Individual Sperm Research Articles

Assessment of Sperm Binding Capacity in the Tubal Reservoir Using a Bovine Ex Vivo Oviduct Culture and Fluorescence Microscopy.

Sperm binding within the oviductal sperm reservoir plays an important role for reproductive success by enabling sperm survival and maintaining fertilizing capacity. To date, numerous in vitro technologies have been established to measure sperm binding capacity to cultured oviductal cells or oviductal explants. However, these methods do not accurately represent the microenvironment and complex multi-molecular nature of the oviduct. In this paper, we describe a novel protocol for assessing sperm binding capacity in the tubal sperm reservoir using an ex vivo oviduct culture in the bovine model. This protocol includes the staining of frozen-thawed bovine spermatozoa with the DNA-binding dye Hoechst 33342, the co-incubation of stained sperm in closed segments of the oviduct and the visualization and quantification of bound spermatozoa by fluorescence microscopy. By generating overlays of multiple Z-stacks of randomly selected regions of interest (ROIs), spermatozoa bound in the sperm reservoir can be visualized and quantified within the 3D arrangement of the oviductal folds. This method, which is applicable to multiple species, can be used to assess individual sperm binding capacity in males for prognostic purposes as well as to assess the impact of diseases and medications on the formation of the sperm reservoir in the oviduct in humans and animals.

Mixture Analysis of Associations between Occupational Exposure to Polycyclic Aromatic Hydrocarbons and Sperm Oxidative DNA Damage.

This study aimed to determine (i) associations between levels of the polycyclic aromatic hydrocarbon (PAH) mixture with 16 targeted PAH compounds in the personal breathing zone area and sperm oxidative DNA damage, (ii) associations between levels of individual PAH compounds and sperm oxidative DNA damage, (iii) oxidative stress as the mode of action for the genotoxic effects on sperm, and (iv) any dose-response relationship between exposure to the PAH mixture and/or individual PAH compounds and sperm oxidative DNA damage. Sixteen targeted PAH compounds in the personal breathing zone area of 38 coke-oven workers and 24 control subjects were quantified using gas chromatography-mass spectrometry. Sperm oxidative damage and status were evaluated by measuring levels of sperm 7,8-dihydro-8-oxoguanie (8-oxodGuo), seminal malondialdehyde (MDA) and seminal reactive oxygen species (ROS). Bayesian kernel machine regression with hierarchical variable selection process was employed to determine associations of the PAH mixture and the biomarkers of sperm oxidative damage. A novel grouping approach needed for the hierarchical variable selection process was developed based on PAH bay region and molecular weight. The PAH mixture exhibited a positive trend with increased sperm 8-oxodGuo levels at their lower percentiles (25th-50th). The exposure of the PAH mixture was associated with increased MDA levels in sperm. Bay and bay-like regions of the PAH mixture were the most important group for estimating the associations between the PAH mixture and sperm oxidative stress status. Benzo[a]anthracene was the main individual PAH compound that was associated with increased MDA levels. Sperm oxidative DNA damage induced by occupational exposure to the PAH mixture had a suggestive association with increased MDA levels in coke-oven workers. Finally, the study identified that the individual PAH compound, benzo[a]anthracene, was the primary driver for the suggestive association between the PAH mixture and sperm oxidative damage.

MP21-03 INHIBIN B IS A PREDICTIVE FACTOR OF SPERM RECOVERY IN NON-OBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation I (MP21)1 Sep 2021MP21-03 INHIBIN B IS A PREDICTIVE FACTOR OF SPERM RECOVERY IN NON-OBSTRUCTIVE AZOOSPERMIA Isadora Badalotti-Teloken, Mariangela Badalotti, Guilherme Santiago, Bruna Santos, Bruna Araujo, Ana Karoline Rosa, Alvaro Petracco, and Claudio Teloken Isadora Badalotti-TelokenIsadora Badalotti-Teloken More articles by this author , Mariangela BadalottiMariangela Badalotti More articles by this author , Guilherme SantiagoGuilherme Santiago More articles by this author , Bruna SantosBruna Santos More articles by this author , Bruna AraujoBruna Araujo More articles by this author , Ana Karoline RosaAna Karoline Rosa More articles by this author , Alvaro PetraccoAlvaro Petracco More articles by this author , and Claudio TelokenClaudio Teloken More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002006.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Inhibin B is a glycoprotein produced by seminiferous tubules, predominantly by Sertoli cells, and has been proposed as a non-invasive marker of spermatogenesis. This study aimed to assess the potential of inhibin B as a spermatogenesis marker compared with follicle-stimulating hormone (FSH) and in non-obstructive azoospermic men, its association with testicular cyto-histology, and its potential to predict the presence of sperm at testicular sperm extraction (TESE) procedure. METHODS: Clinical and laboratory data of men treated at a Brazilian human reproduction center were reviewed. To evaluate the correlation between inhibin B and FSH, the patients were divided in groups: non-obstructive azoospermia (NOA), severe oligospermia (SO, sperm concentration <5.106/ml), and mild to moderate oligospermia (MO, sperm concentration 5-14.9.106/ml); fertile normozoospermic patients composed the control group (CG). In the NOA group the correlation between inhibin B and testicular cyto-histology (CE–complete spermatogenesis; MA–maturation arrest at level of spermatid; SC–only Sertoli cells, spermatocyte or spermatogonia) was evaluated. Testicular specimens were obtained in 3 different areas of each testi. In patients who underwent TESE, the presence of sperm was evaluated. The statistical analysis was carried out using Kruskal-Wallis, Spearman, and Mann-Whitney U test, multiple regression and ROC curve, considering p<0.05 significant. RESULTS: A total of 246 cases was eligible; NOA n=135 (mean age 36.9 yo), SO n=89 (mean age 36.7 yo), MO n=10 (mean age 37.2 yo) and CG n=11. The median level of inhibin B (pg/ml) was 36.8 in NOA, 74.0 in SO, 109.0 in MO and 196.0 in CG (p<0.0001). The correlation between inhibin B and FSH was significant (r=0,701; p<0.0001). In the cyto-histological comparison, the median value of inhibin B was 87.6 in CE, 42.7 in MA and 22.0 in SC (p<0.0001). Patients with inhibin B ≥88,0 have a 73.3% chance of having spermatozoa or spermatid in cyto-histology, and those with inhibin B ≤22.0 have a 76.3% chance of not having these cells (p<0.0001). In the TESE results, the median level of inhibin B was 80.0 in the group with sperm and 27.6 in the group with no sperm. Inhibin B ≥88,0 also has an 85% positive predictive value for having spermatozoa in TESE (p=0.047). CONCLUSIONS: This study shows a cutoff point for inhibin B ≥ 88.0 for a good prognosis of finding sperm in azoospermic individuals for assisted reproduction through TESE. Furthermore, an inhibin B ≤22 points to a bad prognosis, suggesting that the patients look further into infertility treatment alternatives, such as sperm bank. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e346-e347 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Isadora Badalotti-Teloken More articles by this author Mariangela Badalotti More articles by this author Guilherme Santiago More articles by this author Bruna Santos More articles by this author Bruna Araujo More articles by this author Ana Karoline Rosa More articles by this author Alvaro Petracco More articles by this author Claudio Teloken More articles by this author Expand All Advertisement Loading ...

Correlation and Regression Between Age and Body Weight on Semen Quality of Limousin Bulls in Artificial Insemination Center, Lembang, West Java

This research aimed to determine the correlation and to find the best regression model for predicting semen quality (semen volume, individual motility, and sperm concentration) based on age and body weight in Limousin bulls. This research was conducted in December 2019 in Artificial Insemination Center, Lembang, West Java. The method used was a survey with census data collection, namely all Limousin bulls with semen collected were used in this study. The data were analyzed for correlation and simple and multiple regression using R program. The results showed that the average of age, body weight, semen volume, individual motility and sperm concentration were 58.36 ± 40.19 months, 807.12 ± 91.59 kg, 5.86 ± 1.31 ml, 67.59 ± 4.11%, and 1006.23 ± 145.28 million/ml, respectively. Age with semen volume, body weight with semen volume, and combination of age and body weight with semen volume, had a medium positive correlation (P&lt;0.05), with a correlation coefficient of 0.52, 0.58 and 0.58, respectively. The regression model with the highest coeficient of determination value (R2) was between body weight and semen volume with regression equation of Ŷ = -0.8129 + 0.008269X and of R2 = 0.34. In conclusion, semen volume of Limousin bull could be estimated using their age and body weight, while individual motility and sperm concentration might not be able to be estimated using their age and body weight.

Increased de novo DNA Methylation Enzymes in Sperm of Individuals with Varicocele.

Objective Chronic genital heat-stress associated with varicocele leads to DNA hypo-methylation of spermatozoa. The objective of this study was comparing level of DNA methyl-transferases (DNMTs) in sperm of men suffering varicocele with fertile individuals. Materials and Methods In this case-control study, semen samples were obtained from 35 infertile men with varicocele (grade II or III) and 26 fertile men. Sperm parameters were assessed according to World Health Organization (WHO) protocol. DNMTs enzymes level were assessed by flow cytometer and fluorescence microscope. mRNAs expression of these DNMTs were also assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). Results DNMT1 and DNMT3A proteins were mainly localized in equatorial and mid-piece regions of sperm head, respectively, while DNMT3B protein appeared to be localized mainly in equatorial and anterior regions of sperm head. In contrast to DNMT1, expression and percentage of DNMT3A and DNMT3B at RNA and protein levels were significantly higher in the varicocele group compared to the fertile group (P<0.05). In addition, significant correlations were found between sperm concentration and motility as well as DNMT1 and DNMT3B proteins levels in the infertile individuals with varicocele (P<0.05). Additionally, significant correlations were observed between abnormal sperm morphology with DNMTs proteins in the infertile individuals with varicocele. ConclusionUnlike DNMT1, which is involved in maintenance of DNA methylation at both RNA and protein levels, expression of de novo methylation enzymes (DNMT3A and DNMT3B) at both levels were increased in the varicocele group compared to the fertile group. Based on literature, this increase might be due to the dual roles played by DNMT3A and DNMT3B, as methyl-transferases in normal condition as well as dehydroxymethylases in stress condition, like varicocele. Although, this hypothesis needs further validation.

TYROSINE KINASE GENE POLYMORPHISMS ASSOCIATE WITH FRESH SEMEN QUALITY FROM DAIRY BULL FRIESIAN HOLSTEIN

This research aims to identify polymorphisms in TEK genes to identify any related possibility to fresh semen quality of FH bull using the PCR method. A total of 14 samples of bull’s whole blood were collected and also the quality of each bull’s fresh semen. DNA amplification was carried out using primer Forward (TEK_F) 5'-TAGATTGTCGCTTGCCTGGG-3 'and Reverse (TEK_R) 5'-CCTGTGCCGACAGGTTTACT-3'. Analysis of the DNA sequence results was carried out using BioEdit and NCBI BLAST software. The results showed that of the 7 samples producing 262 bp and found polymorphisms in the TEK gene sequence in 23 gene bank databases. In the analysis of the relationship between the motility of individual spermatozoa with mutations, r count&gt; r table (0.806&gt; 0.754) or significance value &lt;5% significance level (0.029 &lt;0.050). In the analysis of the relationship between semen concentration and mutation, r count&gt; r table (0.897&gt; 0.754) or significance level &lt;5% significance level (0.006 &lt;0.050) is obtained.

Motility, Viability, Spermatozoa Abnormality, and pH of Bali Cattle Semen in Another-Yellow Water Driller Stored in a Different Time

This study aims to determine the quality of Bali cattle spermatozoa diluted using sugarcane juice-egg yolk. This research was carried out in the stables and Laboratory of the Faculty of Agriculture, University of Timor in August-September 2020. Semen was collected from an adult male bali cattle, aged ± 4.5 years in healthy condition. The method used in this study is an experimental method using a completely randomized design consisting of 4 treatments and 5 replications. Each treatment was T1: 0.075 ml semen + 0.3 ml of egg yolk sugarcane juice diluent and stored for 24 hours, T2: semen 0.075 ml + 0.3 ml of diluent for egg yolk cane juice and stored for 48 hours, T3: semen 0.075 ml + 0.3 ml of diluent for egg yolk sugarcane juice and stored for 72 hours, T4: semen 0.075 ml + 0 ,3 ml of egg yolk sugarcane juice diluent and stored for 96 hours. The variables measured were individual motility, viability, spermatozoa abnormalities, and semen pH. The results showed that the T4 treatment on a shelf life of 96 hours still showed a good average value with individual spermatozoa motility values of 45%, spermatozoa viability of 77.3%, spermatozoa abnormalities of 12.5% and semen pH 6.6. It can be concluded that the use of cane juice-egg yolk diluent has a positive impact because it is able to maintain the viability of spermatozoa for 96 hours.&#x0D; &#x0D;

P–096 Real-time ranking of single spermatozoa using artificial vision analysis of complex motility patterns during ICSI aimed at improving fertilization and blastocyst development

Abstract Study question Can real-time artificial vision identify beneficial movement patterns of single spermatozoa in a cohort visualized in PVP during ICSI possibly enhancing fertilization and embryo development? Summary answer Artificial vision seems able to identify advantageous movement patterns of individual spermatozoa having a significant impact on both normal fertilization and blastocyst formation. What is known already Spermatozoa isolated from poor semen may reduce the quality of embryo development and blastocyst formation. Normal motility is dependent on general sperm morphology and characteristic movement of the flagellum enabling forward mobility. Spermatozoa roll as they swim. It is known that this rotational motion around their longitudinal axis promotes rheotaxis, which is a mechanism that allows the sperm to navigate to the site of fertilization. Therefore, it is possible that the characteristics of the rotational movement are related to sperm quality. Study design, size, duration Non-intervention study based on a cohort of 132 videos of in-vitro fertilization treatments with ICSI during which the sperm selection process was recorded up to sperm injection. The study was performed at one IVF center within a 6-month period. Injected spermatozoa and their corresponding oocytes were individually assessed from fertilization to blastocyst formation. Videos, where spermatozoa selected for injection could not be identified, were excluded. Relevant outcomes included normal fertilization (2PN), and blastocyst formation. Participants/materials, setting, methods Using a digitizer attached to an optical microscope (640 x 480 pixels), videos were recorded to include the sperm selection process, immobilization, and subsequent injection following standard ICSI protocols. Individual spermatozoa motility features were extracted using a proprietary computer-vision algorithm (SID, IVF 2.0 LTD). The rotational movements of spermatozoa were inferred by computing the variations of the mean intensity of the sperm in the video-sequence across time (MI). Main results and the role of chance Based on SID’s analysis, we found statistically significant differences between the median prominences of the MI of those injected spermatozoa that resulted in successful fertilization in comparison to those with failed fertilization (p-value=0.029, 28 negative fertilization, and 71 positive fertilization) using a one-tailed t-Student test with a significance level of 5%. We also found statistically significant differences between the median prominences of the MI of those spermatozoa that resulted in blastocysts in comparison with the spermatozoa-oocyte cohorts which didn’t reach the blastocyst stage (p-value 0.004, 51 with negative blastocyst formation and 48 with blastocyst formation). Limitations, reasons for caution The size of this database is modest, therefore a larger study with multiple clinics will be necessary to confirm the findings. Large prominence does not necessarily assurance successful fertilization or blastocyst formation since there may be other factors such as oocyte quality or the ICSI technique. Wider implications of the findings: Objective assessment of sperm rotational movement is difficult to quantify and to be objectively assessed during standard sperm selection. Real-time artificial vision tools such as SID could assist embryologists during the sperm selection process for ICSI. Trial registration number NA

P–046 Application of hyperspectroscopy as a new sperm diagnostic and selection technique for ICSI based on hyperspectral signature of single spermatozoa – a proof of concept

Abstract Study question Is hyperspectral imaging of individual sperm cells an appropriate candidate technique with potential to identify molecular characteristics of single spermatozoa prior to microinjection? Summary answer Preliminary tests have allowed us to obtain the images, set up the methodology and the correct segmentation of each sperm into its signature spectra. What is known already Although image techniques such as fluorescence microscopy and Raman spectroscopy have been used to identify biomarkers on sperm cells, their translation to the fertility clinic is highly complex and unfeasible. Hyperspectroscopy has not yet been introduced in the reproductive field, but it has been successfully used in other medical disciplines to differentiate cell types or pathological versus healthy tissue. It relies on the combination of information from optical images and electromagnetic spectra produced by cells at different wavelengths. Therefore, it is an interesting candidate for objective non-destructive sperm selection before ICSI according to the presence of specific biomarkers. Study design, size, duration Pilot, prospective, observational study in the IVIRMA Valencia fertility clinic, designed as a proof of concept. Discarded semen samples were used for the technical setup and the preliminary tests of image acquisition. Hyperspectral images were obtained of 12 spermatozoa: two sperm cells were imaged 10 times each, while the other 10 sperm were imaged one time each. Participants/materials, setting, methods Samples were prepared on glass-bottom ICSI plates in PVP droplets according to routine clinical practice. Images were obtained using the camera HinaLea 4200M coupled to the Olympus IX73 inverted microscope. Hypercubes were extracted using MATLAB 2016a, and the most informative wavelength was chosen. Matrox Copilot software was used to process images and select the regions of interest (ROI) equivalent to one sperm. MATLAB was used to obtain the information for the pixels within each ROI. Main results and the role of chance Hypercube size for each image was 608x968x299 pixels. Each hypercube obtained provided information for 299 different wavelengths, from 402.8 to 998.3 nm. Once the hypercubes were obtained from each image, the spectra at 520 nm wavelength was selected as the one at which the pixel provided the most information. The background of each uploaded image was successfully removed by creating a mask, which was then used to extract and characterize all the spectra from each pixel of each sperm image. The result of this proof of concept, though measure qualitatively, is that the information obtained from hyperspectral imaging of individual sperm is highly reproducible and successful in showing overlapping spectra resulting from 10 different images of the same specimen. Spectra resulting from images of different sperm cells showed tangible differences on their composition demonstrating unique biochemical features of each. The next step will be to quantitatively measure the reproducibility and sensitivity of the technique in a larger sample, to then study the potential of single sperm hyperspectral imaging as a sperm selection tool for ICSI. Limitations, reasons for caution These are preliminary results obtained from the development of an appropriate methodology to adapt the acquisition of hyperspectral images in the clinic’s IVF laboratory. Thus, the group will continue to evaluate the reproducibility, sensitivity, and informative capability of the technique before contemplating its use on samples destined to perform cycles. Wider implications of the findings: Having developed a laboratory and image processing methodology that allows us to successfully segment each obtained image according to the hyperspectral information of every pixel of each individual sperm, we could move forward in testing the potential of these hyperspectral signatures to identify sperm markers related to successful reproductive outcomes. Trial registration number Not applicable

O-041 Functional sperm diagnostics: The evidence behind commercially available tests

Abstract text Despite the fact that the semen analysis has remained the cornerstone of the male infertility evaluation for decades, it has significant limitations. Among them are the inability to completely predict reproductive potential and the inability to analyze the fitness of individual sperm. There remains a real need for novel tests that can determine which interventions for male infertility are the most likely to result in a significant improvement in reproductive potential, to analyze the chance of success or failure with ART and to determine if there exists a sub population of sperm whose use could lead to optimal chances of success with fertility treatments. In this talk we will cover what the limitations of the current semen analysis are, what novel tests are available and delve into the impact of future technologies such as microfluidics, machine learning and others to optimize the diagnostic armamentarium of male fertility assessment. We will also focus on tools that could, potentially, non-invasively image sperm and report reproductive fitness while maintaining viability for ART. Trial registration number: Study funding: Funding source:

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

The future of sperm: a biovariability framework for understanding global sperm count trends

The past 50 years have seen heated debate in the reproductive sciences about global trends in human sperm count. In 2017, Levine and colleagues published the largest and most methodologically rigorous meta-regression analysis to date and reported that average total sperm concentration among men from ‘Western’ countries has decreased by 59.3% since 1973, with no sign of halting. These results reverberated in the scientific community and in public discussions about men and masculinity in the modern world, in part because of scientists’ public-facing claims about the societal implications of the decline of male fertility. We find that existing research follows a set of implicit and explicit assumptions about how to measure and interpret sperm counts, which collectively form what we term the Sperm Count Decline hypothesis (SCD). Using the study by Levine and colleagues, we identify weaknesses and inconsistencies in the SCD, and propose an alternative framework to guide research on sperm count trends: the Sperm Count Biovariability hypothesis (SCB). SCB asserts that sperm count varies within a wide range, much of which can be considered non-pathological and species-typical. Knowledge about the relationship between individual and population sperm count and life-historical and ecological factors is critical to interpreting trends in average sperm counts and their relationships to health and fertility.

Synergistic interactions of sperm impairing bacteria: Impact on pregnancy outcome in mouse model

Earlier in our laboratory, the role of various individual sperm impairing microorganisms on sperm parameters and female infertility has been elucidated at higher doses. As, multiple bacterial species tend to exert more pathogenic effect in comparison to single organism hence, present study was carried out to evaluate that if consortia of these sperm impairing organisms can lead to infertility in female mice at sub fertility dose. For this, impact of individual bacterial strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Klebsiella pneumoniae and consortia of Escherichia coli and Pseudomonas aeruginosa, Escherichia coli and Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae was examined on the motility, viability of mouse spermatozoa and fertility outcome. The results showed that the individual bacterial strains of E. coli, S. marcescens and K. pneumoniae could led to immobilization of spermatozoa by agglutination and P. aeruginosa led to immobilization of spermatozoa without agglutination. Also, all of them led to 100% sperm death in 45 min of incubation. In case of consortia of bacterial strains, the results showed sperm agglutination in all the cases and they were able to induce 100% sperm death at 30 min of incubation time. Further, in vivo studies were carried out to evaluate the impact of individual bacterial strains and consortia of bacterial strains on the fertility outcome in female Balb/c mice. For this, female mice were administered intravaginally with 101 cfu/20µl of individual bacterial strains or consortia of strains for 10 consecutive days or PBS. The results showed that both individual bacterial strains and consortia of bacterial strains were able to efficiently colonize the mouse vagina. Further, control group receiving phosphate buffer saline and groups receiving individual bacterial strains showed all the pregnancy related changes viz. abdominal distension, string of pearls on palpation as well as delivery of pups on completion of gestation period and delivery of pups. The histological examination of reproductive organs viz. uterus and ovary, of the female mice receiving PBS or individual bacterial strains showed the formation of corpus luteum in the ovary and the formation of decidua’s in the uterus, indicative of pregnancy. However, mice receiving consortia of bacterial strains did not show any pregnancy related changes throughout the experiment. Thus, these results indicate that the presence of consortia of sperm impairing microorganisms in vaginal milieu is efficient in provoking infertility even at subfertility doses.

SEASONAL VARIATIONS ON SEMEN CHARACTERISTICS OF FATTAILED AND GARUT RAMS UNDER TROPICAL CONDITIONS

This study investigated the influences of rainy and dry seasons on semen characteristics of fat-tailed and Garut rams under tropical conditions. A total of 4 healthy rams (2 fat-tailed rams and 2 Garut rams) aged 3 to 4 years old were used in this study. The semen was collected from each ram during November 2016 to February 2017 (rainy season) and May to August 2017 (dry season). The measurement of semen characteristics including semen volume (SV), sperm concentration (SpC), total sperm (TSp), individual sperm motility (ISM), post-thawing sperm motility (PTSM), and frozen semen production (FSP). The rainy season resulted higher SV (P= 0.021), TSp (P= 0.005), ISM (P= 0.028), and FSP (P= 0.005) than the dry season. On the other hand, fat-tailed rams had higher (P0.001) SV, TSp, and FSP compared to Garut rams. There was no interaction (P0.05) between season and breed on all semen characteristic parameters found in the present study. It can be concluded that the semen collection during the rainy season had a beneficial impact to produce high-quality semen for artificial insemination program than in the dry season. Fat-tailed rams have better semen characteristics compared to Garut rams.

Automated motility and morphology measurement of live spermatozoa.

Automated sperm analysis has wide applications in infertility diagnosis. Existing systems are not able to measure sperm count and both motility and morphology of individual live spermatozoa. Morphology measurement requires invasive staining, making the spermatozoa after morphology measurement not applicable to infertility treatment. To evaluate the reproducibility and reliability of automated measurement of individual live sperm's motility and morphology. Fresh semen samples were obtained from twenty male partners attending for fertility investigations. The system firstly measured motility for all the spermatozoa within the field of view under a low magnification (20×), then a spermatozoa of interest is selected by the user and automatically relocated by the system after switching to a high magnification (100×) for morphology measurement. Reproducibility of sperm measurements was evaluated by intraclass correlation coefficients on consecutive measurement. Reliability of motility and morphology measurement was evaluated by tracking error rate and limits of agreement, respectively, with manual measurement as benchmark. Measurement of all motility and morphology parameters had intraclass correlation coefficients higher than 0.94. Sperm motility measurement had a tracking error rate of 2.1%. Limit of agreement analysis indicated that automated measurement and manual measurement of sperm morphology were interchangeable. Automated measurement of all morphology parameters was not statistically different from manual measurement, as confirmed by the paired sample t test. Automated motility and morphology measurement of single sperm revealed high reproducibility and reliability. The system also achieved a high efficiency for motility and morphology measurement. In addition to the intracytoplasmic sperm injection (ICSI) samples with polyvinylpyrrolidone (PVP), the developed sperm measurement technique is also effective for analyzing semen and washed samples. The system provides a valuable tool for quantitative measurement and selection of single spermatozoa for ICSI. It can also be used for sperm motility and morphology analysis in andrology laboratories.

Analysis of the Functional Aspects of Sperm and Testicular Oxidative Stress in Individuals Undergoing Metabolic Surgery.

Metabolic surgery is a recommended treatment for obese patients that results in BMI reduction; however, the observed impact of this therapy on male fertility is inconsistent. This research aimed to study the effects of BMI changes after metabolic surgery on seminal analysis, sex hormonal profile, sperm functional integrity, and the seminal plasma lipid peroxidation levels. A prospective study was performed in 15 patients for whom metabolic surgery was recommended. The patients were evaluated by the techniques proposed in this study before and after the surgical procedure for 12 months. In each analysis, the male sex hormonal profile, semen analysis, sperm functional integrity, and seminal lipid peroxidation levels were assessed. The surgery resulted in BMI reduction and improvement in seminal characteristics and male sex hormone profile. The semen analysis showed increases in volume, sperm progressive motility, and in sperm morphology and a decrease in immotile sperms. Sperm mitochondrial activity and sperm DNA integrity were improved, and the levels of seminal lipid peroxidation were decreased. The hormonal profile showed lower levels of estradiol and highest levels of luteinizing hormone (LH), sex hormone-binding globulin (SHBG), and testosterone. BMI changes resulting from this treatment and its metabolic consequences can be associated with changes in the male fertile potential, leading to an improvement in the seminal quality, male sex hormone profile, sperm functional aspects, and levels of seminal lipid peroxidation, thus decreasing the testicular oxidative stress.

Altered mitochondrial function in spermatozoa from patients with repetitive fertilization failure after ICSI revealed by proteomics.

Unexplained fertilization failure (FF), occurring in 1-3% of intracytoplasmic sperm injection (ICSI) cycles, results in both psychological and financial burden for the patients. However, the molecular causes behind FF remain largely unknown. Mass spectrometry is a powerful technique to identify and quantify proteins across samples; however, no study so far has used it to dissect the proteomic signature of spermatozoa with FF after ICSI. To investigate whether sperm samples from patients suffering repetitive FF after ICSI display alterations in their protein content. Seventeen infertile men were included: 5 patients presented FF in ≥3 consecutive ICSI cycles, while 12 patients had a fertilization rate >75% (controls). Individual sperm samples were subjected to 2D-LC-MS/MS. Both conventional and novel statistical approaches were used to identify differentially abundant proteins. Additionally, analysis of mitochondrial and proteasomal abundance and activity were performed, using Western blot, FACS analysis of JC-1 staining and AMC-peptide fluorometric assay. Four proteins presented lower abundance (FMR1NB, FAM209B, RAB2B, and PSMA1) in the FF group compared to controls, while five mitochondrial proteins presented higher abundance in FF (DLAT, ATP5H, SLC25A3, SLC25A6, and FH) (p<0.05). The altered abundance of mitochondrial DLAT and proteasomal PSMA1 was corroborated by Western blot. Of relevance, novel stable-protein pair analysis identified 73 correlations comprising 28 proteins within controls, while different mitochondrial proteins (ie, PDHA2, PHB2, and ATP5F1D) lost >50% of these correlations in specific FF samples pointing out specific mitochondrial deregulations. This is the first proteomic analysis of spermatozoa from patients who resulted in fertilization failure after ICSI. The altered proteins, most of them related to mitochondrial function, could help to identify diagnostic/prognostic markers of fertilization failure and could further dissect the molecular paternal contribution to reach successful fertilization. Sperm samples from patients with FF after ICSI present altered abundance of different proteins, including mainly mitochondrial proteins.

Cryopiece, a novel carrier with faster cooling rate, high recovery rate and retrieval rate, for individual sperm cryopreservation.

BackgroundCryopreservation of extremely few spermatozoa is still a major challenge for male fertility preservation. This study aims to evaluate the cooling rate, recovery rate, and retrieval rate, along with other parameters of spermatozoa that cryopreserved using Cryopiece, a novel carrier, for individual sperm cryopreservation.MethodsSemen samples from 60 fertile donors were collected, and each semen sample was screened for motile sperm and mixed with cryoprotective agent (CPA), and then frozen using Cryopiece, micro-straw, and mini-straws. The cooling rate, retrieval rate, and recovery rate, morphology, DNA fragmentation index (DFI) and mitochondrial membrane potential (MMP), were compared among the un-frozen sperm and the sperm cryopreserved using these carriers.ResultsCryopiece possessed the fastest cooling rate. After freeze-thaw, the average retrieval rate of sperm cryopreserved using Cryopiece was 96.25%, and the average recovery rate was 64.40%, which were higher than that of sperm cryopreserved using the other two carriers (71.42% and 54.30% for micro-straw, and 63.54% and 58.04% for mini-straw, respectively). There was no significant impact on DFI after sperm cryopreservation, and no significant difference in morphology between sperm cryopreserved using these carriers was observed. Though MMP of sperm changed significantly after cryopreservation, micro-straw maintained sperm MMP better than Cryopiece and mini-straw did, while no significant difference was observed in MMP between sperm cryopreserved using Cryopiece and mini-straw.ConclusionsCryopiece produced satisfying retrieval and recovery rates in sperm cryopreservation and should be an ideal carrier for cryopreservation of small number of sperm.

Seasonal Variations in Semen Quality of Pasundan Bulls in their Native Tropical Environment

The success of artificial insemination highly depends on the semen quality, in which it may significantly associate with the environmental factor. However, the information regarding the seasonal effects on semen quality of Pasundan bulls is still scarce. This study aimed to examine seasonal variation in semen quality of Pasundan bulls in their native environment. A total of 203 ejaculates produced by two Pasundan bulls aged at four years old were used in this study. The bulls were kept under standard management conditions in Lembang Artificial Insemination Center. The results showed that mean values of semen volume, sperm concentration, total sperm number, individual sperm motility, post-thawing sperm motility and frozen semen production of Pasundan bulls were 5.7 mL, 1.17 billion/ml, 6.57 billion/ejaculate, 62.1,0 40.6 and 272% doses/ejaculate, respectively. Semen volume, individual sperm motility and post-thawing sperm motility were not affected by different seasons (P>0.05). Whereas, the significant reduction of sperm concentration (P<0.05), total sperm number (P<0.01) and frozen semen production (P<0.01) were observed in the dry season as compared to the rainy season. The minor change in ambient temperature, maximum ambient temperature, relative humidity and temperature-humidity index were detected between seasons. On the other hand, about 52% greater sunshine duration was detected in the dry season compared to the rainy season. In conclusion, the semen quality of Pasundan bulls is reduced during the dry season, which is considerably associated with the increase in sunshine duration.

Male obesity impacts DNA methylation reprogramming in sperm

BackgroundMale obesity has profound effects on morbidity and mortality, but relatively little is known about the impact of obesity on gametes and the potential for adverse effects of male obesity to be passed to the next generation. DNA methylation contributes to gene regulation and is erased and re-established during gametogenesis. Throughout post-pubertal spermatogenesis, there are continual needs to both maintain established methylation and complete DNA methylation programming, even during epididymal maturation. This dynamic epigenetic landscape may confer increased vulnerability to environmental influences, including the obesogenic environment, that could disrupt reprogramming fidelity. Here we conducted an exploratory analysis that showed that overweight/obesity (n = 20) is associated with differences in mature spermatozoa DNA methylation profiles relative to controls with normal BMI (n = 47).ResultsWe identified 3264 CpG sites in human sperm that are significantly associated with BMI (p < 0.05) using Infinium HumanMethylation450 BeadChips. These CpG sites were significantly overrepresented among genes involved in transcriptional regulation and misregulation in cancer, nervous system development, and stem cell pluripotency. Analysis of individual sperm using bisulfite sequencing of cloned alleles revealed that the methylation differences are present in a subset of sperm rather than being randomly distributed across all sperm.ConclusionsMale obesity is associated with altered sperm DNA methylation profiles that appear to affect reprogramming fidelity in a subset of sperm, suggestive of an influence on the spermatogonia. Further work is required to determine the potential heritability of these DNA methylation alterations. If heritable, these changes have the potential to impede normal development.