Computer-aided Sperm Analysis System Research Articles

An objective quality indicator for evaluating filtering of microscopic video in CASA system

Computer-Aided Sperm Analysis (CASA) has received great attention in the diagnosis of male reproductive health for its objectivity and accuracy. But its accuracy may be greatly affected by algorithms used in microscopic video preprocessing, especially algorithms for microscopic videos filtering. Owing to the Peak Signal to Noise Ratio (PSNR) and Structural Similarity (SSIM) indicators are not suitable for microscopic images, objective indicators for evaluating filtering of microscopic videos are scarce at present, leading to filtering algorithms are chosen arbitrarily. In addition, it is critical to establish quality control indicators for CASA from the perspective of image processing, but they are rarely mentioned for the reason that algorithms adopted in image processing aren’t public. To reduce influences of filtering on diagnostic results, it is imperative to establish an objective quality control indicator for evaluating filtering of microscopic video from the perspective of image preprocessing. The principle of formatting sperm tracking trajectory and the rules of grading trajectory continuity were introduced firstly. Then, impacts of filtering algorithms on the continuity of sperm trajectory were investigated and the relation between the continuity of sperm trajectory and the fluctuation of sperm segmentation thresholds was further explored. Finally, an objective indicator for appraising the quality of filtering for microscopic video was established based on SADTM (Sum of Absolute Difference between Threshold and the Mean).The proposed indicator can objectively evaluate the quality of microscopic video filtering and provide a guideline for algorithm selection, laying foundation for quality control for CASA.

Ensembling noisy segmentation masks of blurred sperm images

Background:Sperm tail morphology and motility have been demonstrated to be important factors in determining sperm quality for in vitro fertilization. However, many existing computer-aided sperm analysis systems leave the sperm tail out of the analysis, as detecting a few tail pixels is challenging. Moreover, some publicly available datasets for classifying morphological defects contain images limited only to the sperm head. This study focuses on the segmentation of full sperm, which consists of the head and tail parts, and appear alone and in groups. Methods:We re-purpose the Feature Pyramid Network to ensemble an input image with multiple masks from state-of-the-art segmentation algorithms using a scale-specific cross-attention module. We normalize homogeneous backgrounds for improved training. The low field depth of microscopes blurs the images, easily confusing human raters in discerning minuscule sperm from large backgrounds. We thus propose evaluation protocols for scoring segmentation models trained on imbalanced data and noisy ground truth. Results:The neural ensembling of noisy segmentation masks outperforms all single, state-of-the-art segmentation algorithms in full sperm segmentation. Human raters agree more on the head than tail masks. The algorithms also segment the head better than the tail. Conclusions:The extensive evaluation of state-of-the-art segmentation algorithms shows that full sperm segmentation is challenging. We release the SegSperm dataset of images from Intracytoplasmic Sperm Injection procedures to spur further progress on full sperm segmentation with noisy and imbalanced ground truth. The dataset is publicly available at https://doi.org/10.34808/6wm7-1159.

Dietary Moringa oleifera leaves to male rainbow trout (Oncorhynchus mykiss) broodstock: Effects on sperm quality and reproductive performance

Moringa oleifera leaves (MOL), a superfood rich in protein and vitamins, is native to the Indian subcontinent. Moringa were added to the broodstock trout diet to examine their influence on the reproductive performance of male broodstock rainbow trout (Oncorhynchus mykiss). Females were fed with a commercial broodstock trout diet without any inclusion of moringa leaves. In 12 circular fiberglass tanks, 36 male rainbow trout aged 3+ (1786.36 ± 35.36 g; 56.08 ± 0.94 cm) were divided into four groups (n = 9) (control, MOL-1, MOL-2, and MOL-3) with three parallels each. Fourteen weeks of feeding trials were performed where powdered moringa leaf was used in the diets at various levels: 0% (control), 4% (MOL-1), 8% (MOL-2), and 16% (MOL-3). Sperm quality parameters and fertilization success were investigated. Sperm motility parameters were examined at 10.5 °C using a computer-aided sperm analysis (CASA) system, and all samples were analyzed on the same day. Sperm volume and seminal plasma osmolality did not differ significantly across groups (p > 0.05). Spermatozoa concentration (6.71 ± 0.25, 7.54 ± 0.31, 8.44 ± 0.55, and 7.66 ± 0.35 × 109 cells ml−1 in control, MOL-1, MOL-2, and MOL-3 groups, respectively), spermatocrit ratio (18 ± 2.55%, 25 ± 3.16%, 38 ± 6.44%, and 28 ± 5.15%), and motility duration differed significantly (p < 0.05) among groups. Total motility percentage did not change, but progressive motility (74.72 ± 5.94%, 88.52 ± 4.35%, 89.36 ± 1.76%, and 90.47 ± 2.67%) was considerably higher in all three moringa groups than in control one (p < 0.05). Average Path Velocity (VAP), Straight Line Velocity (VSL), and Curvilinear Velocity (VCL) significantly differ between control and treatment groups (p < 0.05). The fertilization rate in the MOL-2 group was similar to the control and MOL-3 groups, while significant differences were observed from the eyeing stage, with the highest result observed in the MOL-2 group (p < 0.05). Hatching rates were also highest in the MOL-2 group (81.65 ± 0.49%), followed by MOL-3 (79.08 ± 1.27%) and MOL-1 (74.40 ± 2.32%), with the control group having the lowest hatching rate (74.24 ± 0.98%) (p < 0.05). Progressive motility, VAP, VSL, and VCL have significantly enhanced in the moringa-treated groups than the control one, hence boosting the fertilization success. The present study revealed that moringa leaves in the diet improve the sperm quality parameters and enhance the reproductive performance in broodstock male rainbow trout (Oncorhynchus mykiss). Therefore, 8% moringa leaf can be recommended as a supplement diet for male rainbow trout broodstock.

Effect of High Viscosity on Energy Metabolism and Kinematics of Spermatozoa from Three Mouse Species Incubated under Capacitating Conditions

In order to sustain motility and prepare for fertilization, sperm require energy. The characterization of sperm ATP production and usage in mouse species revealed substantial differences in metabolic pathways that can be differentially affected by capacitation. Moreover, spermatozoa encounter different environments with varying viscoelastic properties in the female reproductive tract. Here, we examine whether viscosity affects sperm ATP levels and kinematics during capacitation in vitro. Sperm from three mouse species (Mus musculus, M. spretus, M. spicilegus) were incubated under capacitating conditions in a modified Tyrode's medium containing bicarbonate, glucose, pyruvate, lactate, and bovine serum albumin (mT-BH) or in a bicarbonate-free medium as a non-capacitating control. Viscosity was increased with the inclusion of polyvinylpyrrolidone. ATP was measured with a bioluminescence kit, and kinematics were examined with a computer-aided sperm analysis system. In M. musculus sperm, ATP declined during capacitation, but no differences were found between non-capacitating and capacitating sperm. In contrast, in M. spretus and M. spicilegus, ATP levels decreased in capacitating sperm. Increasing viscosity in the medium did not modify the timing or proportion of cells undergoing capacitation but did result in additional time- and concentration-dependent decreases in ATP in M. spretus and M. spicilegus under capacitating conditions. Additionally, increased viscosity altered both velocity and trajectory descriptors. The limited impact of capacitation and higher viscosity on M. musculus sperm ATP and kinematics could be related to the low intensity of postcopulatory sexual selection in this species. Responses seen in the other two species could be linked to the ability of their sperm to perform better under enhanced selective pressures.

Alkaline semen diluent combined with R848 for separation and enrichment of dairy goat X-sperm.

A large number of female goats are needed for the dairy goat industry; therefore, the development of a method to ensure the birth of more females than males in a single pregnancy will lead to economic benefits. Increasing the number of X-sperm would be an effective way to increase the proportion of female offspring. In this study, goat semen was incubated at pH 7.4 in alkaline diluent combined with resiquimod (R848) and the number of X-sperm was enriched by the swim-up method. The percentage of X-sperm was determined using the double TaqMan qPCR method. Sperm total motility, progressive motility, average path velocity, straight-line velocity, and curvilinear velocity were measured using a computer-aided sperm analysis system, and the functional parameters of the sperm plasma membrane, the acrosome, mitochondrial activity, ATP content, and reactive oxygen species levels were also measured. Lastly, the ratio of female embryos was determined by in vitro fertilization, and the number of female kids and the pregnancy rate of does was assessed by artificial insemination. The results showed that dilution of semen in an alkaline buffer containing R848 could enrich the number of X-sperm to 85.57% ± 3.27%. The progressive motility, average path velocity, straight-line velocity, curvilinear velocity, mitochondrial activity, and ATP level of the collected X-sperm-enriched semen were significantly reduced, but its total motility, plasma membrane, and acrosome were not affected. The in vitro fertilization experiments showed that the rate of female embryo production using X-sperm-rich seminal fluid could reach 83.25% (174/209), which was significantly higher than the proportion of female embryos in the control group, 47.71% ± 1.80% (104/218). As determined by artificial insemination, the number of female kids in the test group increased by 62.79% (243/387), which was significantly higher than that in the control group (47.65%, 193/405). There was no significant difference in pregnancy rate between the test group and the control group (71.71% vs. 78.48%). Therefore, this study demonstrated that use of a pH 7.4 diluent containing R848 is a simple and effective method of X-sperm enrichment for dairy goat production. Its application would allow does to produce more female offspring for herd expansion and milk production.

LensHooke® X1PRO comparison with laboratory based manual semen analyses in a Chilean men’s population.

For over 30 years, CASA-Mot technology has been used for kinematic analysis of sperm motility in different mammalian species, but insufficient attention has been paid to the technical limitations of commercial computer-aided sperm analysis (CASA) systems. Counting chamber type and frame rate are two of the most important aspects to be taken into account. Counting chambers can be disposable or reusable, with different depths. In human semen analysis, reusable chambers with a depth of 10 µm are the most frequently used, whereas for most farm animal species it is more common to use disposable chambers with a depth of 20 µm . The frame rate was previously limited by the hardware, although changes in the number of images collected could lead to significant variations in some kinematic parameters, mainly in curvilinear velocity (VCL). A frame rate of 60 frames s−1 is widely considered to be the minimum necessary for satisfactory results. However, the frame rate is species specific and mus...

Study on cryopreservation of Guanzhong dairy goat semen with bovine semen seminal plasma

There are phospholipase and yolk agglutinase in the seminal plasma of dairy goat. The existence of these enzymes will hydrolyze lecithin in yolk into lysophosphatides and cause the change of pH value of diluent, resulting in toxic and side effects on sperm. In order to explore the effect of bovine seminal plasma replacing different doses of dairy goat seminal plasma (0%, 25%, 50%, 100%) on semen freezing quality of Guanzhong dairy goat, we used computer-aided sperm analysis (CASA) system, specific fluorescent probe detection technology, hypo osmotic swelling test (HOST), oxidation index determination and in vivo fertilization verification test to study the semen cryopreservation of Guanzhong dairy goat. The results showed that if we used bovine seminal plasma instead of 100% dairy goat seminal plasma, the sperm motility, plasma membrane functionality and acrosome integrity rate were the highest after thawing, reaching 67.04%, 38.12% and 68.83% respectively, which was significantly higher than that of other groups (P < 0.05); In terms of oxidation indexes, the values of SOD and GSH-PX in sperm of dairy goats were 199.78 U/mL and 133.55 U/L, respectively, which were significantly higher than those in other groups (P < 0.05), and the values of ROS and MDA were 397.58 U/mL and 5.16 nmol/L, respectively, which were significantly lower than those in other groups (P < 0.05); The results of artificial insemination also showed that using bovine seminal plasma instead of 100% of dairy goat seminal plasma could significantly improve the fertilization ability of sperm. The quality of frozen Guanzhong dairy goat sperm can be improved by using bovine seminal plasma instead of dairy goat seminal plasma and using bovine diluent.

A new fluorescent method to determine honey bee sperm motility parameters with computer-aided sperm analysis

Honey bees are a keystone species, playing an important role in the food cycle. Improving honey bee reproduction will aid in the replacement of lost colonies. Fertility potential and reproductive health are dependent on semen and sperm quality. Current data on drone semen parameters are limited to semen volume, sperm concentration, sperm viability, and the assignment of sperm motility grade scores. The assessment of drone sperm motility is of importance to determine fertility potential and colony health. This study aimed to establish a quantitative and a qualitative method to measure drone sperm quality, and the fertility potential of Apis mellifera capensis subspecies of South Africa. Firstly, an improved five-point semi-quantitative manual motility index score was used to classify drone sperm motility. Secondly, it was possible to accurately analyse drone sperm motility qualitatively using a fluorescent technique in conjunction with a computer-aided sperm analysis (CASA) system. Manual motility index scores corresponded with total motility percentages as determined by using a CASA system. Furthermore, low values for motility kinematic parameters, particularly velocity parameters, were obtained in samples with both low motility index scores and low total motility percentages. Additionally, total sperm motility percentage and velocity parameters positively correlated with sperm total progressivity. This study provides in-depth data on honey bee drone sperm motility and motility kinematic parameters, which can serve as a reference for future studies on honey bee sperm and possibly related species.

Sperm activation in Black Sea Trout ( Salmo trutta labrax ): Effect of ions and osmolality on spermatozoa motility

In this study, sperm characteristics of Black Sea trout (Salmo trutta labrax) and effects of ions, their concentrations and osmolality of the solution on sperm activation were evaluated. Two different activation solutions with ionic and non-ionic osmolality levels were investigated. After the definition of conditions where osmolality played a role, activation solutions containing calcium ion (Ca2+) in different molarity levels were tested under single and combined solutions to determine their relations (Sperm motility percentage was determined by using computer-aided sperm analysis system (CASA) for each activation solution). It was found that Black Sea trout sperm motility is initiated with iso-osmolality and activation solution with non-ionic osmolality had no effect on motility whereas ionic osmolality was effective on motility. The best solution determined in this study for the Black Sea trout is proved to have the same osmolality as the seminal plasma of the handled fish prepared with NaCl containing 2–32 mM CaCl2.

Effect of Glutathione on Sperm Quality in Guanzhong Dairy Goat Sperm During Cryopreservation.

In the process of cryopreservation of dairy goat semen, it will face many threats such as oxidative damage, which will affect the motility and plasma membrane function of sperm. As an endogenous antioxidant in animals, glutathione (GSH) can significantly improve the quality of thawed sperm when added to the frozen diluent of semen of pigs and cattle. In this study, different concentration gradients of GSH [0 mmol/L (control), 1, 2, 3, 4 mmol/L] were added to the frozen diluent of Guanzhong dairy goat semen. By detecting the sperm motility parameters, acrosome intact rate and plasma membrane intact rate after thawing, the effect of GSH on the cryopreservation of dairy goat semen was explored. Sperm motility parameters were measured with the computer-aided sperm analysis (CASA) system (total power, TM; forward power, PM; linearity, LIN; average path speed, VAP; straight line speed, VSL; curve speed, VCL; beat cross frequency, BCF). The sperm acrosome integrity rate after thawing was detected by a specific fluorescent probe (isothiocyanate-labeled peanut agglutinin, FITC-PNA), and the sperm plasma membrane integrity rate after thawing was detected by the hypotonic sperm swelling (HOST) method. Reactive oxygen species (ROS) kit, malondialdehyde (MDA) kit, superoxide dismutase (SOD) kit, glutathione peroxidase (GSH-PX) kit were used to detect various antioxidant indicators of thawed sperm. in vitro fertilization experiment was used to verify the effect of adding glutathione on sperm fertilization and embryo development. The results showed that when the concentration of glutathione was 2 mmol/l, the sperm viability, plasma membrane intact rate, and acrosome intact rate were the highest after thawing, reaching 62.14, 37.62, and 70.87% respectively, and they were all significantly higher. In terms of antioxidant indexes; the values of SOD and GSH-PX were 212.60 U/ml and 125.04 U/L, respectively, which were significantly higher than those of the control group; The values of ROS and MDA were 363.05 U/ml and 7.02 nmol/L, respectively, which were significantly lower than the control group. The addition of 2 mmol/L glutathione significantly improves the fertilization ability of sperm. In short, adding 2 mmol/l glutathione to the semen diluent can improve the quality of frozen Guanzhong dairy goat sperm.

P–029 Identification of spermatozoa by unsupervised learning from video data

Abstract Study question Can artificial intelligence (AI) algorithms identify spermatozoa in a semen sample without using training data annotated by professionals? Summary answer Unsupervised AI methods can discriminate the spermatozoon from other cells and debris. These unsupervised methods may have a potential for several applications in reproductive medicine. What is known already Identification of individual sperm is essential to assess a given sperm sample’s motility behaviour. Existing computer-aided systems need training data based on annotations by professionals, which is resource demanding. On the other hand, data analysed by unsupervised machine learning algorithms can improve supervised algorithms that are more stable for clinical applications. Therefore, unsupervised sperm identification can improve computer-aided sperm analysis systems predicting different aspects of sperm samples. Other possible applications are assessing kinematics and counting of spermatozoa. Study design, size, duration Three sperm-like paint images were manipulated using a graphic design tool and used to train our AI system. Two paintings have an ash colour background and randomly distributed white colour circles, and one painting has a predefined pattern of circles. Selected semen sample videos from a public dataset with videos obtained from 85 participants were used to test our AI system. Participants/materials, setting, methods Generative adversarial networks (GANs) have become common AI methods to process data in an unsupervised way. Based on single image frames extracted from videos, a GAN (SinGAN) can be trained to determine and track locations of sperms by translating the real images into localization paintings. The resulting model showed the potential of identifying the presence of sperms without any prior knowledge about data. Main results and the role of chance Visual comparisons of localization paintings to real sperm images show that inverse training of SinGANs can track sperms. Converting colour frames into grayscale frames and using grayscale synthetic sperm-like frames showed the best visual quality of generated localization paintings of sperm frames. Feeding real sperm video frames to the SinGAN at different scaling factors, which is defining the resolution of the input image, showed different quality levels of generated sperm localization paintings. A sperm frame given to the algorithm with a scaling factor of one leads to random sperm tracking, while the scales two to four result in more accurate localization maps than scaling levels five to eight. In contrast, scales from six to eight result in an output close to the input frame. The proposed method is robust in terms of the number of spermatozoa, meaning that the detection works well for samples with a low or high sperm count. For visual comparisons, visit our Github page: https://vlbthambawita.github.io/singan-sperm/. The sperm tracking speed of our SinGAN using an NVIDIA 1080 graphic processing unit, is around 17 frames per second, which can be improved by using parallel video processing capabilities. This shows the capability of using this method for real-time analysis. Limitations, reasons for caution Unsupervised methods are hard to train, and the results need human verification. The proposed method will need quality control and must be standardized. Unsupervised sperm tracking SinGAN may identify blurry bright spots as non-existing sperm heads which may restrict the use of SinGAN sperm tracking for sperm counting. Wider implications of the findings: Assessment of semen samples according to the WHO guidelines is subjective and resource-demanding. This unsupervised model might be used to develop new systems for less time-consuming and more accurate evaluation of semen samples. It may also be used for real-time analysis of prepared spermatozoa for use in assisted reproduction technology. Trial registration number N/A

Association of Lifestyle Factors and Sperm Motility in Adults from an Ethnic Minority Region of Southwest China

Objectives: To understand sperm motility in adults and its association with lifestyle in an ethnic minority area in Southwest China. Methods: A hospital-based cross-sectional study to assess sperm motility in male adults was conducted at the Reproductive Health Center from January 2018 to May 2019. The data was collected with a questionnaire and semen quality was analyzed with Computer-Aided Sperm Analysis system (CASA). Analysis of covariance (ANCOVA) was used to measure the relationship between lifestyle factors and sperm motility. Results: A total of 349 people were recruited. Dietary celery intake was significantly related to the increase of sperm progressive motility and total motility (β = 7.00, 95% CI: 1.59, 12.42 and β = 7.26, 95% CI: 1.45, 13.07, respectively). Cola consumption was associated with increased sperm progressive motility (β = 9.71, 95% CI: 1.46, 17.96). Frequent use of plastic bags for meat food storage (β = -5.56, 95% CI: -10.61, -0.51), industry work (β = -5.64, 95% CI: -11.21, -0.07), organic disease (β = -6.14, 95% CI: -11.00, -1.28) and sedentary lifestyle (β = -5.92, 95% CI: -10.66, -1.17 for 3-5 h/d and β = -6.04, 95% CI: -11.60, -0.47 for ≥5 h/d, respectively) were related with the decreased sperm progressive motility. Meanwhile, using plastic bags for meat food storage (β = -6.37, 95% CI: -11.79, -0.95), industry work (β = -7.96, 95% CI: -13.94, -1.98) and sedentary lifestyle (β = -5.51, 95% CI: -10.60, -0.42 for 3-5 h/d and β = -6.03, 95% CI: -12.01, -0.06 for ≥5 h/d, respectively) were also risk factors for total motility. Conclusions: Some modifiable lifestyle factors such as job title, cola consumption, dietary celery intake, plastic bags for meat food storage, and sedentary hours were linked to male sperm motility, indicating that changing these lifestyles may improve it.

Standardized Laboratory Procedures, Quality Control and Quality Assurance Are Key Requirements for Accurate Semen Analysis in the Evaluation of Infertile Male.

Semen analysis is a basic test for evaluating male fertility potential, as it plays an essential role in driving the future management and treatment of infertility in couples. Manual semen analysis includes the evaluation of both macroscopic and microscopic parameters, whereas automated semen analysis is conducted through a computer-aided sperm analysis system and can include additional parameters that are not evaluated by manual analysis. Both quality control (QC) and quality assurance (QA) are important to ensure reproducible results for semen analysis, and represent fundamental checks and balances of all stages (pre-analytical, analytical, and post-analytical) of semen analysis. To ensure accuracy and precision, the laboratory technicians' performance should be evaluated biannually. This narrative review aims to describe standardized laboratory procedures for an accurate assessment of semen parameters that incorporate both QC and QA practices.

Comparison of commercially available chamber slides for computer-aided analysis of human sperm

ABSTRACT Despite the increasing use of computer-aided sperm analysis (CASA) in clinical practice, there is still no golden standard for the type of slide to be used with these systems. Counting chamber depth and loading method, can profoundly influence motility and concentration estimates, thereby undermining the validity and accuracy of CASA. To contribute toward standardized sperm analysis, this study compared different commercially available capillary-filled slides including 10 and 20 µm deep Leja slides (Leja10 and Leja20); 10, 16 and 20 µm deep CellVision slides (CV10, CV16 and CV20); and drop-loaded slides including slide and coverslip (SCS) with a depth of 20.1 µm and the Makler chamber with a depth of 10 µm for sperm analysis when using CASA. The Sperm Class Analyzer (SCA) CASA system was used to assess concentration, motility, and detailed kinematic parameters of 20 normozoospermic human samples using the different chamber slides. Results were evaluated by the repeated measures ANOVA and Intraclass correlation coefficients. The Makler chamber showed significantly (P < 0.05) higher concentrations than other slides. However, there was no significant difference in the percentage of sperm in different motility groups among the slides. CV10, Leja10 and Makler showed significantly higher curvilinear-, average path- and straight-line velocity (VCL, VAP, VSL) values than other slides. In conclusion, despite the objectiveness of the assessments by CASA systems, there are still some discrepancies in the results of sperm concentration, motility and other kinematic parameters when using different commercially available slides. The possible negative influence of the sperm quality misdiagnosis on the selection of treatment strategy in a clinical setting, emphasizes the need for further standardization and quality control of the commercially available chamber slides for use with CASA. Furthermore, this study found more consistent results for capillary-filled chambers compared to drop-loaded slides, suggesting a superior method when using CASA. Abbreviations: DNA: Deoxyribonucleic acid; CASA: Computer-aided semen analysis; SCA: Sperm Class Analyzer; WHO: World Health Organization; CV: CellVision; SCS: Slide and Coverslip; SD: Standard deviation; ANOVA: Analysis of Variance; PR: Progressive; NP: Non-progressive; IMT: Immotile; VCL: Curvilinear velocity; VAP: Average path velocity; VSL: Straight-line velocity; Lin: Linearity; STR: Straightness; WOB: Wobble; ALH: Amplitude of lateral head displacement; BCF: Beat cross frequency

A preliminary study of sperm identification in microdissection testicular sperm extraction samples with deep convolutional neural networks.

Sperm identification and selection is an essential task when processing human testicular samples for in vitro fertilization. Locating and identifying sperm cell(s) in human testicular biopsy samples is labor intensive and time consuming. We developed a new computer-aided sperm analysis (CASA) system, which utilizes deep learning for near human-level performance on testicular sperm extraction (TESE), trained on a custom dataset. The system automates the identification of sperm in testicular biopsy samples. A dataset of 702 de-identified images from testicular biopsy samples of 30 patients was collected. Each image was normalized and passed through glare filters and diffraction correction. The data were split 80%, 10%, and 10% into training, validation, and test sets, respectively. Then, a deep object detection network, composed of a feature extraction network and object detection network, was trained on this dataset. The model was benchmarked against embryologists' performance on the detection task. Our deep learning CASA system achieved a mean average precision (mAP) of 0.741, with an average recall (AR) of 0.376 on our dataset. Our proposed method can work in real time; its speed is effectively limited only by the imaging speed of the microscope. Our results indicate that deep learning-based technologies can improve the efficiency of finding sperm in testicular biopsy samples.

In vitro effects of imidacloprid on honey bee sperm: evaluation using computer-aided sperm analysis (CASA)

Pesticides are considered as the most important factors of pollinators’ decline. Imidacloprid (IMD), belonging to Neonicotinoids class, is widely used as a powerful insecticide but generating important negative effects on honey bees. The current study investigated the direct effect of IMD on bee spermatozoa. The experimental design consisted of in vitro co-incubation of bee gametes with three different concentrations of IMD 1, 10, and 25 μM for 15 min. Conventional microscopic and Computer-aided sperm analyses (CASA) were concomitantly used with a dual goal: to evaluate objectively the effects of IMD on sperm parameters on the one hand and to report computer kinematic parameters in the control group, without IMD treatment, on the other hand. The results revealed apparent negative effects in a dose-dependent manner with motility collapsing completely at 25 μM of IMD. The current results highlighted the direct impact of IMD on bee spermatozoa and revealing the potential subsequent effects on bee reproduction. This study reported also drone computer sperm parameters that could serve as a reference in the studied region. Bee sperm velocities showed the lowest values ever reported in the different animal species. The CASA system appeared as objective and a sensitive method to detect subtle toxic effects on bee sperm, and this opens real perspectives particularly in studying existing correlations between CASA parameters and fertility outputs of different environmentally toxic molecules.

Smartphone based sperm counting - an alternative way to the visual assessment technique in sperm concentration analysis

Sperm Counting is the first phase of the infertility diagnosis. Computer Aided Sperm Analysis (CASA) and Visual Assessment (VA) are two evaluation techniques employed in analyses. VA is carried out by observing sperm on counting chambers. Therefore, diagnosis strongly depends on the skills and experiences. CASA isolates the human factor by utilizing the computer based techniques. However, it is more costly than VA and requires exhausted parameter settings. In this study, we present a novel approach that uses smartphone and computer for sperm counting analysis. Smartphone is used to obtain images similar to VA technique. Then, sample videos are transferred to the computer side where we developed the Computerized Sperm Counting Software (CSCS) to count the sperm using counting chambers and eliminate human effects. CSCS consists of four modules: (1) Data Acquisition and Organization, (2) Regions of Interest (ROI) detection, (3) Motile/Immotile Sperm Detection, (4) Counting. Smartphone based data acquisition provided less costly design contrary to CASA systems. ROI extraction was realized by a combinational approach of line detection and segmentation methods. Background and Foreground extractions were employed in immotile and motile sperm detection process, respectively. Additionally, active contour was implemented to enhance the segmentation of immotile sperm. As the final step, detected sperms were counted by pixel based blob analysis. According to experimental results, the proposed smartphone based sperm concentration analysis can be adapted in laboratories due to its modularity, functionality, accuracy and cost when compare to CASA and VA based sperm counting analysis.

A novel copy number variation in CATSPER2 causes idiopathic male infertility with normal semen parameters.

Are genetic abnormalities in CATSPER (cation channel of sperm) genes associated with idiopathic male infertility with normal semen parameters and, if so, how do they affect male fertility? A novel copy number variation (CNV) in CATSPER2 causes idiopathic male infertility with normal semen parameters by disrupting the ability of sperm to penetrate viscous media, undergo hyperactivation and respond to progesterone. CATSPER is the principle Ca2+ channel mediating extracellular Ca2+ influx into spermatozoa. Although several case reports have suggested a causal relationship between CATSPER disruption and human male infertility, whether genetic abnormalities in CATSPER genes are associated with idiopathic male infertility with normal semen parameters remains unclear. Spermatozoa were obtained from men attending the reproductive medical center at Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, China between January 2014 and June 2016. In total, 120 men from infertile couples and 20 healthy male donors were selected to take part in the study, based on their normal semen parameters. CATSPER and KSPER currents were assessed using the whole-cell patch-clamp technique. Whole-genome sequencing and TaqMan® CNV assays were performed to identify genetic variations. The expression levels of genes encoding the CATSPER complex were measured by quantitative real-time PCR and Western blot. Sperm motion characteristics and hyperactivation were examined with a computer-aided sperm analysis (CASA) system. Sperm responses to progesterone, assessed as increases in CATSPER current and intercellular Ca2+ concentrations ([Ca2+]i), as well as inducement of penetration ability and acrosome reaction, were examined by means of whole-cell patch-clamp technique, single-sperm [Ca2+]i imaging, penetration into methylcellulose assay and chlortetracycline staining, respectively. An infertile man with complete disruption of CATSPER current was identified. This individual has a novel CNV which disrupts one gene copy in the region 43894500-43950000 in chromosome 15 (GRCh37.p13 Primary Assembly, nsv3067119), containing the whole DNA sequence of CATSPER2. This CNV affected the expression of CATSPER2, resulting in dramatically reduced levels of CATSPER2 proteins in the individual's spermatozoa. Although this individual exhibited normal semen parameters, his spermatozoa showed impaired penetration ability, deficient hyperactivation, and did not respond to progesterone, in terms of monovalent current potentiation, [Ca2+]i increase, penetration ability enhancement and acrosome reaction inducement, which may explain the individual's idiopathic infertility. N/A. Our novel findings require more cases to support the CATSPER2 CNV identified in this study as a common cause of idiopathic male infertility in patients with normal semen parameters. Therefore, caution must be taken when extrapolating the use of this CNV as a potential biomarker for idiopathic male infertility. The findings from the unique human CATSPER 'knockout' model in this study not only confirm the essential roles of CATSPER in mediating progesterone response and regulating hyperactivation in human spermatozoa but also reveal that disruption of CATSPER current is a significant factor causing idiopathic male infertility. This study was funded by National Natural Science Foundation of China (81771644 and 31400996 to T.L.; 31230034 to X.Z.); National Basic Research Program of China (973 Program, 2015CB943003 to X.Z.); National Key Research and Development Program of China (2016YFC1000905 to T.L.); Natural Science Foundation of Jiangxi, China (20121BBG70021 and GJJ12015 to X.Z.; 20161BAB204167 and 20171ACB21006 to T.L.) and the open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07 to T.L.). The authors have no conflicts of interest to declare.

Exposure of environmental Bisphenol A in relation to routine sperm parameters and sperm movement characteristics among fertile men

Although several human studies have examined bisphenol A (BPA) exposure in relation to routine sperm parameters, evidence of BPA’s effects on sperm movement characteristics is limited. We examined associations of BPA exposure with sperm parameters including sperm movement characteristics among fertile men. The cross-sectional study was conducted in Sandu County, Guizhou Province, China. Subjects provided semen samples analyzed by computer-aided sperm analysis (CASA) system and urine samples for BPA assay. They were invited to complete an in-person interview with a structured questionnaire to obtain demographics, lifestyle factors, etc. In final analyses, 500 subjects were included. We used multivariate linear regression analyses to estimate associations between BPA and sperm parameters after adjusting for potential confounders. BPA was detected in 73.6% of urine samples, with a geometric mean of 0.44 μg/gCreatinine. Compared with subjects of undetected BPA, subjects with detected BPA had increased Linearity (LIN, β: 2.19, 95% confidence interval (CI): 0.37, 4.0), Straightness (STR, β: 1.47, 95% CI: 0.19, 2.75), Wobble (WOB, β: 1.75, 95% CI: 0.26, 3.25), reduced Amplitude of lateral head displacement (ALH, β: −0.26, 95% CI: −0.5, −0.02) and Mean angular displacement (MAD, β: −2.17, 95% CI: −4.22, −0.11). Subjects in the highest tertile of creatinine-adjusted BPA group had lower sperm concentration than those with undetected BPA. Dose-response relationships of BPA with LIN, WOB, ALH, MAD and sperm concentration were demonstrated by statistically significant trends across tertiles of creatinine-adjusted BPA concentrations. Similar results were obtained using unadjusted BPA concentrations. Exposure to environmental BPA would decrease sperm concentration and sperm swing characteristics (ALH and MAD), and increase sperm velocity ratios (LIN, STR and WOB), which might mediate further effects on impaired male fecundity.

Bottlenose dolphin (Tursiops truncatus) sperm revisited: Motility, morphology and ultrastructure of fresh sperm of consecutive ejaculates

Computer aided sperm analysis systems allow detailed examination of sperm motility and morphology variables, which are important for the understanding of the spermatology of a species and the development of assisted reproductive techniques. Cetacean biology is too complex to study in the wild and data from captive individuals provide an important alternative for the conservation of these charismatic animals. The present study evaluates ejaculate and sperm characteristics, including sperm motility, kinematic variables and quantitative sperm morphology and ultrastructure, of consecutive ejaculates from Atlantic bottlenose dolphins (Tursiops truncatus). Sperm concentrations and total and progressive motilities were greater in the second than the first ejaculate, with all ejaculates being of very high quality (6.9–1127 × 106/ml sperm concentration, 75% to 91% total motility and 89% to 96% normal sperm). Most sperm in an ejaculate (≥84%) were highly (VCL>150 μm/s) and progressively motile with very few abnormal sperm. The sperm have small heads, a short but very bulky midpiece and a long tail. Detailed sperm morphometrics using CASA indicated there were similarities from one ejaculate to the next. The large mitochondria with extensive cristae mitochondriales are tightly packed in the midpiece resulting in a large midpiece volume. All the semen and sperm characteristics indicate high quality sperm and support the assumption that a multimale mating system is present in T. truncatus.