Individual Sperm Research Articles

P-108 Real-time deep learning based multi object tracking of spermatozoa in fresh samples

Abstract Study question Can real-time deep learning model track hundreds of spermatozoa simultaneously? Summary answer The state-of-the-art deep learning detection model YOLOv5 shows possibilities of multi-sperm tracking with high sensitivity and precision. What is known already Computer-aided sperm analysis (CASA) systems can be used for the evaluation of sperm motility by applying tracking algorithms. However, the presence of cellular debris and/or cell aggregations in human semen samples makes the tracking of spermatozoa difficult, resulting in unreliable motility assessment. Thus, there is a need for an improved methodology. There are several studies on detecting spermatozoa in real-time, such as DeepSperm, which is trained and tested on bull semen samples. However, differences in characteristics between human and animal spermatozoa imply the requirement of a multi-sperm tracking system adapted to human spermatozoa. Study design, size, duration We used the open-access VISEM dataset consisting of video recordings of human semen samples from 85 participants. We selected three videos with low sperm counts to perform manual annotations (bounding boxes around sperms) and create a training dataset. Only the first 30 seconds of each video were extracted for annotations. Then, all the spermatozoa of the three videos were annotated with bounding boxes using LabelBox. The annotations will be made public in a future study. Participants/materials, setting, methods We used two different object detection models, YOLOv5 Nano and YOLOv5 XLarge to detect spermatozoa and performed transfer learning without layer freezing. Each model was trained for a maximum of 300 epochs, stopping if the validation loss did not improve for the previous 100 epochs. Leave-one-out cross-validation was performed to obtain the presented results. Precision, sensitivity, and mean average precision (mAP) are the quantitative metrics measured to evaluate our detection models. Main results and the role of chance Our models are able to detect a large number of human spermatozoa simultaneously in a given video in real-time. The model YOLOv5 Nano took around 45 min and the YOLOv5 XLarge model took around 270 min to train on one fold. In terms of training and detection, the Nano model is faster because it has a lower number of trainable parameters compared to the XLarge model, but at the expense of precision. YOLOv5 Nano has capability of predicting 200 frames per second (fps), and YOLOv5 XLarge can predict 56 fps. Both models show high prediction rates which are greater than the real-time prediction 25 fps threshold. Performance-wise, the Nano model shows an average recall value of 0.8545 which is better than the average recall value of 0.7632 of XLarge model. In contrast, the XLarge model shows a higher precision value of 0.8020 compared to the low precision of 0.7149 of the Nano model. Furthermore, the XLarge model has an average mAP (mAP_0.5) of 0.7632 which is larger than the mAP value of the Nano model of 0.7027. The current prediction errors in this stage might be a result of the small training dataset. Limitations, reasons for caution The low amount of data makes validating the models difficult, especially evaluating how well they generalize to unseen samples. Furthermore, the training data consisted of samples containing low sperm counts, making the performance on samples with high sperm concentration uncertain. Wider implications of the findings Sperm tracking is integral to achieving accurate and less subjective motility assessment. The detections can be used to analyse the individual spermatozoa, leading to better performance. Deep feature extraction, trajectory prediction, and trajectory extraction can be used for future studies like generating synthetic spermatozoa for training generalizable machine learning models. Trial registration number not applicable

O-044 ICSI in the lab: from vintage to AI

Abstract With the advent of in vitro fertilization (IVF) by Patrick Steptoe and Bob Edwards in the late 70s, the first conception outside of the human body resulted in the birth of Louise Brown. Although a terrific success, limitations of IVF surfaced, represented by the unexpected complete fertilization failure with suboptimal or dysfunctional spermatozoa. This prompted curiosity toward individual spermatozoa for a deeper understanding of its role aimed at enhancing the interaction between complementary gametes. Techniques were designed to manipulate the oocytes, such as stripping, partially digesting, or cracking the zona pellucida (ZP). These methods were palliative solutions to overcome fertilization failure and were often plagued by polyspermy. As a result, more direct approaches were implemented to overcome the ZP, such as subzonal injection (SUZI) that although more consistent, was still unable to overcome the shortcomings of dysfunctional spermatozoa. This laid the foundation for the utilization of ICSI that, whilst attempted by some investigators, became popular when Gianpiero Palermo serendipitously inserted one spermatozoon into the ooplasm during SUZI. Consistent fertilization then followed by injecting a cohort of oocytes by ICSI in SUZI cycles, and replacement of these embryos led to 4 pregnancies described in the first clinical ICSI report. To minimize oocyte damage, the procedure was further refined by inducing a deep invagination of the oolemma toward the 9 o’clock position, granting higher chances of post-injection survival. What set apart ICSI from other forms of ART was that any sperm sample, regardless of quality/quantity, would yield fertilization. Indeed, ICSI is the sole insemination method used with epididymal and testicular spermatozoa and has therefore revolutionized fertility treatment of azoospermic men. Indeed, even immotile testicular spermatozoa can still fertilize and yield successful pregnancies, albeit at a lower rate than their motile counterpart. Also, for these semen sources, aggressive sperm immobilization was introduced to enhance sperm membrane permeabilization and grant optimal fertilization results. Furthermore, ICSI has transformed the field of reproductive medicine by assisting other reproductive techniques, such as testing embryos for single gene defects to reduce the occurence of sperm DNA contamination, or overcoming the cryostress-induced changes of the ZP during cryopreservation allowing the oocyte to be fertilized at a higher rate. Oocyte cryopreservation now empowers women in their reproductive age to ordain their childbearing future. ICSI has proven to be the ultimate technique to overcome male infertility and has broadened its indication by yielding consistent fertilization and successful pregnancies in most circumstances, ensuring that men have the chance of fathering their own progeny. To date, ICSI is applied in several countries, and in some, is performed as the preferred/sole insemination method contributing to the birth of millions of babies worldwide. Thus far, no concerning differences have been seen in the health of ICSI versus standard IVF offspring, or even naturally conceived. In fact, it has been currently established that young adults of both genders born through ICSI retain their reproductive health. Despite its growing popularity, ICSI does not always succeed but still provides an invaluable platform to deepen our knowledge of gamete biology and helps to investigate/overcome some of the most severe and persistent forms of infertility. For example, combined with assisted gamete treatment, ICSI allows couples plagued by sperm-bound oocyte-activation-deficiency to achieve pregnancy. The need to increase access and curtail costs of reproductive care has led to the testing of automation in ART. This is also occurring with ICSI and to date, different automated modules have been proposed for oocyte denudation, sperm tracking, and robotic ICSI. Concurrently, there has been an interest in experimenting with artificial intelligence in the IVF laboratory to minimize human shortcomings and ensure that the best spermatozoon is chosen.

P-125 Effectiveness of IMSI technique in enhancing blastocyst formation in male factor infertility

Abstract Study question does the use of IMSI technique in sperm selection during ICSI in case of male factor enhance blastocyst formation? Summary answer The use of IMSI technique enhanced the ferritization and blastulation rates as well the blastocyst quality in comparison with the conventional ICSI technique What is known already Prior to sperm injection the morphology of the sperm cell is evaluated with high magnification Diffraction Interference Contrast (DIC) microscopy. ICSI is performed using morphologically well-shaped spermatozoa selected within the limits of the conventional ICSI inverted microscope magnification of x400. However, it became evident that the morphology of the individual spermatozoon used for microinjection into the oocyte is associated to both fertilization and pregnancy outcome.The impact of nuclear vacuoles in the head of spermatozoa on pregnancy outcome was analyzed too, showing that microinjection of vacuolated sperm reduced the pregnancy rate and was associated with a higher risk for early abortion. Study design, size, duration A prospective cohort observational randomized sibling-oocyte study A total of 300 infertile couples undergoing ICSI were included in the study from August 2020 to July 2021. Ethical approval from Alexandria University, faculty of Medicine. Participants/materials, setting, methods A total of 300 infertile couples undergoing ICSI, Male factor infertility according to the World Health Organization (WHO) criteria 2010, patients with Azoospermia.and sperm concentration less than 0.1 million/mL were excluded.The oocytes of each patient were randomly divided into two groups in 1 to 1 ratio. One group was injected with sperms selected by normal magnification (3357) oocytes and the other group was injected with sperms selected by high magnification (IMSI) technique (3384) oocytes. Main results and the role of chance Statistical results according to number of cases showed that IMSI is significantly higher than ICSI in the number of fertilized oocytes (Mean± SD was 9.46±4.10vs. 9.02 ± 4.20, P ≤ 0.001), number of blastocysts (Mean± SD was 4.74±3.42 vs. 4.47±3.27, P = 0.035) and number of good quality blastocysts (Mean± SD was 1.97±2.57 vs. 1.75±2.50, P = 0.009), with No Significant difference in the number of injected oocytes.Comparison between ICSI and IMSI according to rates related to number of oocytes. Statistical results according to rates showed that IMSI is significantly higher than ICSI in fertilization rate (Mean± SD was 84.25 ± 16.57 vs. 80.01 ± 17.95, P ≤ 0.001), and the rate of good quality blastocysts (Mean± SD was 17.04 ± 18.47 vs. 15.59 ± 18.71, P = 0.04). No Significant difference in the blastulation rate P = 0.106. Comparison between ICSI and IMSI according to total number of injected oocytes (n = 6741) Statistical results according to total number of injected oocytes showed that IMSI is significantly higher than ICSI in fertilized oocytes (83.9% vs. 80.6% P = 0.001), and blastocysts with good quality (17.4% vs. 15.7%, P = 0.05). No Significant difference in the total number of blastocysts P = 0.075. Limitations, reasons for caution implantation, pregnancy and abortion rates were not included in the study Wider implications of the findings Similar to the present result and agreement to our suggestion several studies showed higher fertilization and blastocyst formation with the use of IMSI techniques and others showed higher implantation and pregnancy rates and lower miscarriage rates.Our recommendation is to use IMSI technique during sperm selection for treating infertile couples Trial registration number not applicable

P-272 Automatic Tracking of the ICSI procedure using Deep Learning

Abstract Study question Can deep learning be used to detect and track spermatozoa and the different parts of an ICSI procedure? Summary answer Deep learning can be used as a tool to assist and organize the contents of an ICSI procedure. What is known already Sperm tracking has been a topic of research and practice for many years, especially in the context of computer-aided sperm analysis (CASA). Recent studies have proposed using deep learning algorithms to track spermatozoa for spermatozoon selection in human and animal samples. One critical part of performing ICSI involves the selection of the “best” spermatozoon for injection, but other parts of the procedure may also be of importance. However, as far as we know, tracking using deep learning has not been applied to the ICSI procedure, where detecting instruments and the oocyte could also be helpful in post-analysis and training. Study design, size, duration The study was performed using three anonymized videos of the ICSI procedure. The frames of the videos were manually annotated by data scientists and verified by an embryologist. The annotations were bounding boxes around specific parts of the ICSI procedure, including sperm, pipettes, and the oocyte. We trained a YOLOv5 model on the collected data, where two videos were used for training and one video for validation. Participants/materials, setting, methods The videos of the ICSI procedure were captured at 200x magnification with a DeltaPix camera at Fertilitetssenteret in Oslo, Norway. ICSI was performed using a Nikon ECLIPSE TE2000-S microscope connected with Eppendorf TransferMan 4m micromanipulators. The spermatozoa were immobilised in 5 µl Polyvinylpyrrolidone (PVP; CooperSurgical). The videos had a resolution of 1920x1080 and were resized to 640x640 before being processed by the YOLOv5 model. The data will be made public in a later study. Main results and the role of chance Mean average precision (mAP) with the threshold of 0.5 (mAP@.5) is the main quantitative parameter measured in the YOLOv5 model. All the experiments were performed using three-fold cross-validation, where we present the average metrics calculated over the three folds. Overall, the method showed an average mAP@.5 of 0.50 across all predicted classes, which means that the method can track the different components with good accuracy. Looking closer at the individual classes, we see that instruments like the holding pipette and ICSI pipette are detected with high accuracy with a mAP@.5 of 0.87 and 0.94, respectively. The oocyte is also easily tracked with a mAP@.5 of 0.92. The first polar body is well detected with a mAP@.5 of 0.65. The model has issues detecting and tracking individual sperm (both outside and within the pipette), where the method achieved a mAP@.5 of 0.46 for tracking sperm outside the pipette and 0.03 for the sperm inside the pipette. The low score of detecting the sperm in the pipette can be explained by the often unclear visibility of the sperm through the pipette and the low number of training samples. Limitations, reasons for caution The limited sample size makes the generalizability of the method difficult to determine. A more extensive evaluation is necessary. Moreover, as the currency study focuses on tracking, patient information and clinical outcome were not included in the analysis. Wider implications of the findings Deep learning has the potential to aid embryologists to perform successful ICSI through tracking and detection of spermatozoa, pipettes, and the oocyte. This could potentially lead to better internal quality control and teaching possibilities, and hopefully better results. Trial registration number not applicable

Sperm freezability is neither associated with the expression of aquaporin 3 nor sperm head dimensions in dromedary camel (Camelus dromedarius)

The expression of aquaglyceroporin 3 (AQP-3) has been demonstrated in the spermatozoa of several mammalian species and its role has been associated with cryotolerance. Post-thaw sperm quality from individual dromedary males with different response to freezing-thawing process was evaluated through sperm head morphometry. In order to understand the cellular mechanisms affected by cryoinjury we have explored the presence and distribution of sperm AQP-3 using western blotting and immunocytochemistry. WB showed different intensity of the specific signal bands at 28 kDa. Immunofluorescence assessments allowed us to identify five different and clear AQP-3 distribution patterns of labelling in the sperm plasma membrane; acrosome, post-acrosome, mid-piece, and principal and final tail. Although expression of AQP-3 varied among male ejaculates, the individual sperm response to freeze-thawing was not associated with AQP-3 expression. Thus, AQP3 expressions do not seem like a reliable predictor of sperm response to freeze-thawing process in this species. This work is the first to describe the morphometric characteristics of the heads of dromedary spermatozoa. No correlation was found between sperm head dimensions and sperm quality variables after freeze-thawing suggesting that dromedary camel sperm head morphometry is also not a reliable predictor of cryosurvival.

Island-specific evolution of a sex-primed autosome in a sexual planarian

The sexual strain of the planarian Schmidtea mediterranea, indigenous to Tunisia and several Mediterranean islands, is a hermaphrodite1,2. Here we isolate individual chromosomes and use sequencing, Hi-C3,4 and linkage mapping to assemble a chromosome-scale genome reference. The linkage map reveals an extremely low rate of recombination on chromosome 1. We confirm suppression of recombination on chromosome 1 by genotyping individual sperm cells and oocytes. We show that previously identified genomic regions that maintain heterozygosity even after prolonged inbreeding make up essentially all of chromosome 1. Genome sequencing of individuals isolated in the wild indicates that this phenomenon has evolved specifically in populations from Sardinia and Corsica. We find that most known master regulators5–13 of the reproductive system are located on chromosome 1. We used RNA interference14,15 to knock down a gene with haplotype-biased expression, which led to the formation of a more pronounced female mating organ. On the basis of these observations, we propose that chromosome 1 is a sex-primed autosome primed for evolution into a sex chromosome.

Shielding effect of melatonin improves seminal quality and oxidative stress indices during chilled storage of ram semen.

Supplementation of antioxidant to semen extender maintains seminal quality by reducing oxidative stress during preservation time period. The main aim of this study was to investigate the effect of different concentrations of melatonin supplementation on liquid storage of Magra ram semen. This study was performed on adult Magra ram (n = 8), and seminal ejaculates (48) were collected and evaluated for various macroscopic and microscopic seminal quality parameters for further processing. After preliminary evaluation, ejaculates of each collection session were mixed and divided into four equal aliquots. All the aliquots were diluted (1:10) with Tris-citric fructose egg yolk extender contained sans melatonin served as control, whereas the other three aliquots were supplemented with 0.5, 1 and 2mM MLT which were grouped as MLT0.5, MLT1 and MLT2, respectively. Thereafter, the samples were stored at 4 ºC for 72h, and various seminal parameters (individual sperm progressive motility, viability, abnormalities, plasma membrane functionality) along with oxidative stress parameters (total antioxidant capacity (TAC), malondialdehyde (MDA)) were evaluated at 0, 24, 48 and 72h of preservation. The results indicated that the mean percent values for progressive sperm motility, viability, plasma membrane functionality and TAC were significantly higher (p < 0.05) in treated groups with the highest values in MLT1 group. Significantly lower (p < 0.05) percentage of total sperm abnormalities and MDA level was observed in treatment groups compared to control group. The results report that supplementation of 1mM melatonin efficiently maintains the seminal quality and ameliorates oxidative stress during preservation at 4 ºC to 72h.

Effect on physiological parameters and semen quality upon oral administration of fresh onion juice to V-line rabbit buck during severe heat stress

Severe heat stress is recognized as a factor that severely influences the semen quality and antioxidant status of rabbits. In this context, fresh onion juice (FOJ) may be a safe and useful option to improve reproductive performance. This research was designed to evaluate the anti-stress effects of FOJ on physiological and semen parameters in heat-stressed bucks. Thirty-six V-line rabbit bucks were randomly distributed into three groups. The bucks received FOJ orally at different doses [0 (water), 1.5, and 3 ml/kg live body weight] every 2 days over a period of 2 months, with 3 weeks as an adaptation period. FOJ treatments significantly improved semen characteristics, such as libido, mass and individual sperm motility, semen concentration, sperm viability, and acrosome reaction with increased initial seminal fructose, via the oral administration of 1.5 and 3 mL FOJ/kg body weight, compared with the findings in control bucks. Seminal plasma antioxidant status was significantly enhanced by FOJ treatments. It was concluded that the oral administration of FOJ under severe heat stress can improve bucks’ semen characteristics and sex hormone concentrations except for testosterone, and it is considered a good strategy for improving the heat resistance of rabbit bucks, possibly due to its antioxidant activity.

Computer software (SiD) assisted real-time single sperm selection associated with fertilization and blastocyst formation

Research questionIs it possible to explore an association between individual sperm kinematics evaluated in real time and spermatozoa selected by an embryologist for intracytoplasmic sperm injection (ICSI), with subsequent normal fertilization and blastocyst formation using a novel artificial vision-based software (SiD V1.0; IVF 2.0, UK)? DesignICSI procedures were randomly video recorded and subjected to analysis using SiD V1.0, proprietary software developed by our group. In total, 383 individual spermatozoa were retrospectively analysed from a dataset of 78 ICSI-assisted reproductive technology cycles. SiD software computes the progressive motility parameters, straight-line velocity (VSL) and linearity of the curvilinear path (LIN), of each sperm trajectory, along with a quantitative value, head movement pattern (HMP), which is an indicator of the characteristics of the sperm head movement patterns. The mean VSL, LIN and HMP measurements for each set of spermatozoa were compared based on different outcome measures. ResultsStatistically significant differences were found in VSL, LIN and HMP among those spermatozoa selected for injection (P < 0.001). Additionally, LIN and HMP were found to be significantly different between successful and unsuccessful fertilization (P = 0.038 and P = 0.029, respectively). Additionally, significantly higher SiD scores were found for those spermatozoa that achieved both successful fertilization (P = 0.004) and blastocyst formation (P = 0.013). ConclusionThe possibility of carrying out real-time analyses of individual spermatozoa using an automatic tool such as SiD creates the opportunity to assist the embryologist in selecting the better spermatozoon for injection in an ICSI procedure.

English

This work was conducted to study the breeding soundness of semen producing bulls, their semen quality to functional tests and the inter relationship of these functional tests with spermatozoa motility percentage for the semen produced at National Animal Genetic Improvement Institute (NAGII). A total of 14 breeding bulls (Boran = 4, Crosses of 75% Holstein Frisian &times; 25% Boran = 4 and Holstein Frisian = 6) with respective ejaculates of 35, 33 and 57 for semen quality evaluations were considered. After physical examination of bulls for breeding soundness, semen samples were tested for functional and structural (spermatozoa acrosome integrity, viability and hypo osmotic swelling reactivity) tests and spermatozoa motility percentage using subjective and objective (computer assisted semen analysis; CASA) evaluation methods, to see the correlation of functional tests with spermatozoa motility percentage. And in the study, nevertheless, significant differences between Cross and HF breeds were not recorded for the breeding soundness evaluation parameters and for most of the semen quality parametric test values; significantly (P &lt; 0.05) minimal and highest parametric values of the breeding soundness evaluation and semen quality were recorded in Boran and HF breeds, respectively. All the functional semen quality evaluation tests (HOST, acrosome integrity, sperm morphological defect and viability) were related in one or the other to the objective CASA individual spermatozoa motility evaluation; which were not true with subjective individual spermatozoa motility estimation. Therefore, conducting stringent breeding soundness evaluation for these breeds and evaluating their individual spermatozoa motility percentage using CASA system instead of subjective motility estimation can screen good quality semen and subsequently make possible to predict their fertility potential. Key words: Boran, correlation, cross-breed, Holstein-Frisian, semen.

Morphological differences in tardigrade spermatozoa induce variation in gamete motility

BackgroundFertilization is an event at the beginning of ontogeny. Successful fertilization depends on strategies for uniting female and male gametes that developed throughout evolutionary history. In some species of tardigrades, investigations of reproduction have revealed that released spermatozoa swim in the water to reach a female, after which the gametes are stored in her body. The morphology of the spermatozoa includes a coiled nucleus and a species-specific-length acrosome. Although the mating behaviour and morphology of tardigrades have been reported, the motility of male gametes remains unknown. Here, using a high-speed camera, we recorded the spermatozoon motilities of two tardigrades, Paramacrobiotus sp. and Macrobiotus shonaicus, which have longer and shorter spermatozoa, respectively.ResultsThe movement of spermatozoa was faster in Paramacrobiotus sp. than in M. shonaicus, but the beat frequencies of the tails were equal, suggesting that the long tail improved acceleration. In both species, the head part consisting of a coiled nucleus and an acrosome did not swing, in contrast to the tail. The head part of Paramacrobiotus sp. spermatozoa swung harder during turning; in contrast, the tail of M. shonaicus moved more widely than the head. Finally, after mating, the spermatozoa that reached the female aggregated around the cloaca while waiting to enter her body in both tested species.ConclusionsThis study provides results for the first observations and analyses of individual spermatozoon motility in tardigrades. A comparison of the spermatozoon movements of the two tardigrades suggested that the motilities of the male gametes were affected by morphological differences, where the longer spermatozoa swam faster and the shorter ones showed more stable swimming. Swimming was mainly induced by tail movement, but the long head of Paramacrobiotus sp. spermatozoa might be especially important for turning. In addition, observations of mated female cloacae suggested that the head parts of the spermatozoa were required for aggregation around the cloaca of a mated female.

Collection and evaluation of indigenous buck semen at the coastal region of Bangladesh

Evaluation of semen characteristics is an important and prior for semen preservation. The aim of this study was to collection and evaluation of indigenous buck semen in the coastal region of Bangladesh. The semen was collected from bucks through artificial vagina method. The colour, odour, volume, viscosity, mass activity, consistency, concentration and individual sperm motility were analysed and recorded after collection from pre-selected four bucks. The colour and odour of all buck (B) semen were creamy white to milky white and fishy smell, respectively. In this study, we found that the average volume of B-1, B-2, B-3 and B-4 were 0.74, 0.98, 0.42 and 0.60 ml, respectively. The average grading of viscosity of B-1, B-2, B-3 and B-4 were 3.2, 3.8, 2.6 and 3.0, respectively. The average grading of mass activity of B-1, B-2, B-3 and B-4 were 3.6, 3.2, 2.4 and 3.4, respectively. The consistency of B-1, B-2, B-3 and B-4 were 4.2, 4.8, 2.8 and 4.0, respectively. The concentration of B-1, B-2, B-3 and B-4 were 1.58, 1.94, 0.62 and 1.54 ×109 per ml of semen volume. The average percentage of individual sperm motility of B-1, B-2, B-3 and B-4 were 81, 71, 66 and 80%, respectively. Viscosity, mass activity, consistency, concentration and individual sperm motility were significantly (p &lt; 0.05) correlate with each other. It may be concluded that the data about semen of the bucks are in acceptable level for preservation. Further study will be designed for the evaluation of viability and motility of sperm before and after freezing as liquid semen.

Effect of taurine in tris egg yolk citrate extender on quality of surti buck semen at refrigerated temperature

Semen was collected from four Surti bucks (>24 months-old) twice a week for 8 weeks (16 ejaculates per buck) during November, 2020 to March, 2021. Ejaculates of all four bucks were pooled and divided into five equal aliquots. The aliquots were diluted with tris-egg yolk-citrate (TEYC) extender containing 0 (control T1), 25 (T2), 50 (T3), 75 (T4) and 100 mM (T5) taurine with final concentration of 200x106 sperm/ml (pH 6.5 to 6.8) in each. At 24, 36 and 48 h of refrigerated storage, individual sperm motility, per cent live sperm and HOST reacted sperm were significantly (p<0.01) higher in T3 (50 mM) and abnormal sperm % was significantly (p<0.01) lower in T3 (50 mM) and T4 (75 mM) as compared to other groups. With increasing duration of storage, significant (p<0.01) increasing trend for abnormal sperm per cent and decreasing trend for rest of the characters were observed. Motility showed significant (p<0.01) positive correlation with live spermatozoa, abnormal spermatozoa and HOST-reacted spermatozoa. Live count showed significant (p<0.01) negative correlation with abnormal spermatozoa and significant (p<0.01) positive correlation with HOST-reacted spermatozoa. Live spermatozoa showed significant (p<0.01) negative correlation with abnormal spermatozoa and HOST non-reacted spermatozoa. Abnormal spermatozoa showed significant (p<0.01) negative correlation with HOST-reacted spermatozoa. It was concluded that supplementation of 50 mM taurine in TEYC extender maintained optimum quality of buck semen at refrigerated temperature up to 48 h.

Simultaneous Morphology, Motility, and Fragmentation Analysis of Live Individual Sperm Cells for Male Fertility Evaluation

A new technique for sperm analysis is presented, measuring DNA fragmentation, morphology with virtual staining, and motility, all three criteria on the same individual unstained live cell. The method relies on quantitative stain‐free interferometric imaging, providing unique topographic structural and content maps of the cell, becoming available for the first time for clinical use, together with deep‐learning frameworks and least‐squares linear approximation. In the common clinical practice, only motility evaluation can be carried out on live human cells, while full morphological evaluation and DNA fragmentation assays require different staining protocols, and therefore cannot be performed on the same cell, resulting in inconsistencies in fertility evaluation. A clinic‐ready interferometric module is used to acquire dynamic sperm cells without chemical staining, together with deep learning to evaluate all three scores per cell with accuracy of 93.1%, 88%, and 90% for morphology, motility, and DNA fragmentation, respectively. It is shown that the expected number of cells that pass all three criteria based on the current evaluations performed separately does not correspond with the number of cells that pass all criteria, demonstrating the importance of the suggested method. The proposed stain‐free evaluation method is expected to decrease uncertainty in infertility diagnosis, increasing treatment success rates.

DNA repair and response to sperm DNA damage in oocytes and embryos, and the potential consequences in ART: a systematic review.

Sperm DNA damage is considered a predictive factor for the clinical outcomes of patients undergoing ART. Laboratory evidence suggests that zygotes and developing embryos have adopted specific response and repair mechanisms to repair DNA damage of paternal origin. We have conducted a systematic review in accordance with guidelines from Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) to identify and review the maternal mechanisms used to respond and repair sperm DNA damage during early embryonic development, how these mechanisms operate and their potential clinical implications. The literature search was conducted in Ovid MEDLINE and Embase databases until May 2021. Out of 6297 articles initially identified, 36 studies were found to be relevant through cross referencing and were fully extracted. The collective evidence in human and animal models indicate that the early embryo has the capacity to repair DNA damage within sperm by activating maternally driven mechanisms throughout embryonic development. However, this capacity is limited and likely declines with age. The link between age and decreased DNA repair capacity could explain decreased oocyte quality in older women, poor reproductive outcomes in idiopathic cases and patients who present high sperm DNA damage. Ultimately, further understanding mechanisms underlying the maternal repair of sperm DNA damage could lead to the development of targeted therapies to decrease sperm DNA damage, improved oocyte quality to combat incoming DNA insults or lead to development of methodologies to identify individual spermatozoa without DNA damage.

Sperm Inspection for In Vitro Fertilization via Self-Assembled Microdroplet Formation and Quantitative Phase Microscopy.

We present a new method for the selection of individual sperm cells using a microfluidic device that automatically traps each cell in a separate microdroplet that then individually self-assembles with other microdroplets, permitting the controlled measurement of the cells using quantitative phase microscopy. Following cell trapping and droplet formation, we utilize quantitative phase microscopy integrated with bright-field imaging for individual sperm morphology and motility inspection. We then perform individual sperm selection using a single-cell micromanipulator, which is enhanced by the microdroplet-trapping procedure described above. This method can improve sperm selection for intracytoplasmic sperm injection, a common type of in vitro fertilization procedure.

Single sperm vitrification with permeable cryoprotectant-free medium is more effective in patients with severe oligozoospermia and azoospermia

Testicular sperm extraction (TESE) is an invasive surgery for achieving the spermatozoa in cases with azoospermia. In these patients, the number of retrieved spermatozoa is limited and the optimal cryo-storage is very critical for their fertility preservation. Therefore, single sperm vitrification has been introduced for preservation of low number of spermatozoa.The goal was to assess the efficacy of sperm freezing medium (SFM) and sucrose medium as cryoprotectants for single sperm vitrification in cases with severe oligozoospermia and azoospermia.A total of 20 ejaculates from severe oligozoospermia and 20 testicular samples from azoospermia were processed. Twenty-five sperm cells were collected using ICSI injection pipette and transferred to a cryoprotectant droplet placed on the Cryotech, then vitrified by plunging in liquid nitrogen. Sperm motility, viability, fine-morphology, mitochondrial activity and DNA fragmentation index (DFI) were assessed before and after vitrification.Sperm motility, viability and the percentage of cells with mitochondrial activity were significantly decreased after vitrification in both severe oligozoospermic and testicular samples in either cryoprotectants. However, the rates of post-warm sperm motility and the cells with mitochondrial activity increased significantly in sucrose medium in both severe oligozoospermic and testicular samples compared to SFM. In testicular samples, the DFI of spermatozoa vitrified in SFM was significantly higher than those vitrified with sucrose medium.Sperm motility, viability, mitochondrial activity, and DNA integrity were better preserved in sucrose medium than SFM after single cell vitrification. The presented method may be a useful candidate for successful freezing of individual sperm cells in clinical setting.

Hexavalent sperm-binding IgG antibody released from vaginal film for development of potent on-demand nonhormonal female contraception

Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana-expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody "Fab-IgG-Fab" (FIF). The Nicotiana-produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana-produced FIF into a polyvinyl alcohol-based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.

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

This article was published in Cell J (Yakhteh), Vol 23, No 4, 2021, on pages 389-396, corresponding author asked us to add another address (5. Biomedical and Pharmaceutical Research Unit, QU Health, Qatar University, Doha, Qatar) to the fourth author's affiliation. The authors would like to apologies for any inconvenience caused.

DNA profiling of single sperm cells after whole genome amplification

Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analyzing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.