Semen analysis and sperm function testing

Despite controversy regarding the clinical value of semen analysis, male fertility investigation still relies on a standardized analysis of the semen parameters. This is especially true for infertility clinics in both developing and developed countries. Other optional tests or sophisticated technologies have not been widely applied. The current review addresses important changes in the analysis of semen as described in the new World Health Organization （WHO） manual for semen analysis. The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality. Apart from the above mentioned changes, the initial evaluation and handling methods remain, in most instances, the same as in previous editions. Furthermore, the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology. WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995. The external qualitY control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training. This review reports on current sperm functional tests, such as the induced acrosome reaction, and sperm-zona pellucida binding assays, as well as the impact of sperm quality in terms of DNA integrity, and the relationship of sperm function tests to sperm morphology.

Current status and potential of morphometric sperm analysis

The spermatozoon is the most diverse cell type known and this diversity is considered to reflect differences in sperm function. How the diversity in sperm morphology arose during speciation and what role the different specializations play in sperm function, however, remain incompletely characterized. This work reviews the hypotheses proposed to explain sperm morphological evolution, with a focus on some aspects of sperm morphometric evaluation; the ability of morphometrics to predict sperm cryoresistance and male fertility is also discussed. For this, the evaluation of patterns of change of sperm head morphometry throughout a process, instead of the study of the morphometric characteristics of the sperm head at different stages, allows a better identification of the males with different sperm cryoconservation ability. These new approaches, together with more studies employing a greater number of individuals, are needed to obtain novel results concerning the role of sperm morphometry on sperm function. Future studies should aim at understanding the causes of sperm design diversity and the mechanisms that generate them, giving increased attention to other sperm structures besides the sperm head. The implementation of scientific and technological advances could benefit the simultaneous examination of sperm phenotype and sperm function, demonstrating that sperm morphometry could be a useful tool for sperm assessment.

Influence of several uropathogenic microorganisms on human sperm motility parameters in vitro

Aim: The effects of certain uropathogenic microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus, Staphylococcus epidermidis and Mycobacterium tuberculosis) on human sperm motility characteristics were studied in vitro. Methods: In 10 healthy fertile men, ejaculates were aseptically obtained by masturbation and With a swim-up technique, a sperm suspension of high motility and purity was obtained. Several uropathogenic bacteria were obtained from outpatients with genitourinary tract infections. The sperm suspension was incubated with the pathogens at a bacteria: sperm ratio of 50:1 at 37℃. The sperm mobility parameters were estimated with a computerassisted sperm analyzer (CASA) provided with a multiple-exposure photography system (Madi Corp., Zhejiang, China). Measurements were carried out at 0, 2 and 4 hours of incubation. Results: Staphylococcus aureus significantly decreased the sperm motility and viability, but Staphylococcus epidermidis, Mycobacterium tuberculosis and Neisseria gonorrhoeae did not. Conclusion: Staphylococcus aureus has an inhibitory effect on human sperm motility in vitro.

Sperm speed is associated with sex bias of siblings in a human population

Recent studies investigating possible causes of male subfertility have largely focused on how lifestyle or environmental factors impact on the process of spermatogenesis, Markedly, fewer studies have investigated those risk factors that result in reduced sperm quality, such as poor sperm motility. The speed at which sperm swim is a major predictor of fertility and is extremely variable in human populations. It has been hypothesized that offspring sex may be adaptively manipulated to maximize the offspring＇s reproductive fitness （e.g., parents with genes for good male fertility traits, such as high sperm speed, would produce primarily sons and fewer daughters because the offspring will inherit advantageous male fertility genes）. Conversely, parents with poor male fertility genes would produce primarily daughters, We tested whether there was an association between how fast a man＇s sperm swam and the sex bias of his siblings in a sample of men attending clinic for fertility investigations with their partner and with a wide range of semen characteristics, including sperm speed. We found that the sex bias of a man＇s siblings is associated with his sperm speed; men with female-biased siblings had significantly slower sperm （judged using computer-assisted sperm analysis （CASA）） than men from male-biased sibships. This observation suggests family composition is an important factor that needs to be considered in future eDidemiological and clinical studies of human fertility,

UNE COLORATION SIMPLE ET RAPIDE DU FROTTIS DU SPERME

Objective To introduce a rapid simple staining method for sperm morphology.Methods Liquifiedsemen was on the glass, fixed by methanol, treated with phosphonic acid buffer, stained by A, B staining solution separately, and rinsed by running water. Results After staining, the whole sper-matozoid was very clear. The cephalic zone of spermatozoid head , acrosome area , was colored pink; its caudal zone colored violet red or violet blue. The body and tail of spermatozoid colored pink or light blue. Normal and abnormal morphology of spermatozoid could be differentiated easily. The sloughed spermatogenic cells and white blood cells could be differentiated also. Conclusion This rapid staining method of semen smear can produce the same effect of other usual staining method , but the staining time is shorter and the procedure is simpler.

Deep learning methods for noisy sperm image classification from convolutional neural network to visual transformer:a comprehensive comparative study

Background With the gradual increase of infertility in the world,among which male sperm problems are the main factor for infertility,more and more couples are using computer-assisted sperm analysis(CASA)to assist in the analysis and treatment of infertility.Meanwhile,the rapid development of deep learning(DL)has led to strong results in image classification tasks.However,the classification of sperm images has not been well studied in current deep learning methods,and the sperm images are often affected by noise in practical CASA applications.The purpose of this article is to investigate the anti-noise robustness of deep learning classification methods applied on sperm images.Methods The SVIA dataset is a publicly available large-scale sperm dataset containing three subsets.In this work,we used subset-C,which provides more than 125,000 independent images of sperms and impurities,including 121,401 sperm images and 4,479 impurity images.To investigate the anti-noise robustness of deep learning classification methods applied on sperm images,we conducted a comprehensive comparative study of sperm images using many convolutional neural network(CNN)and visual transformer(VT)deep learning methods to find the deep learning model with the most stable anti-noise robustness.Results This study proved that VT had strong robustness for the classification of tiny object(sperm and impurity)image datasets under some types of conventional noise and some adversarial attacks.In particular,under the influence of Poisson noise,accuracy changed from 91.45%to 91.08%,impurity precison changed from 92.7%to 91.3%,impurity recall changed from 88.8%to 89.5%,and impurity F1-score changed 90.7%to 90.4%.Meanwhile,sperm precision changed from 90.9%to 90.5%,sperm recall changed from 92.5%to 93.8%,and sperm F1-score changed from 92.1%to 90.4%.Conclusion Sperm image classification may be strongly affected by noise in current deep learning methods;the robustness with regard to noise of VT methods based on global information is greater than that of CNN methods based on local information,indicating that the robustness with regard to noise is reflected mainly in global information.

Study on the Accuracy of Different CASA Systems in the Quality Detection of Fresh Boar Semen

[Objectives]This study was conducted to compare and analyze the accuracy of computer-aided sperm analysis(CASA)and manual method for detecting the quality of fresh boar semen at room temperature.[Methods]Statistical methods such as analysis of variance,ZB score and Z score were used to compare the accuracy of five different brands of CASA systems and manual method to detect the vitality and density of fresh boar semen at room temperature.[Results]After setting the parameters of five CASA systems the same as follows:VCL(curvilinear velocity),VSL(straight-line velocity)≥5μm/s,STR(straightness)=VSL(straight-line velocity)/VAP(average path velocity)≥25%,the sperm motility of six parts of boar semen was tested at normal temperature using different brands of special fixed-volume slides with a uniform chamber height[(20±2)μm].There were no significant differences in sperm vitality detected by the five CASA systems(P>0.05).The ZB scores of the vitality obtained by observers or instrument engineers who did not have a job certificate from the quality inspection department showed that the results of three observers or instrument engineers were unsatisfactory(∣ZB∣>3),but there were no significant differences in vitality between the CASA systems and the inspector with a job certificate(P>0.05).Regarding sperm density detection,when the sperm density was less than 280×106/ml,there were no significant differences between the results displayed by the instruments and the results of the manual hemocytometer counting(P>0.05).[Conclusions]The accuracy of the CASA systems set to uniform parameters was consistent with the accuracy of the visual vitality obtained by an inspector with a job certificate.When the semen was diluted with 3%NaCl solution to a sperm density<280×10^6/ml,the sperm density detected by the CASA systems was consistent in reliability with that obtained by the hemocytometer detection.The CASA systems are faster and more efficient and objective than manual detection,and have the advantages of strong operability and easy promotion.

Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex

This study was designed to determine the ability of computer-assisted sperm morphometry analysis （CASA-Morph） with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa （SX and SY, respectively）. Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average （P 〈 0.001） although with important differences between bulls. A simultaneous evaluation of all the measured features by discriminant analysis revealed that nuclear area and average fluorescence intensity were the variables selected by stepwise discriminant function analysis as the best discriminators between SX and SY. In the second experiment, the sperm nuclear morphometric results from CASA-Morph in nonsexed （mixed SX and SY） and sexed （SX） semen samples from four bulls were compared. FISH allowed a successful classification of spermatozoa according to their sex chromosome content. X-sexed spermatozoa displayed a larger size and fluorescence intensity than nonsexed spermatozoa （P 〈 0.05）. We conclude that the CASA-Morph fluorescence-based method has the potential to find differences between X- and Y-chromosome-bearing spermatozoa in bovine species although more studies are needed to increase the precision of sex determination by this technique.

Orchiopexy:one procedure,two diagnoses-different male infertility outcomes

Infertility,affecting one in six couples,is often related to the male partner’s congenital and/or environmental conditions or complications postsurgery.This retrospective study examines the link between orchiopexy for undescended testicles(UDT)and testicular torsion(TT)in childhood and adult fertility as assessed through sperm analysis.The study involved the analysis of semen samples from 7743 patients collected at Soroka University Medical Center(Beer Sheva,Israel)between January 2009 and December 2017.Patients were classified into two groups based on sperm concentration:those with concentrations below 5×10^(6)sperm per ml(AS group)and those above(MN group).Medical records and surgical histories were reviewed,categorizing orchiopexies by surgical approach.Among 140 individuals who had undergone pediatric surgery,83(59.3%)were placed in the MN group and 57(40.7%)in the AS group.A higher likelihood of being in the MN group was observed in Jewish compared to Arab patients(75.9%vs 24.1%,P=0.006).In cases of childhood UDT,45(78.9%)patients exhibited sperm concentrations below 5×10^(6)sperm per ml(P<0.001),and 66(76.7%)had undergone unilateral and 18(20.9%)bilateral orchiopexy.Bilateral orchiopexy was significantly associated with lower sperm concentration,total motility,and progressive motility than unilateral cases(P=0.014,P=0.001,and P=0.031,respectively).Multivariate analysis identified UDT as a weak risk factor for low sperm concentration(odds ratio[OR]:2.712,P=0.078),with bilateral UDT further increasing this risk(OR:6.314,P=0.012).Jewish ethnicity and TT diagnosis were associated with a reduced risk of sperm concentrations below 5×10^(6)sperm per ml.The findings indicate that initial diagnosis,surgical approach,and ethnicity markedly influence male fertility outcomes following pediatric orchiopexy.

Morphometric and kinematic sperm subpopulations in split ejaculates of normozoospermic men

This study was designed to analyze the sperm kinematic and morphometric subpopulations in the different fractions of the ejaculate in normozoospermic men. Ejaculates from eight normozoospermic men were collected by masturbation in three fractions after 3-5 days of sexual abstinence. Analyses of sperm motility by computer-assisted sperm analysis （CASA-Mot）, and of sperm morphometry by computer-assisted sperm morphometry analysis （CASA-Morph） using fluorescence were performed. Clustering and discriminant procedures were performed to identify sperm subpopulations in the kinematic and morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three kinematic subpopulations （slow with low ALH [35.6% of all motile spermatozoa], with circular trajectories [32.0%], and rapid with high ALH [32.4%]）, and three morphometric subpopulations （large-round [33.9% of all spermatozoa], elongated [32.0%], and small [34.10%]）. The distribution of kinematic sperm subpopulations was different among ejaculate fractions （P 〈 0.001）, with higher percentages of spermatozoa exhibiting slow movements with low ALH in the second and third portions, and with a more homogeneous distribution of kinematic sperm subpopulations in the first portion. The distribution of morphometric sperm subpopulations was also different among ejaculate fractions （P〈 0.001）, with more elongated spermatozoa in the first, and of small spermatozoa in the third, portion. It is concluded that important variations in the distribution of kinematic and morphometric sperm subpopulations exist between ejaculate fractions, with possible functional implications.

A comparative study of the morphometry of sperm head components in cattle, sheep, and pigs with a computer-assisted fluorescence method

The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle （Bos taurus）, sheep （Ovis aries）, and pigs （Sus scrofa）. Semen smears of twenty ejaculates from each species were fixed and labeled with a propidium iodide-Pisum sativum agglutinin （PI/PSA） combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull 〉 ram 〉 boar. However, for the other morphometric parameters （length, width, and perimeter）, there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species.

Comparison of different statistical approaches to evaluate morphometric sperm subpopulations in men

This study was designed to characterize morphometric sperm subpopulations in normozoospermic men by using different statistical methods and examining their suitability to classify correctly different sperm nuclear morphologies present in human ejaculates. Ejaculates from 21 normozoospermic men were collected for the study. After semen collection and analysis, samples were prepared for morphometric determination. At least 200 spermatozoa per sample were assessed for sperm morphometry by computer-assisted sperm morphometry analysis （CASA-Morph） using fluorescence. Clustering and discriminant procedures were performed to identify sperm subpopulations from the morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three morphometric subpopulations （large-round 30.4%, small-round 46.6%, and large-elongated 22.9%）. In the second analysis, using discriminant methods, the classification was made independently of size and shape. Three morphological categories according to nuclear size （small 〈10.90 μm^2, intermediate 10.91-13.07 μm^2, and large 〉13.07 μm^2） and four categories were defined on 400 canonical cells （100 × 4） from 10 men according to sperm nuclear shape （oval, pyriform, round, and elongated）. Thereafter, the resulting classification functions were used to categorize 4200 spermatozoa from 21 men. Differences in the class distribution were observed among men from both clustering and discriminant procedures. It was concluded that the combination of CASA-Morph fluorescence-based technology with multivariate cluster or discriminant analyses provides new information on the description of different morphometric sperm subpopulations in normal individuals, and that important variations in the distribution of morphometric sperm subpopulations may exist between men, with possible functional implications.

Effect of low-dose fenvalerate on semen quality capacitation in adult mice

Background Fenvalerate （FEN） has been demonstrated to be a reproductive toxicant in humans and rodents. However,little is known about whether short-term exposure to low-dose FEN produces reproductive toxicity.Methods We administered FEN （0.009 375, 0.1875, 3.750, or 45.00 mg·kg-1d-1 by gavage for 30 days） to male ICR mice and compared reproductive toxicity parameters between groups receiving different concentrations of FEN.Reproductive toxicity was evaluated by computer-assisted semen quality analysis （CASA）, chlortetracycline （CTC） assay,and histopathology.Results The sperm morphology and testis histology of FEN-exposed mice （all doses） were similar to that in controlling mice. Exposure to FEN at a concentration of 0.1875 mg·kg-1d-1 decreased sperm path straightness （STR） and linearity （LIN） （both P〈 0.05）, but had no significant impact on average path velocity （VAP）, straight line velocity （VSL）, curvilinear velocity （VCL）, lateral amplitude （ALH）, beat cross frequency （BCF）, or progressive motility （MOT）. FEN reduced the rate of mouse sperm capacitation in a dose-dependent manner.Conclusion The present results demonstrate that exposure to low-dose FEN for 30 days reduces semen quality and sperm capacitation in adult mice.