Use Of Sperm Research Articles

P-042 Enhancing human sperm motility using high-frequency ultrasound

Abstract Study question Is high-frequency ultrasound effective in the treatment of low-motility sperm for use in assisted reproduction? Summary answer Our study reveals that immotile and poorly motile sperm respond to ultrasound treatment and show up to 266% boost to motility parameters upon ultrasound exposure What is known already Sperm motility is central to both natural and assisted reproduction. This crucial importance has led to the utilization of chemical drugs such as pentoxifylline, theophylline, and other phosphodiesterase inhibitors to improve motility. However, these drugs are embryotoxic with a potentially harmful effect on sperm longevity. In a recent study, we showed that sperm cells at the population level showed a 32% boost to motility after 15 seconds of exposure to ultrasound. However, to translate these findings into clinical applications, it is crucial to identify which subset of sperm within a population is most affected by the treatment. Study design, size, duration We conducted single-cell study aimed at comprehensively assessing how different sperm motility grades are affected by ultrasound exposure. We utilized a droplet acoustofluidic system using which sperm cells were isolated in droplets and their responses to ultrasound were monitored. A total of 239 individual sperm from three biologically independent human samples were included in this study and parameters such as sperm motility, viability, DNA integrity, and mitochondrial membrane potential (MMP) upon ultrasound exposure were analysed. Participants/materials, setting, methods Fresh human semen samples were collected from healthy donors with the approval of the Monash University Human Research Ethics Committee. Three human donors contributed to a total of sixteen independent experiments performed in this work. The manual tracking plugin in ImageJ was used to measure sperm motility. The LIVE/DEAD™ Sperm Viability Kit (Invitrogen, USA) was used to evaluate sperm viability and JC-1 (Invitrogen, USA) was used to monitor the MMP. Main results and the role of chance We classified the sperm cells based on their pre-exposure straight-line velocity (VSL) into three categories of grade C (non-progressive, VSL < 5 µm s-1), grade B (slow progressive, 5 ≤ VSL< 25 µm s-1), and grade A (rapid progressive, 25 µm s-1 ≤ VSL) cells. Our single-cell analysis results show that 20 seconds of ultrasound exposure boosts the motility of grade C sperm up to 266% and as a result, 90% of total sperm are graded as progressively motile after exposure. Through ANOVA tests, we showed that non-progressive sperm cells show a boost to all of the motility parameters more than the other grades. In addition, ultrasound exposure renders 34% of live immotile sperm motile with 24% of the cells graded as progressive and the remaining 10% showed a twitching motility after exposure. We also showed that ultrasound exposure induces flagellum deformation to 16% of total live immotile sperm cells, providing opportunities to detect live sperm in testicular samples using this approach. Finally, we explored the non-invasiveness of the ultrasound platform and showed that there is no detrimental effect on sperm DNA integrity and viability post exposure. Limitations, reasons for caution A larger sample size could provide a higher statistical confidence to our study. Treatment of patient samples (testicular or asthenozoospermic) and evaluation of embryo safety must be included in future studies to evaluate the effectiveness of this method for clinical use. Wider implications of the findings Our acoustic mechanotherapy method could enhance sperm motility to potentially benefit assisted reproduction outcomes. It also enables new opportunities for detecting live sperm in testicular samples. Trial registration number not applicable

The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

AbstractThe in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit “classical” features of mPTP; specifically, they are resistant to cyclosporin A‐mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

Micro/Nanorobotics in In Vitro Fertilization: A Paradigm Shift in Assisted Reproductive Technologies.

In vitro fertilization (IVF) has transformed the sector of assisted reproductive technology (ART) by presenting hope to couples facing infertility challenges. However, conventional IVF strategies include their own set of problems such as success rates, invasive procedures, and ethical issues. The integration of micro/nanorobotics into IVF provides a prospect to address these challenging issues. This article provides an outline of the use of micro/nanorobotics in IVF specializing in advancing sperm manipulation, egg retrieval, embryo culture, and capacity future improvements in this swiftly evolving discipline. The article additionally explores the challenges and obstacles associated with the integration of micro/nanorobotics into IVF, in addition to the ethical concerns and regulatory elements related to the usage of advanced technologies in ART. A comprehensive discussion of the risk and safety considerations related to using micro/nanorobotics in IVF techniques is likewise presented. Through this exploration, we delve into the core principles, benefits, challenges, and potential impact of micro/nanorobotics in revolutionizing IVF procedures and enhancing affected person outcomes.

Changes in Testosterone Levels Following Surgical Sperm Retrieval in Men with Non-Obstructive Azoospermia: Systematic Review and Meta-Analysis.

Surgical sperm retrieval (SSR) is used to extract spermatozoa for use with intracytoplasmic sperm injection in men with obstructive and non-obstructive azoospermia (NOA). The procedure may lead to segmental devascularization, postoperative fibrosis, and atrophy with a subsequent decrease in testosterone. The aim of the study is to investigate the impact of SSR on serum levels of total testosterone (TT), follicle-stimulating hormone (FSH), luteinizing hormone (LH) testicular volume, and sexual function in infertile azoospermic men. In this systematic review and meta-analysis (SRMA), we searched articles in "PubMed" and "Scopus" exploring the impact of SSR on TT, FSH, LH, and testicular volume. The full-text articles were screened to assess eligibility before data extraction, quality assessment, and meta-analysis. Seventeen studies meeting the inclusion criteria were finally analyzed and included 1,685 infertile, azoospermic men. Patients underwent SSR and were followed in the postoperative period (one week to 32 months). The analysis showed a significant reduction in TT (mean difference [MD] 3.81 nmol/L, 95% confidence interval [CI] 0.55:7.06; p=0.02) compared to pre-SSR values. We also observed insignificant differences in serum FSH (MD 5.08 IU/L, 95% CI -5.6:15.8; p=0.35), LH (MD -2.96 IU/L, 95% CI -6.31:0.39; p=0.08), and no change in testicular volume (MD 0.07 mL, 95% CI -1.92:2.07; p=0.94) after SSR. Sexual dysfunction was associated with hypogonadism, depression, and anxiety, especially in men with unsuccessful SSR and Klinefelter syndrome. The results of this SRMA indicate a significant reduction in TT after SSR. Sexual dysfunction after testicular sperm extraction and the potential negative impact of future SSR repeat should be considered during preoperative counseling.

Effects of sperm separation methods before intrauterine insemination on pregnancy outcomes and live birth rates: Differences between the swim-up and microfluidic chip techniques.

Various sperm preparation techniques have been developed to obtain functionally, genetically and morphologically high-quality competent spermatozoa for use in assisted reproductive technologies, which may affect treatment options and thus pregnancy outcomes and live birth rates. We aimed to compare swim-up washing procedure (SWP) and microfluidics sperm sorting (MSS) with regard to sperm separation, pregnancy outcomes and live birth rates in infertile couples receiving intrauterine insemination. A total of 326 couples with unexplained infertility who underwent intrauterine insemination were enrolled in this retrospective cohort study and were divided into 2 groups according to sperm preparation technique. The MSS and SWP methods were used to prepare sperm in 178 and 148 patients, respectively. The median sperm concentration reduced significantly from 51 (30-100) million/mL to 20 (10-40) million/mL in the MSS group, and from 45 (26-80) million/mL to 25 (11-48) million/mL in the SWP group (both P < .001). Median motility increased significantly from 30.43 ± 17.79 to 57.48 ± 20.24 in the MSS, and from 32.89 ± 13.92 to 43.91 ± 20.11 in SWP (both P < .001). There was a difference between groups after preparation regarding sperm concentration (better with SWP) and motility (better with MSS) (P = .018 and P < .001, respectively). A total of 86 (26.4%) pregnancies were observed in participants and the clinical pregnancy rate was 23% in the MSS group and 30.4% in the SWP group (P = .133). Fifty-one infants were born alive and a great majority (n = 47) were delivered at term. Multivariate logistic regression analysis showed that higher duration of infertility was independently associated with lower live birth success (odds ratio: 0.811, 95% confidence interval: 0.662-0.996; P = .045). Other variables, including female age, type and reason of infertility, number of cycles, and sperm motility and concentration, were found to be nonsignificant (P > .05). We observed nonsignificant worse reproductive results using microfluid sperm selection in comparison to the pellet swim-up technique (live birth rate = 12% vs 20%). Our evidence is of limited quality due to the retrospective design of this study and sufficiently powered RCTs are needed to evaluate whether sperm selection based using a microfluidic chip is better, similar, or worse than the pellet swim-up technique.

Automated Orientation Control of Motile Deformable Cells

Automated manipulation of deformable objects is challenging due to the object&#x2019;s deformation behavior. Different from still deformable objects such as wires and cloth, biological organisms such as sperm and worms are both deformable and motile, requiring the control of both deformation and motion. This paper reports automated orientation control of live sperm, as an example of motile deformable cells. Robotic manipulation of human sperm was performed by using a glass micropipette, which is a standard clinical tool, to rotate individual motile sperm. Sperm rotation must be performed before immobilization, as required in clinical cell surgery for infertility treatment. To control tail deformation during sperm rotation, a path planner was designed based on kinematic analysis and manipulation point update. To deal with the intrinsic motion of a motile sperm, a motorized stage was controlled to compensate for sperm swimming motion, and an observer was designed to decouple sperm orientation from its wiggling motion. A sliding mode controller was designed to cope with stiffness variances along the sperm tail and among different sperm. Deep neural networks were developed for robust sperm tail detection, and Kalman filter was used to predict tail motion. Experimental results demonstrated that automated sperm manipulation achieved an orientation error of 0.8<inline-formula> <tex-math notation="LaTeX">$^{\circ}$</tex-math> </inline-formula> and operation time of 6.8 s, both significantly less than those of manual operation. The designed observer was effective to reduce sperm orientation error by reducing the disturbance from sperm wiggling motion. The developed sliding mode controller outperformed the PID controller in operation time, reducing the time of oocyte exposure to the ambient environment. <i>Note to Practitioners</i>&#x2014;This work tackled the challenge of rotating a fast-swimming and deformable sperm in clinical cell surgeries. Automated manipulation of deformable objects has wide applications in industrial and service settings such as manipulating wires and folding cloth. However, the intrinsic motion of a motile sperm and the lack of a rotational degree of freedom in standard micromanipulators pose difficulties to automated sperm manipulation. In this paper, we propose automation techniques for sperm orientation control. For sperm tail detection, deep learning was used to handle the variances of shape and length among different sperm. A path planning strategy and a controller were designed to achieve automated rotation of motile sperm, with its deformation and motion both controlled. The developed methods can be generalized to the manipulation of other deformable objects such as wires, cables and cloth. These objects exhibit significant variance of mechanical properties, and calibration is often time-consuming. The designed controller can be used to manipulate deformable objects with robustness to varied mechanical parameters. Path planning was designed by updating the manipulation point based on the object&#x2019;s deformation behavior, and is suitable in manipulation where constraints are imposed such as the object&#x2019;s strain.

Human Sperm as an In Vitro Model to Assess the Efficacy of Antioxidant Supplements during Sperm Handling: A Narrative Review.

Spermatozoa are highly differentiated cells that produce reactive oxygen species (ROS) due to aerobic metabolism. Below a certain threshold, ROS are important in signal transduction pathways and cellular physiological processes, whereas ROS overproduction damages spermatozoa. Sperm manipulation and preparation protocols during assisted reproductive procedures-for example, cryopreservation-can result in excessive ROS production, exposing these cells to oxidative damage. Thus, antioxidants are a relevant topic in sperm quality. This narrative review focuses on human spermatozoa as an in vitro model to study which antioxidants can be used to supplement media. The review comprises a brief presentation of the human sperm structure, a general overview of the main items of reduction-oxidation homeostasis and the ambivalent relationship between spermatozoa and ROS. The main body of the paper deals with studies in which human sperm have been used as an in vitro model to test antioxidant compounds, including natural extracts. The presence and the synergic effects of different antioxidant molecules could potentially lead to more effective products in vitro and, in the future, in vivo.

Contaminants of Emerging Concern (CECs) and Male Reproductive Health: Challenging the Future with a Double-Edged Sword.

Approximately 9% of couples are infertile, with half of these cases relating to male factors. While many cases of male infertility are associated with genetic and lifestyle factors, approximately 30% of cases are still idiopathic. Contaminants of emerging concern (CECs) denote substances identified in the environment for the first time or detected at low concentrations during water quality analysis. Since CEC production and use have increased in recent decades, CECs are now ubiquitous in surface and groundwater. CECs are increasingly observed in human tissues, and parallel reports indicate that semen quality is continuously declining, supporting the notion that CECs may play a role in infertility. This narrative review focuses on several CECs (including pesticides and pharmaceuticals) detected in the nearshore marine environment of False Bay, Cape Town, South Africa, and deliberates their potential effects on male fertility and the offspring of exposed parents, as well as the use of spermatozoa in toxicological studies. Collective findings report that chronic in vivo exposure to pesticides, including atrazine, simazine, and chlorpyrifos, is likely to be detrimental to the reproduction of many organisms, as well as to sperm performance in vitro. Similarly, exposure to pharmaceuticals such as diclofenac and naproxen impairs sperm motility both in vivo and in vitro. These contaminants are also likely to play a key role in health and disease in offspring sired by parents exposed to CECs. On the other side of the double-edged sword, we propose that due to its sensitivity to environmental conditions, spermatozoa could be used as a bioindicator in eco- and repro-toxicology studies.

Characterization of proteases in the seminal plasma and spermatozoa of llama

Camelids' semen has peculiar characteristics that differentiate it from other species, including the highly viscous aspect of seminal plasma that greatly difficult sperm manipulation and the development of techniques such as cryopreservation, artificial insemination, and/or in vitro fertilization. The presence of proteases in the seminal plasma is responsible for semen liquefaction, and sperm functionality to achieve fertilization. The enzymatic and molecular composition of the semen of llama remains unknown. Therefore, the goal of the study was to characterize the protease activity and composition of the seminal plasma and sperm of llama semen. The proteolytic activity was performed using gelatine zymography and the composition by mass-spectrometry.Metallo-proteases were the major source of gelatinolytic activity in seminal plasma, while serine-peptidases were the main enzymes of sperm cells. Matrix Metalloproteinase 2 (MMP2) was the most prominent metallo-protease of llama seminal plasma characterized under the exposure of different inhibitors (EDTA and benzamidine) and by a specific immunodetection. Moreover, the prostate and epididymis were identified as potential sites of its synthesis and secretion.Outstandingly, this metalloproteinase was undetectable in llama sperm. Regarding, the molecular composition of semen by mass-spectrometry, 4 metallo-, 9 serine-, 8 threonine-, and 1 aspartic-peptidases were identified alongside 15 regulators in the sperm cell; where 24 were directly or indirectly interacting. Whereas 6 metallo-, 12 serine-, 3 cysteine-, and 1 aspartic-peptidases were identified, besides 7 inhibitors and 5 regulators in llama seminal plasma where 30 of them were directly or indirectly interconnected. This is the first study describing a partial degradome of llama seminal plasma and spermatozoa suggesting significant differences especially the absence of MMP2 in spermatozoa in contrast to data observed in other species. The characterization of proteases in llama semen will provide a better understanding of the molecular mechanisms involved in the in vivo or in vitro fertilization process in this species.

Using GnRH agonists in combination with hCG in antagonist ART cycles: A SCOPING Review of recent evidences.

Assisted Reproductive technology encompasses all techniques involving ovarian stimulation to produce high-quality oocytes and manipulation of both oocytes and sperm in vitro to produce embryos for the purpose of reproduction. The final maturation of oocytes induced by a "trigger" is a crucial step with the potential to affect in vitro fertilization outcomes. Human chorionic gonadotropin has traditionally been used as a substitute for luteinizing hormone to induce final oocyte maturation and meiosis. However, this practice may cause a potentially fatal iatrogenic complication known as ovarian hyperstimulation syndrome, which can cause significant morbidity and, in rare cases, death in otherwise healthy women. Thus, gonadotropin releasing hormone agonists have been promoted as a safer alternative for inducing oocyte maturation, albeit at the expense of luteal phase defect. Since then, various combinations of gonadotropin releasing hormone agonists and human chorionic gonadotropin have been tried. This scoping review evaluates these trigger combinations in various types of responders.

Robotic Manipulation of Sperm as a Deformable Linear Object

The robotic manipulation of deformable linear objects is a classic and challenging topic. Apart from synthetic objects, such as wires and cables, linear objects are also commonly found in biological cells and organisms. Biomanipulation of such objects is hampered by difficulties, such as limited degrees of freedom of micromanipulators and varied mechanical properties of the biological entities to manipulate. This article presents a robotic manipulation of human sperm, which are deformable cells with a linear shape. The shape and movement of the cell are recapitulated by our developed geometric and kinematic models. Under unfixed constraints between the end-effector and the cell, path planning is designed to update the manipulation point to control cell deformation. A state transition function is formulated in path planning to handle the stiffness variations of sperm without force sensing. A model-predictive controller is designed to minimize the orientation error and manipulation path length. To detect sperm tail for visual feedback, an accuracy of 98% was achieved via deep neural networks. The robotic manipulation of human sperm was performed using a standard clinical setup of a glass micropipette to rotate a sperm to the target orientation. Experimental results showed that robotic sperm rotation achieved an orientation error of 0.8 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$^{\circ }$</tex-math></inline-formula> , a tail curvedness of 0.14 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\mu$</tex-math></inline-formula> m <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$^{-1}$</tex-math></inline-formula> , and an operation time of 5.6 s, all significantly less than those of the manual approach. The less orientation error and tail curvedness after robotic rotation led to a significantly lower speed of sperm entering the micropipette during sperm aspiration, resulting in a higher success rate of 97% (versus 76% after manual rotation) for aspiration control.

Robotic Rotational Positioning of End-Effectors for Micromanipulation

Precise rotational positioning of end-effectors under microscopy is crucial for robotic micromanipulation. However, the end-effector is presently limited to a fixed orientation, which is manually set before a given micromanipulation task, lacking accuracy and versatility of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in situ</i> reorientation. In this article, we present a unified framework for rotationally positioning the end-effector in three dimensions by establishing a general rotational model, developing a detection method within the limited field of view under microscopy, and designing a three-loop control strategy that adapts to different experimental requirements and model parameters. In experiments, a standard angled micropipette was used as the end-effector to verify the validity of the proposed methods. The performance was evaluated experimentally where the micropipette was robotically rotated to an arbitrarily desired orientation with an average orientation error less than <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$2^\circ$</tex-math></inline-formula> . In the experiments of sperm manipulation, the <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in situ</i> micropipette orientation control capability improved the success rate of sperm immobilization and achieved dexterous robotic sperm orientation for facile aspiration.

Diurnal rhythm of human semen quality: analysis of large-scale human sperm bank data and timing-controlled laboratory study.

STUDY QUESTIONCan we identify diurnal oscillations in human semen parameters as well as peak times of semen quality?SUMMARY ANSWERHuman semen parameters show substantial diurnal oscillation, with most parameters reaching a peak between 1100 and 1500 h.WHAT IS KNOWN ALREADYA circadian clock appears to regulate different physiological functions in various organs, but it remains controversial whether diurnal rhythms occur in human semen parameters.STUDY DESIGN, SIZE, DURATIONThe medical record of a provincial human sperm bank (HSB) with 33 430 semen samples collected between 0800 and 1700 h from 1 March 2010 to 8 July 2015 was used to analyze variation in semen parameters among time points. A laboratory study was conducted to collect semen samples (n = 36) from six volunteers at six time points with identical time intervals (2 days plus 4 h) between 6 June and 8 July in 2019, in order to investigate the diurnal oscillation of semen parameters in vivo, with a strictly controlled abstinence period. Therefore, the sperm bank study with a large sample size and the in vivo study with a strictly controlled abstinence period in a 24-h time window could be compared to describe the diurnal rhythms in human semen parameters.PARTICIPANTS/MATERIALS, SETTING, METHODSSamples were obtained from potential HSB donors and from participants in the laboratory study who were volunteers, recruited by flyers distributed in the community. Total sperm count, sperm concentration, semen volume, progressive motility and total motility were assessed using computer-aided sperm analysis. In addition, sperm chromatin integrity parameters (DNA fragmentation index and high DNA stainability) were assessed by the sperm chromatin structure assay, and sperm viability was measured with flow cytometry in the laboratory study.MAIN RESULTS AND THE ROLE OF CHANCEThe 33 430 samples from the HSB showed a temporal variation in total sperm count, sperm concentration, semen volume, progressive motility and total motility (all P < 0.001) between 0800 and 1700 h. Consequently, the eligibility of semen samples for use in ART, based on bank standards, fluctuated with time point. Each hour earlier/later than 1100 h was associated with 1.14-fold risk of ineligibility. Similarly, the 36 samples taken during the 24-h time window showed diurnal oscillation. With the pre-collection abstinence period strictly controlled, most semen parameters reached the most favorable level between 1100 and 1500 h.LIMITATIONS, REASONS FOR CAUTIONSome of the possible confounding factors, such as energy intake, which might influence semen quality or diurnal rhythms, were not adjusted for in the analyses. In addition, the findings should be considered with caution because the study was conducted in a specific population, time and place, while the timing of oscillations could differ with changing conditions.WIDER IMPLICATIONS OF THE FINDINGSThe findings could help us to estimate semen quality more precisely and to obtain higher quality sperm for use in ART and in natural conception.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by the National Natural Science Foundation of China (81871208) and National Key R&D Program of China (2017YFC1002001). There are no conflicts of interest to declare.TRIAL REGISTRATION NUMBERN/A.

What is the importance of sperm subpopulations?

The study of sperm subpopulations spans three decades. The origin, meaning, and practical significance, however, are less clear. Current technology for assessing sperm morphology (CASA-Morph) and motility (CASA-Mot) has enabled the accurate evaluation of these features, and there are many options for data classification. Subpopulations could occur as a result of the stage of development of each spermatozoon in the subpopulation. Spermatogenesis might contribute to the production of these subpopulations. Insights from evolutionary biology and recent molecular research are indicative of the diversity among male gametes that could occur from unequal sharing of transcripts and other elements through cytoplasmic bridges between spermatids. Sperm cohorts exiting the gonads would contain different RNA and protein contents, affecting the spermatozoon physiology and associations with the surrounding environmental milieu. Subsequently, these differences could affect how spermatozoa interact with the environmental milieu (maturation, mixing with seminal plasma, and interacting with the environmental milieu, or female genital tract and female gamete). The emergence of sperm subpopulations as an outcome of evolution, related to the reproductive strategies of the species, genital tract structures, and copulatory and fertilization processes. This kind of approach in determining the importance of sperm subpopulations in fertilization capacity should have a practical impact for conducting reproductive technologies, inspiring and enabling new ways for the more efficient use of spermatozoa in the medical, animal breeding, and conservation fields. This manuscript is a contribution to the Special Issue in memory of Dr. Duane Garner.

The effects of cryogenically preserved sperm on the fertilization, embryonic development and hatching success of lumpfish C. lumpus

Lumpfish (C. lumpus) are used as cleaner fish in the Atlantic salmon (Salmo salar) farming industry to remove parasitic sea lice. At present, wild lumpfish broodstock are used which puts strain on wild populations. By successfully cryopreserving lumpfish sperm, the number of wild males required will be reduced and it enables the long-term storage of sperm for use in breeding programmes. The present study compared the use of fresh sperm and sperm which was cryogenically frozen for 24 h to test whether it is a viable method of preservation. The fresh and frozen sperm from 5 males was used (in equal volumes) to fertilize eggs pooled from 5 females and the difference between fertilization success, percentage of eggs which reached the eyed stage, and the hatching success was measured. A group of 100 hatched larvae were on-grown for two weeks to test whether there was a difference weight between treatment groups. The results of the trial showed that fresh sperm produced a significantly higher percentage of fertilized eggs (fresh 92.6 ± 0.8%, frozen 77.9 ± 1.8%, mean ± SEM), a higher percentage of eggs surviving to the eyed stage (fresh 93.9 ± 0.5% and frozen 80.8 ± 1.4%) and had a more successful hatch rate (fresh 72.3 ± 6.6% and frozen 63.6 ± 5.0%). There was no difference in mean weight (± SEM) of the two-week old larvae between treatments (fresh 0.63 g ± 0.024, frozen 0.59 g ± 0.028). In conclusion, this study showed that the same volume of cryogenically preserved lumpfish sperm produced fewer viable lumpfish larvae than fresh sperm. Despite this difference, the use of cryopreserved sperm did produce relatively high results at each stage of testing.

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

Impaired transcription of human endogenous retroviruses in the sperm with exception of syncytin 1: short communication.

Human endogenous retroviruses (HERVs), remnants of ancestral infections, represent 8% of the human genome. HERVs are co-opted for important physiological functions during embryogenesis; however, little is known about their expression in human gametes. We evaluated the transcriptional levels of several retroviral sequences in human spermatozoa. We assessed, through a Real-Time PCR assay, the transcription levels of the pol genes of HERV-H, -K and -W families and of env genes of syncytin (Syn)1 and Syn2 in the spermatozoa from 8 normospermic subjects. The entity and distribution of their expressions were compared to values found in white blood cells (WBCs) from 16 healthy volunteers. The level of HERV transcripts was significantly lower in spermatozoa than in WBCs for HERV-H-pol, HERV-K-pol, HERV-W-pol, and Syn2.In contrast, the level of expression of Syn1 in the sperm was similar to that found in WBCs and it was significantly higher than the mRNA concentrations of other HERV genes in spermatozoa. Our findings show, for the first time, the presence of several retroviral mRNAs in the sperm, although in low amounts. The higher concentration of Syn1 suggests that it could play a key role in the fusion process between gametes during fertilization and, perhaps, be involved in embryo development. Further studies could clarify whether aberrant HERV expressions, in particular of Syn1, negatively affect fertilization and embryo growth and whether sperm manipulation procedures, such as cryopreservation, may potentially influence HERV transcription in the human male gamete.

Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

Simple SummaryThe use of assisted reproductive techniques, which involve the manipulation of sperm and oocytes in the laboratory, support owner production of valuable animals’ offspring. However, several limitations remain underlining the need to further optimize existing protocols as well as to develop new strategies. For example, the required conditions to make equine spermatozoa competent to fertilize an oocyte in vitro (IVF) have not been established. Therefore, our initial goal was to optimize different conditions associated with frozen equine sperm manipulations in order to improve their quality. We observed that simple factors such as sample concentration, incubation period and centrifugation time affect the sperm motility. Since in vivo fertilization involves the interaction between spermatozoa and epithelial cells in the mare’s oviductal tract, our next goal was to mimic this environment by establishing primary cultures of oviductal cells. Using this in vitro system, we were able to select a sperm population capable of fertilization. In short, this study provides a novel protocol that improves the yield of fertilization-capable sperm obtained from equine frozen spermatozoa.Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we established an in vitro OEC culture model to select a sperm population with potential fertilizing capacity in this species. We demonstrated E-cadherin and cytokeratin expression in cultures of OECs obtained. When sperm–OEC cocultures were performed, the attached spermatozoa were motile and presented an intact acrosome, suggesting a selection by the oviductal model. When co-cultures were incubated in capacitating conditions a greater number of alive (p < 0.05), capacitated (p < 0.05), with progressive motility (p < 0.05) and with the intact acrosome sperm population was observed (p < 0.05) suggesting that the sperm population released from OECs in vitro presents potential fertilizing capacity. Improvements in handling and selection of cryopreserved sperm would improve efficiencies in ARTs allowing the use of a population of higher-quality sperm.

Cryopreservation of small numbers of human spermatozoa in a Stripper tip: Report of the first live-birth worldwide

PurposeThe aim is to report the first live-birth following ICSI using spermatozoa previously vitrified in a Stripper Tip. Principal resultsA 34-year-old cryptozoospermic man was enrolled in a sperm vitrification program. After failure of conventional freezing technique, spermatozoa were vitrified using two carriers: a commercially-available, Cell Sleeper, and a “home-made” one, Stripper Tip. This man and his 30-year-old wife underwent an ICSI attempt using vitrified-warmed spermatozoa from these devices. All frozen-warmed spermatozoa were quickly recovered. Among the oocytes retrieved, six were injected with sperm from the Cell Sleeper, and seven with sperm from the Stripper tip, leading to 4 embryos in each case. Two embryos, arising from sperm frozen in the Stripper tip, were transferred, resulting in a healthy live-birth. ConclusionsThis is the first successful delivery following the use of spermatozoa frozen in an original device, the Stripper Tip, giving a promising prospect for managing severe male infertilities.

Ultrasonic Cutting of Frozen Semen Straws to Optimize the Use of Spermatozoa for In Vitro Fertilization.

Simple SummaryThe use of frozen semen is essential for the success of in vitro fertilization in the bovine embryo transfer industry. Therefore, we aimed to maximize the use of a single frozen semen straw by employing a cutting protocol. To the best of our knowledge, the current study is the first to apply ultrasonic cutting to frozen semen. The mechanical damage of spermatozoa was studied in frozen bovine semen by assaying sperm motility, acrosome abnormalities, and developmental competence. These findings may help to optimize the utilization of frozen semen for in vitro fertilization in the bovine industry.The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.