysis remains the primary diagnostic tool for male infertility. Unlike concentration and motility, sperm morphology shows a much higher correlation with paternal genetic integrity. However, it poses challenges due to inter-observer and inter-laboratory variability. The 2023 semen proficiency test highlighted these inconsistencies, reporting deviations of up to 13% among laboratories and acceptability ranging from 0% to 43%. This inconsistency is linked to complex staining procedures and interpretive subjectivity. To address these challenges, our study evaluates a novel AIOM-assisted platform, employing a live sperm morphology deep learning model. We aim to explore the platform’s reproducibility and reliability. Study design, size, duration In the first stage, liquefied semen specimens were split into three aliquots, each assigned to one of three operators (operators 1–3). Each operator assessed sperm morphology using Diff-Quik and live sperm morphology using the LensHooke® X12 semen analysis system (Bonraybio, Taichung). In the second stage, a single well-trained operator conducted sperm morphology assessments for three replicates (replicates 1-3) using the two methods. Interclass-correlation coefficients (ICCs) were calculated to assess consistency between replicates and operators. Participants/materials, setting, methods Semen samples (n = 30) were collected post 2-5 day abstinence and liquefied at room temperature for 30 minutes. Specimens were diluted to 100 million/mL with HTF, and underwent Diff-Quik staining according to WHO’s instruction. Aliquot of 3 μL semen specimen was loaded to 10-μm LensHooke® CS3 slide and analyzed by X12 analysis using live sperm morphology algorithm. Main results and the role of chance X12 utilizes a deep learning algorithm to automatically annotate sperm morphology, focusing on parameters such as sperm head, midpiece, tail, and excess residual cytoplasm, ultimately generating outputs for normal morphology and the teratozoospermic index (TZI) based on WHO strict criteria. Compared to manual Diff-Quik analysis, X12 showed higher consistency across all parameters, with ICCs above 0.85, unlike the manual method’s lower ICCs (below 0.70). The determination of abnormal midpiece had the lowest agreement between operators [ICC (95% CI): 0.15 (-0.61–0.58)]. In contrast, X12 demonstrated high agreement in all parameters, with ICC values consistently exceeding 0.85. When comparing the agreement of normal morphology rates between the two methods, a notable difference was observed [(Manual method: 0.59 (0.22–0.8) versus X12: 0.8 (0.61–0.9)]. To minimize inter-operator variability, a highly trained operator conducted three replicates following the manual method, thereby reducing deviations between measures. Remarkably, a single assessment of live sperm morphology produced results that were comparable to the mean average of repeated measures obtained through manual methods. In conclusion, the new platform for sperm morphology assessment, contingent on AIOM technology, is a reproducible and reliable tool in male infertility diagnosis. Limitations, reasons for caution This study’s limitations include its laboratory-based nature and a small sample size. Further research should include a more diverse and larger patient cohort to validate the efficacy and accuracy of AIOM in live sperm morphology assessment. Wider implications of the findings AIOM’s integration of automated interpretation simplifies sperm morphology assessment, reducing sample requirements and is smear-free. This advancement not only enhances diagnostic testing consistency but also eases andrologists’ workloads. This platform can contribute to standardized sperm analysis and improved laboratory management. Trial registration number Not applicable

P-062 The impact of isolated teratozoospermia on cumulative live birth rates in IVF and ICSI treatment: a retrospective analysis of 5254 completed cycles

Abstract Study question To assess fecundity using cumulative live birth rate (CLBR) per egg collection to determine if IVF and ICSI outcomes are significantly influenced by sperm morphology. Summary answer The presence of isolated teratozoospermia does not have a significant impact on the CLBR of IVF treatment. What is known already There is ongoing debate regarding the clinical significance. Some studies indicate a pivotal role of sperm morphology in successful pregnancies, others cast doubt on its reliability as a predictive factor. Despite the application of stringent grading criteria, concerns remain about the subjective nature of assessing sperm morphology. Discrepancies in success rates between normozoospermic and isolated teratozoospermic groups shown by previous research findings underscore the uncertainty surrounding this matter. Inconsistent findings in previous research may be explained by limited sample sizes and a focus on immediate successes, neglecting subsequent frozen cycles. This study seeks to address the overlooked aspects of teratozoospermia. Study design, size, duration A retrospective study was conducted at Waterstone Clinic, involving 5254 IVF/ICSI autologous cycles completed between 2009-2022 cycles (completed meaning all embryos were either transferred or disposed of). Cycles were categorised according to sperm morphology and divided into two groups: the isolated teratospermic (ITS) group (1218 cycles) defined as having less than 4% normal morphologic forms with all other semen parameters being normal, and the normal sperm (NS) group (4036 cycles). Participants/materials, setting, methods The primary outcome measures, included fertilisation rate, number of cryopreserved embryos per egg collection, and CLBR. Employing multivariate statistics, the analysis explored the impact of interferons on CLBR in relation to variables such as sperm morphology, female age, egg number, and aetiology. Main results and the role of chance The female base characteristics were similar between the two groups in terms of age (37.1 vs 37.1 y/o), oocyte number (9.4 vs. 9.7) and FSH level (7.6 vs. 7.6 IU/L). Statistically significant differences were observed in fertilisation rates (61.7% vs. 65.8%, p < 0.001) and fertilisation failure (5.9% vs. 3.3%, p < 0.001) from IVF treatments when comparing the ITS and the NS groups. However, there was not any significant difference in the number of cryopreserved embryos between the two groups. Additionally, the CLBR did not show any significant difference between the two groups for IVF (35.8% vs. 37.8%, p > 0.05) or ICSI treatments (38.1% vs. 33.2%, p > 0.05). ICSI did not confer any benefit in improving the CLBR outcome when compared to conventional IVF, when adjusted for female age, number of oocytes collected, and aetiology. Limitations, reasons for caution While this study is retrospective it encompasses a substantial cohort. A potential for bias due to the selection criteria for IVF and ICSI exists. The intrinsic subjectivity in sperm morphology assessment introduces additional potential bias. Wider implications of the findings Despite the lack of support from CLBR to advocate for choosing ICSI in patients with ITS, this study suggests that couples with ITS experience lower fertilisation and higher fertilisation failure rates with IVF. This observation can serve as a valuable indicator for investigating the occurrence of fertilisation failure. Trial registration number not applicable

Evaluation of structural problems in the application of strict criteria for sperm morphology assessment.

The WHO manual for basic semen analysis and ISO 23162 describe sperm morphology assessment as a standard part of semen analysis. Older studies showed a correlation between morphology results and (artificial) conception. In more recent studies this relationship was less apparent and there is more emphasis on sperm morphology as a marker for healthy spermatogenesis (and general male health). Meantime, many laboratories ceased morphology assessment, probably due to unfamiliarity with this paradigmatic shift and to technical difficulties in the assessment, like the interpretation of morphological criteria. The aim of this study was to identify morphological criteria with high variability in results in the Dutch External Quality Control (EQC) program. Over the period 2015-2020, a total of 72 photos of sperm cells along with dichotomous propositions based on 14 criteria as defined in WHO5 (2010) were distributed in the Dutch EQC program for semen analysis. The EQC results were evaluated for variability per criterion and for trends in time. Between 2015 and 2020, 40 to 60 laboratories assessed the photos. Criteria with low variability between participants were related to acrosomal vacuoles, excessive residual cytoplasm, and tail metrics. In contrast, head ovality, regularity of head and midpiece contours, and alignment of the major axis of the midpiece and head led to the highest variability in outcomes. In general, there was a slightly positive trend (lower variability) in time, except for the criteria with the highest variability (stable or declining trend). This study indicates that there are (high) variabilities in the interpretation of the morphological criteria, leading to inconsistent outcomes of morphology assessment. The results are discussed from the perspective of imperfections in definitions and examples of the criteria as given in the WHO manuals.

Male reproductive function before and after the adjustment of the COVID-19 prevention policy: a multicenter study in China.

At the end of 2022, the adjustment of the coronavirus disease 2019 (COVID-19) pandemic control policy in China resulted in a large-scale increase in public infection. To compare the fertility parameters of male patients before and after the adjustments of the COVID-19 pandemic control policy in China, we collected data on patients' medical histories and laboratory examinations on their first visits between June 2022 and March 2023 in five different hospitals. Data were divided into five groups according to the timeline of the policy adjustment. The data we collected from male patients included semen quality and serum reproductive hormone levels, and intergroup comparisons were made using the Mann-Whitney U and Chi-square tests. In total, 16 784 cases underwent regular semen analysis, 11 180 had sperm morphology assessments, and 7200 had reproductive hormone analyses. The data showed declining trends in semen volume, sperm motility, and the progressive sperm motility rate after the policy adjustment. Subgroup comparison revealed an initial decrease and gradual recovery in progressive motility rate. Sperm morphology analysis showed increased neck and tail abnormalities after the policy adjustment. No significant change in hormone levels was observed. Following the adjustment of the COVID-19 prevention policy in China, a decline in sperm motility and morphology was observed. This trend may gradually recover over 2 months. After the policy adjustment, reproductive hormone levels were relatively stable throughout, except for an increase in luteinizing hormone (LH). These changes in semen parameters suggest that the policy adjustment had a short- to medium-term impact on male reproductive function.

(212) The Questionable Clinical Utility of Sperm Morphology: Rate of Isolated Teratospermia in a Population of Fertile Men

Abstract Introduction Over the last several decades, sperm morphology has represented a standard component of semen analysis. However, its clinical utility is not well-defined, despite continual efforts by the WHO to address critiques of semen morphology criteria through increasingly strict guidelines. Issues with intra- and inter-laboratory technical variability, in addition to the subjective nature of the morphology assessment, have been thoroughly discussed in existing literature. Furthermore, current literature has highlighted the low predictive value of sperm morphology abnormalities in assisted fertility treatment (ART) and natural pregnancy outcomes. As of 2020, the American Society for Reproductive Medicine (ASRM) guidelines have noted that morphology alone is not highly predictive or diagnostic of infertility. However, past survey studies have demonstrated the pervasive belief among physicians of the value of sperm morphology, potentially leading to counseling patients towards more expensive ART methods. These concerns call into question the emphasis placed on sperm morphology assessment and its value in the clinical setting. Objective To better understand the impact of sperm morphology on fertility, we assessed sperm morphology in a population of known fertile men. Methods Healthy men over 18 years of age were recruited to provide one semen sample prior to vasectomy from March 2020 through November 2022. Inclusion criteria for the study was as follows: (1) patients were required to have biological children under the age of 5 years old, with no history of difficulty achieving pregnancy, and (2) had undergone no prior fertility procedures. Sperm morphology was analyzed using Papanicolaou stain with Kruger strict criteria by two technicians in an Andrology specialty laboratory per the WHO 5th edition manual. Results There were 83 men who participated in the study, 68 of whom (81.9%) had a complete sperm morphology assessment available. On average, participants were 37 years of age and had a mean BMI of 28 (22.8, 33.2). Of those with a smoking history available (n=61), 38 (62.3%) were non-smokers, 7 (11.5%) were former smokers, and 16 (26.2%) were current smokers. Patients had an average of 3 children with a mean youngest child age of 11.5 months old. There were 38 patients (55.9%) with 3% or lower normal sperm morphology, including two patients who had 0% normal morphology (Table 1). The most common morphologic abnormalities were head shape defects (n=59, 84.3%), followed by coiled tails (n=14, 20.3). Men 3% or lower normal sperm morphology were four times as likely to have both head and tail abnormalities than men with 4% or greater normal sperm morphology (n=8 vs n=2, respectively); half of these men also had lower than normal motility, whereas none of the men with 4% or greater normal sperm morphology had lower than normal motility. Conclusions More than half of fertile male patients (65.6%) had lower than normal sperm morphology in our study. The results of our study further question the clinical relevance of sperm morphology on fertility outcomes and if the current approach in assessing morphology is too strict. Disclosure No.

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.

Modified diff-quick staining for canine sperm morphology

Sperm morphology assessment requires specialized microscopes or stains. Diff-quick (DQ) is considered a universal stain that is cost-effective; however, morphological evaluation of sperm using DQ staining is poor and not encouraging. Therefore, this study investigated modifications to the DQ protocol to improve identification of morphological defects of dog sperm and compared the modified DQ techniques with eosin-nigrosin, Karras, and differential interference contrast microscopy [DIC]). One ejaculate from each of 9 dogs was used. To perform the proposed modified DQ techniques (DQ1 and DQ2), dried semen smears were fixed by immersing for 10 seconds in solution 1 of DQ and 5 minutes each in solutions II and III of the kit. After the third stain solution, slides in DQ1 were rinsed in water whereas slides in DQ2 were not rinsed but were vertically supported to facilitate stain drainage. Results suggested that the standard DQ protocol overestimated normal sperm and detached heads whereas underestimated abnormal heads and total defects compared to DIC, Karras, eosin-nigrosin, and DQ2. Acrosome abnormalities were only detectable with Karras, DIC, and DQ2. In conclusion, prolonging exposure to DQ staining solutions enhanced sensitivity in sperm morphological evaluation, and avoiding rinse as a final step in the DQ protocol improved visualization of certain acrosome defects in dog sperm. Therefore, modified DQ techniques can serve as a viable alternative for dog sperm morphology evaluation in clinical practice.

Effect of incorrect storage of bull semen samples on sperm morphology assessment

Thirteen pairs of bull semen samples were assessed. One of each pair of samples was inadvertently stored in phosphate buffered saline (PBS) that was used for analysis of motility immediately after collection. Other sample of the pair that was correctly stored in buffered formol saline (BFS) was assessed later. Proportion of morphologically normal sperm was different (p = 0.0021) between the 2 storage methods. Most of the abnormalities in the PBS samples were loose and detached heads. Closer examination of these sperm illustrated changes in the composition of the tail, with an apparent loss of the plasma membrane. This serendipitous error allowed documentation of the importance of correct storage of semen samples for morphological assessment of sperm, and the tertiary defects detected with the incorrect storage of semen samples in semen extender. Tertiary defects should always be considered a possibility during morphological assessment of sperm via spermiogram.

Antioxidant Properties of Pistacia vera against the Effects of Phenylhydrazine-Induced Hemolytic Anemia on Male Fertility in Mice

Objective. The reproductive system can be adversely affected by anemia, characterized by elevated free radical production and lipid peroxidation. Conversely, the pistachio is well-known for its antioxidant capabilities. In this study, a mouse model was employed to investigate the safeguarding effects of the aqueous extract of pistachio (Pistacia vera) against oxidative stress induced by phenylhydrazine (PHZ)-triggered hemolytic anemia. Materials and methods. Forty-eight male mice were distributed into six distinct experimental groups (n = 8): control, PHZ, PHZ + 20Pis, PHZ + 80Pis, PHZ + 160Pis, and 80Pis groups. The experimental groups were administered a PHZ dose of 80 mg/kg/48 hr, along with pistachio doses of 20, 80, and 160 mg/kg/day, delivered intraperitoneally. After a period of 35 days, an assessment of sperm count, motility, morphology, and viability was conducted. Additionally, sperm chromatin quality was evaluated through Chromomycin A3, aniline blue, and toluidine blue staining. Hematoxylin–eosin staining was performed to examine the total diameter of seminiferous tubes and spermatogenic cells. Hormonal levels (FSH, LH, T) and antioxidant levels were analyzed using standard kits. Results. The PHZ groups exhibited a noteworthy decrease in various sperm parameters, as well as diminished chromatin quality assays and reduced levels of sex hormones when compared to the control group. Moreover, the morphometric evaluation indicated a substantial reduction in spermatogonia cell number, spermatid count, tubule count, seminiferous diameters, and seminiferous area in the PHZ group. Notably, the Pis group demonstrated the highest concentration of antioxidant contents. Conclusion. The protective impact of P. vera extract against PHZ-induced anemia on the male reproductive system in mice may be attributed to its abundant antioxidant components, which possess therapeutic properties at elevated levels.

Comparison of Special Stains for Analysing the Morphology of Sperm: A Cross-sectional Study

Introduction: In India, male infertility constitutes 40% of infertility cases in males, which has become a serious problem in developing countries. Numerous studies and advanced techniques have been identified in assessing sperm morphology. The detection of sperm morphology is a simple and widely accepted method for determining sperm viability. It assists in selecting appropriate treatments for male infertility in assisted reproductive techniques. Infertility issues can arise in healthy males due to altered sperm morphology. Aim: To analyse the morphology of sperm using basic histopathology stains such as Haematoxylin and Eosin (H&E), Giemsa (G), Eosin-Nigrosin (EN), and Papanicolaou (Pap) stains. Materials and Methods: A cross-sectional study was conducted from April 2022 to June 2022 at the Department of Clinical Pathology, Sree Balaji Medical College and Hospital, Chennai, Tamil Nadu, India. Thirty semen samples were collected from healthy males and stained with H&E, Giemsa, Eosin-Nigrosin, and Papanicolaou stains. The results were observed and tabulated based on sperm shape, size, and motility using Kruger’s strict morphology method. A comparison was made to identify the fastest and most cost-effective method among the four stains. Descriptive statistics were used for the comparison. Results: Among the 30 semen samples, H&E and Papanicolaou stain methods were found to be rapid and cost-effective for analysing sperm morphology compared to the other stains (p-value <0.001). The findings included cases of Normozoospermia (18), Oligozoospermia (9), Necrozoospermia (1), and Teratozoospermia (2). In the Papanicolaou stained samples, the mean sperm head length (μm) was 8.92a±0.5, head width (μm) was 4.48a±0.33, head perimeter (μm) was 27.69a±1.85, head area (μm²) was 33.96a±3.74, tail length (μm) was 44.31a±2.02, sperm length (μm) was 53.24a±2.18, with no cytoplasmic droplets greater than half the size of the head and two vacuoles observed. Papanicolaou stain was the least expensive stain (9.50rs) compared to the other stains used for assessing sperm morphology. Conclusion: Papanicolaou stain is a simple and cost-effective method for analysing sperm morphology compared to other special stains. However, EN Stain is used to detect the morphology of live sperm. Sperm morphology assessment is necessary to monitor the causes of male infertility and determine appropriate treatment modalities.

The effects of male anxiety and depression on IVF outcomes.

What are the effects of male anxiety and depression on IVF outcomes? Men with anxiety had lower final total motile sperm counts (fTMSC) during IVF compared to men without anxiety; however, there were no differences in live birth rates (LBRs). Studies have shown that male anxiety causes low sperm motility, worse sperm morphology, and increased DNA fragmentation, which are known to be influential factors on fertilization rates and embryo quality during IVF. However, data are lacking on whether there is a direct association between male anxiety and/or depression and IVF outcomes. This was a survey-based, retrospective cohort study completed at a single, large hospital-affiliated fertility center with 222 respondents who underwent IVF with or without ICSI. The study was conducted between 6 September 2018 and 27 December 2022. Male partners of couples who underwent IVF or IVF/ICSI completed a Hospital Anxiety and Depression Scale (HADS) questionnaire. They were separated into two groups for both anxiety (HADS-A ≥ 8 or HADS-A < 8) and depression (HADS-D ≥ 8 or HADS-D < 8). Men with an elevated HADS-A or HADS-D score ≥8 were considered to have anxiety or depression, respectively. The primary outcome was LBR. Secondary outcomes included semen parameters at the time of IVF, cycle outcomes, pregnancy outcomes, and prevalence of erectile dysfunction and low libido. There were a total of 222 respondents, of whom 22.5% had a HADS-A ≥ 8 and 6.5% had a HADS-D ≥ 8. The average age of respondents was 37.38 ± 4.90 years old. Antidepressant use was higher in the respondents with a HADS-A or HADS-D ≥ 8 (P < 0.05). Smoking use was similar between groups for both HADS-A and HADS-D (P > 0.05). When adjusted for male BMI, antidepressant use and smoking, men with a HADS-A or HADS-D ≥ 8 had similar rates of erectile dysfunction (adjusted relative risk (aRR) = 1.12 (95% CI 0.60, 2.06)) and low libido (aRR = 1.70 (95% CI 0.91, 3.15)) compared to those with a HADS-A or HADS-D ≤ 8. Men with a HADS-A ≥ 8 were more likely to have a lower fTMSC on the day of oocyte retrieval (11.8 ≥ 8 vs 20.1 < 8, adjusted ß = -0.66 (95% CI -1.22, -0.10)). However, the LBR per embryo transfer (ET) was similar between the HADS-A groups (43.2% ≥8 vs 45.1% <8, adjusted relative risk = 0.90 (95% CI 0.65, 1.06)). Although depression was uncommon in the entire cohort, the HADS-D groups were clinically similar for fTMSC (18.7 ≥ 8 vs 16.0 < 8) and LBR per ET (46.7% ≥8 vs 45.4% <8). Limitations of our study are the survey-based design, the lack of sperm morphology assessment at the time of IVF, our inability to fully assess the HADS-D ≥ 8 cohort due to the small sample size and the large Caucasian demographic. Couples undergoing IVF have an increased likelihood of suffering from anxiety and/or depression. There is currently a debate on whether or not men should be treated with antidepressants while attempting to conceive due to potential detrimental effects on sperm quality. Our study shows that, regardless of antidepressant use, couples with men who did or did not report anxiety and/or depression have similar LBRs when undergoing IVF. Therefore, it is important to assess both partners for mental health and to not withhold treatment due to a concern about a potential impact of antidepressants or anxiety/depression on sperm quality. There was no funding to report for this study. Z.W. is a contributing author for UptoDate. S.S.S. is on the advisory board for Ferring Pharmaceuticals. E.G. was a medical consultant for Hall-Matson Esq, Teladoc, and CRICO and is a contributing author for UptoDate. The remaining authors have nothing to report. N/A.

Comparison of bull sperm morphology evaluation methods under field conditions

Sperm morphological assessment is a critical component in bull breeding soundness evaluations. Although sperm morphology is an important parameter to identify subfertile from fertile bulls, the evaluation can be biased because sperm staining methods used under field conditions may not be exact due to various factors, including artifacts. Objective was to compare 2 sperm morphological evaluation methods. Prebreeding season ejaculates of 1,216 Angus cross bulls collected via electroejaculation were evaluated. For each bull, an unstained (UNS) and an eosin-nigrosin stained (ENS) semen smear were viewed under a phase contrast microscope and a brightfield microscope with an oil immersion lens, both at 1,000 × magnification. Normal and percentage of abnormal sperm were identified by counting 200 sperm. Inter-rater agreements between 2 clinicians for the percentage of abnormal sperm determination and its categories were very good (ENS method, r = 0.84 – 0.96; UNS method, r = 0.76 – 0.96; p &lt; 0.01). No differences (p &gt; 0.1) were observed for abnormal sperm percentage determination and its categories between 2 methods. Correlation was very good between 2 methods for total abnormal sperm percentage determination (r = 0.91; p &lt; 0.01) and its categories (r = 0.84 – 0.96; p &lt; 0.05). Additionally, 60 ejaculates were evaluated by triple stain (TS), ENS, and UNS methods. Agreements between TS (percentage of sperm with damaged membrane) and ENS (percentage of abnormal sperm) and between TS and UNS methods were moderate (r = 0.58; p &lt; 0.05) and fair (r = 0.43; p &lt; 0.05), respectively. Based on our findings, either technique can be used for bull sperm morphological evaluation under field conditions. Considering the ease of semen smear preparation, the UNS method can be a viable alternative to the ENS method.

P-075 Artificial intelligence for sperm analysis: A KNIME-Based automated analysis of sperm morphology

Abstract Study question To evaluate an automated sperm morphology assessment method using the KNIME Analytics Platform compared to manual sperm morphology analysis performed as per WHO guidelines. Summary answer Our method emphasizes how artificial intelligence technologies have the potential to foster standardization of sperm morphology assessment with comparable precision and reliability. What is known already Manual sperm morphology assessment is considered the most difficult parameter to standardize due to its subjective nature, strongly linked to the operator's level of expertise. Indeed, there is a high degree of inter and intra-laboratory variability. Manual examination is time-consuming and laborious. There were many attempts to automate sperm morphology analysis, especially with CASA (Computer Assisted Sperm Analysis) systems, but their performance is still disputable. One of the difficulties in this field of study is the lack of publicly available datasets. Besides, the available databases are only focused on the sperm head morphology. Study design, size, duration A total of 37 semen samples from men attending our laboratory for infertility investigation were included, over a period of one year. For each sample, semen smears were fixed and stained by the Spermoscan® kit. Participants/materials, setting, methods A total of 1000 images of individual spermatozoa were obtained using the MMC® CASA system. The number of images per sample depended on its quality. Three experts have classified these spermatozoa according to modified David classification for sperm morphology. The results were then processed and an algorithm created using teh KNIME Analytics Platform, trained and tested to classify spermatozoa. This workflow uses CNN (convolutional neural network) to perform image classification on our dataset. Main results and the role of chance Of the 1000 images analyzed, we counted : 116 Normal sperm morphology, 67 abnormal post-acrosomal region, 128 abnormal acrosomes, 8 elongated heads, 6 thin heads,10 microcephalic,7 multiple heads, 27 coiled tails, 7 cytoplasmic residues, 17 angulated tails, 6 short tails, 4 multiple tails, and 697 associated abnormalities. For each image, a notebook file containing sperm abnormalities as assessed by the three experts was created, in addition to sperm head, mid-piece and tail dimensions obtained by the CASA system. Our dataset was randomly partitioned into 2 groups: 80% of data formed the training set, and The remaining 20% formed the fully independent test set. The best performance with the KNIME-Based algorithm was achieved for post-acrosomal abnormalities (97 % True positive rate), and the worst for multiple tail abnormalities (69 % True positive rate). Our machine learning model classifies sperm morphology at high accuracy (99.5 %). The overall process occurs in less than 10 seconds. Limitations, reasons for caution Our database included various head, midpiece and tail anomalies. However, the number of images in each category is unequal due to the limited occurrence of some morphological abnormalities. Thus, it is important to increase the number of images in these categories to obtain better results. Wider implications of the findings Our goal is to expand our modified David’s classification-based database. We aim to improve the performance of our model, and to put it at the service of learning and routine use in laboratories. Trial registration number not applicable

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.

MP30-15 THE QUESTIONABLE CLINICAL UTILITY OF SPERM MORPHOLOGY: RATE OF ISOLATED TERATOSPERMIA IN A POPULATION OF FERTILE MEN

You have accessJournal of UrologyCME1 Apr 2023MP30-15 THE QUESTIONABLE CLINICAL UTILITY OF SPERM MORPHOLOGY: RATE OF ISOLATED TERATOSPERMIA IN A POPULATION OF FERTILE MEN David Miller, Lucille Cheng, Anna Wecht, and Kathleen Hwang David MillerDavid Miller More articles by this author , Lucille ChengLucille Cheng More articles by this author , Anna WechtAnna Wecht More articles by this author , and Kathleen HwangKathleen Hwang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The assessment of sperm morphology remains a standard component of the semen analysis however its clinical utility is not well-defined. The WHO guidelines have progressively decreased the percentage of normal morphology over the years and the current provided reference range is 4% or greater. Despite these increasingly strict criteria for the assessment of morphology the clinical relevance remains questionable. Furthermore, accurate repeatability of sperm morphology assessment has been shown to be low due to differences in proficiency between labs and technicians themselves. To better understand the impact of sperm morphology on fertility we assessed sperm morphology in a population of known fertile men. METHODS: Healthy men over 18 years of age were recruited to provide one semen sample prior to vasectomy from 3/2020 to 8/2022. To be included in the study participants were required to have biological children under the age of 5 years old, with no history of difficulty achieving pregnancy, and had undergone no prior fertility procedures. Sperm morphology was analyzed using Papanicolaou stain with Kruger strict criteria by two technicians in an Andrology specialty laboratory per the WHO 5th edition manual. RESULTS: There were 65 men who participated in the study, of which there was a morphology assessment available for 65 patients. The average age of participants was 36 and the mean BMI was 27.9 (20-43.7). Of those with smoking history available, 61% of men were non-smokers, 20% were former smokers, and 13% were current smokers. Patients had an average of 2.7 children with a mean age of the youngest child of 15 months old. The mean sperm density was 57.3 mil/ml, the mean motility was 61%, and mean progressive motility was 76%. The median morphology was 3%. There were 33 patients (51%) with 3% or lower sperm morphology including one patient who had 0% normal morphology (Figure 1). The most common morphologic abnormalities were head shape defects (n=58), followed by coiled tails (n=15) with some patients having both abnormalities. CONCLUSIONS: Over half of fertile male patients had lower than normal sperm morphology in our study. The results of our study further question the clinical relevance of sperm morphology on fertility outcomes and if the current approach in assessing morphology is too strict. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e396 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information David Miller More articles by this author Lucille Cheng More articles by this author Anna Wecht More articles by this author Kathleen Hwang More articles by this author Expand All Advertisement PDF downloadLoading ...

Animal board invited review: An update on the methods for semen quality evaluation in swine - from farm to the lab.

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined.

The effect of sperm DNA fragmentation on in vitro fertilization outcomes of unexplained infertility

BackgroundInfertility is caused by heterogeneous risks, but most of them are unexplained. The sperm DNA Fragmentation Index (DFI) was increasingly acknowledged as a parameter for the evaluation of male infertility. This study aimed to investigate the association between sperm DFI and laboratory and clinical outcomes in a population with unexplained infertility. MethodsThe clinical data of an infertile population was collected for the selection of reproductive patients with unexplained infertility. The authors classified the patients with normal sperm parameters in a control group (DFI < 25%) and an observation group (DFI ≥ 25%) and compared the difference in basal characteristics, laboratory, and clinical outcomes between the two groups. The authors conducted a correlation analysis to examine the relationship between DFI and the number of D3 good-quality embryos, as well as the clinical pregnancy rate and live birth rate. A total of 176 cases were enrolled in the retrospective study. ResultsThe observation group (n = 88) showed advanced male age, lower sperm concentration, progressive motility, and morphology assessment than the control group. In addition, lower No. of D3 good-quality embryos, clinical pregnancy rate, and the live birth rate were shown in the observation group. A negative correlation between the DFI and No. of D3 good-quality embryos (rs = -0.347, p < 0.001) or live birth rate (rs = -0.185, p = 0.028) was shown. ConclusionsSperm DFI was a good indicator for the prediction of D3 good-quality embryos in unexplained infertility couples, but it did not provide sufficient information regarding clinical pregnancy outcome but live pregnancy outcome.

Effect of N-acetyl-L-cysteine on Testicular Tissue in Busulfan-Induced Dysfunction in the Male Reproductive System.

This study aimed to evaluate the protective effect of N-acetyl-L-cysteine (NAC) as an antioxidant on busulfan-induced testicular dysfunction in mice and elucidate its possible mechanism of action. Thirty-two C57BL/6 male mice were randomly divided into four groups (n=8/group) as follows: (1) control group (oral administration of saline [0.1 mL daily] for 35 days); (2) NAC group (oral administration of NAC [10 mg/kg daily] for 35 days); (3) busulfan group (double intraperitoneal injections of 20 mg/kg; total dose of 40 mg/kg); and (4) busulfan+NAC group (after double intraperitoneal injections of 20 mg/kg; total dose of 40 mg/kg, NAC administration [10 mg/kg daily] for 35 days). The testes were removed, weighed, and subjected to sperm parameter analysis and morphology assessment. Reproductive hormone, serum/testicular reactive oxygen species (ROS) level, oxidative stress and antioxidant markers were evaluated. The testicular expression of Nrf2 and HO-1 was examined using RT-qPCR. Busulfan treatment significantly decreased testicular weight, sperm count, and serum testosterone levels. Atrophy and degeneration of germinal epithelium were observed in the busulfan group. NAC administration after busulfan treatment partially attenuated the deterioration of testis weight, sperm quality, serum hormones, histomorphometric changes, and oxidative and antioxidative status. NAC treatment resulted in a considerable improvement in Nrf2 and HO-1 mRNA expression levels. This study provides compelling evidence that NAC as a potent antioxidant has significant protective effects against busulfan-induced male reproductive impairment possibly through modification of the Nrf2/HO-1 signaling pathway.

THE QUESTIONABLE CLINICAL UTILITY OF SPERM MORPHOLOGY: RATE OF ISOLATED TERATOSPERMIA IN A POPULATION OF FERTILE MEN

The assessment of sperm morphology remains a standard component of the semen analysis however its clinical utility is not well-defined. The WHO guidelines have progressively decreased the percentage of normal morphology over the years and currently normal is 4% or greater. Despite these increasingly strict criteria for the assessment of morphology the clinical implications remain questionable. Furthermore, accurate repeatability of sperm morphology assessment has been shown to be low due to differences in proficiency between labs and technicians themselves. To better understand the impact of sperm morphology on fertility we assessed sperm morphology in a population of known fertile men.

Ensembled Deep Learning for the Classification of Human Sperm Head Morphology

Infertility is a growing global health concern, with male factor infertility contributing to half of all cases. Semen analysis is crucial to infertility diagnostics. However, sperm morphology assessment, as a routine part of analysis, is still performed manually and is thus highly subjective. Here, a stacked ensemble of convolutional neural networks (CNNs) is presented for automated classification of human sperm head morphology. By combining traditional CNN models with modern residual and densely connected architectures using a multi‐class meta‐classifier, classification rate improvements of 2.7% (to 98.2%) and 2.3% (to 63.3%) on the HuSHeM and SCIAN‐MorphoSpermGS (SCIAN) datasets, respectively, are achieved. This considerable improvement in prediction performance is achieved as the meta‐classifier improves upon the individual classification rates of the base models by ≈8.5%. The ensembled deep learning model is a powerful step toward an automated sperm morphology analysis, providing new opportunities to standardize clinical practice and reduce treatment costs to improve patient treatment.