Fresh Semen Samples Research Articles

A novel microfluidic system to separate sperm using spermatozoa inherent motion and inertial effect

Sperm separation is an essential part of in vitro fertilization (IVF) process. In conventional procedures, the semen sample is purified from immotile and round cells using centrifugation, which may damage sperm DNA. This study aimed to design a novel microchip to separate the progressively motile spermatozoa using a passive method instead of centrifugation. This microchip is a novel, non-invasive, and two-stage device for auto-selecting the sperm used for IVF. The first stage was designed based on lateral differentiation and rapid divergence to separate the pathways of sperm and round cells. The second stage separates high-quality sperm based on their inherent motion. Before experimenting with fresh human semen samples, preliminary tests were performed using standard particles. The results showed that at the optimized flow rate for separation (1.7 ml/h), the concentration of progressively motile spermatozoa at outlet was significantly increased compared with the initial sample.

Unaffected semen quality parameters in Neospora caninum seropositive Belgian Blue bulls

Neospora caninum is a protozoan parasite that causes abortion, perinatal mortality, and subfertility in cattle worldwide. Despite the presence of the DNA of the parasite in semen of infected bulls, the effect on semen quality has not been extensively studied. This study aimed to investigate the effect of a natural Neospora caninum infection on fresh and frozen semen quality parameters in Belgian Blue bulls. Two hundred and fourteen bulls were serologically screened with an indirect ELISA-test specific for anti-Neospora caninum antibodies, every two months during one year. In addition to serological screening, semen was collected twice weekly using an artificial vagina. The following semen quality parameters were assessed: ejaculate volume, concentration, total motility of fresh semen samples, as well as morphology, total and progressive motility for frozen/thawed semen samples. Bulls were semen sampled throughout the whole year, but only semen samples of bulls that had six consecutive positive or negative ELISA-test results were included in our dataset (n = 98). Generalized linear and binomial mixed models were used for statistical analysis of each outcome variable. In these models the explanatory variables were defined as: age, barn location, mean Temperature Humidity Index (THI) during sperm production (14–42 days before sampling), maximum daily THI at collection, season of sperm production, season at collection and the Neospora caninum antibody test results. Initially, individual explanatory variables were tested in univariable models for each outcome variable. Akaike information criterion (AIC) values were used to select explanatory variables to build a multivariable model, where the Neospora caninum test result was forced in all models. The present study reveals an overall apparent seroprevalence of Neospora caninum of 9,2% in the study population. No significant associations were detected between natural neosporosis, substantiated by ELISA-antibody levels, and any of our tested outcome variables on fresh and frozen/thawed semen samples. Based on the results of the present study, we conclude that Neospora caninum seropositive bulls do not have lower semen quality parameters compared with seronegative bulls.

P-095 Is SARS-CoV-2 really present in human semen? New evidence on this issue

Study questionCan be the SARS-CoV-2 present in human semen samples from patients infected with COVID-19?Summary answerOur research confirms the presence of SARS-CoV-2 in human ejaculate.What is known alreadyIn December 2019 WHO was informed of cases of pneumonia of unknown etiology. This was the beginning of a global pandemic that we are suffering still.COVID-19 disease may damage the male reproductive system. The issue of the presence of SARS-CoV-2 in human semen has been addressed in many studies. Most of them concluded that this virus is not present in human semen. However, until now four groups have reported the presence of SASR-CoV-2.It is quite clear that there is an important controversy about the presence of this virus in semen. Further research is needed to clarify this issue.Study design, size, durationProspective and experimental cohort study to evaluate the presence of SARS-CoV-2 in human semen.Men, COVID-19 disease confirmed, were asked to donate semen sample from July 2020 to March 2021. The patients were recruited with written informed consent.Explicit instructions were given to the correct collection of samples. Participants were interviewed to confirm that collection and transport took place under hygienic conditions. Semen samples were accepted only when instructions were adequately followed.Participants/materials, setting, methodsSemen samples were obtained from 50 men (aged 20-51 years) who were diagnosed with qRT-PCR-COVID19 positive (Ct < 40) in nasopharyngeal swab. Patients were closely followed while in hospital or at home.Each semen sample was divided in three aliquots (1 milliliter each) in order to: 1. Perform a spermiogram, 2. Evaluate SARS-CoV-2 with qRT-PCR-COVID19 and 3. Freeze for later use, if necessary. Patients were asked to provide a new specimen if PCR in semen was positive.Main results and the role of chancePatients included in the study showed different types of pneumonia: mild/moderate (N = 46) and severe (N = 6). Patients with severe symptoms had to be placed in intensive care unit.Average time from admission to hospital to sperm collection was 5,8 days.The study did not find any correlation between fresh semen parameters (volume, pH, count and motility) and severity of illness.SARS-CoV-2 was detected in one of fifty (1/50) fresh semen samples. The patient with SARS-CoV-2 in semen had mild symptoms of pneumonia (fever, headache and muscle pain). The patient was asked about how the semen sample was collected. Standards of hygiene during semen collection were confirmed by patient. Presence of SARS-CoV-2 in patient’s semen sample was verified.Limitations, reasons for cautionIt is possible (though unlikely) that semen sample collection and transportation were incorrect or unhygienic (it was refuted by patient). Besides that, we cannot exclude a false positive in PCR analysis. Although this test has very high specificity with a practical absence of false positives.Wider implications of the findingsAlthough it is evident that the presence of SARS-CoV-2 in human semen is extremely rare, at least four studies confirm this view. In any case the presence of this virus in human semen samples cannot be completely ruled out, on the contrary, further studies are required to confirm this presence.Trial registration number2020-310-1

P-045 Sperm chromatin structure resilience is related to fertilization and blastocyst rates in ICSI cycles involving both oocyte and sperm donors

Abstract Study question Does the structural maintenance of sperm chromatin have an implication in assisted reproduction outcomes? Summary answer Sperm deprotamination is related to fertilizing capacity. Also, the time needed for the reduction of protamine disulphide bridges is a parameter underlying early embryo development. What is known already How male factor affects embryo development has been a controversial topic for decades, with opposite results. A meta-analysis recently showed that whereas sperm genotoxic damage has an impact on IVF success, its repercussion on ICSI remains unclear. Revealing the factors that may predict embryo development is much warranted to understand the sperm contribution in the ICSI era. Sperm DNA is preferentially protamine-condensed and necessarily needs to be switched to histone-condensed chromatin upon fertilization. This study aims to determine the degree of protamination and the disulphide bond integrity to provide new insights into the influence of chromatin resilience on embryo development. Study design, size, duration The study setting included a public university and a private sperm bank. The design was a blinded retrospective study, including samples from 51 sperm donors that were used for a total of 55 cycles conducted with donated oocytes in the fertility clinic. Sperm quality parameters, including those focused on chromatin structure, were evaluated after 0 min and 240 min post-thawing and compared to embryo outcomes. Participants/materials, setting, methods Sperm samples from donors selected by the germplasm bank were collected and used to fertilize oocytes in double-donation cycles (oocyte donation) with standard intracytoplasmic sperm injection. Cryopreserved samples were used to assess sperm viability (SYBR14/PI), incidence of thiol reduction (Dibromobimane, DBB) and chromatin deprotamination (Chromomycin A3, CMA3) through flow cytometry, at 0 min and 240 min after thawing. Positive controls after incubation with Triton-X100, DTT and DTT+NaCl were used to establish the flow cytometry thresholds. Main results and the role of chance In fresh semen samples, sperm concentration was 34.12±18.13 sperm/mL and progressive motility was 37.49±13.22% (mean±standard deviation). At 0 min and 240 min post-thawing, sperm viability was 24.80±12.12% and 18.65±10.37%, respectively, and the death rate was -1.54±1.24%/hour. Percentages of sperm with reduced thiols were 17.37±11.26% and 11.53±9.16% after 0 min and 240 min post-thawing, the reduction rate being -1.46±2.31%. Percentages of sperm with chromatin deprotamination were 21.12±9.03% and 35.34±14.10% after 0 min and 240 min post-thawing, and the deprotamination rate was 14.22±11.90%/hour. ICSI was conducted with an average of 11.86±7.89 oocytes per sperm sample, of which 8.78±5.95 were fertilized; the mean fertilization rate was 75.77±17.61%. For each sperm sample, 5.98±4.93 blastocysts at day 5 were obtained, leading to an average blastocyst rate of 65.17±22.22%. Interestingly, the deprotamination status at 0 min post-thaw was correlated to fertilization rates (Rs= -0.388; P = 0.005) and the decreasing rate of sperm with reduced thiols was correlated to blastocyst rate (Rs= -0.351; P = 0.012). Reduced thiols and deprotamination were also correlated with each other (Rs= -0.320; P = 0.022). Limitations, reasons for caution The main limitation of the present study was the sample size. More donors are thus needed in order for the promising results found in this study to be confirmed. Wider implications of the findings The research conducted herein has evidenced that the resilience of the sperm chromatin structure is related to embryo development outcomes from ICSI cycles conducted with oocyte donors. Trial registration number N/A

Characteristics of fresh and frozen-thawed Uda ram semen extended with Oviplus®, egg yolk and coconut milk

Semen characteristics of Uda sheep and the effects of the egg yolk and coconut milk-based extenders on the quality of semen preserved at 4 oC and -196 oC were evaluated. Semen was collected from six Uda rams using an electro-ejaculator twice a week for ten weeks. Fresh semen samples were pooled and analyzed macroscopically and microscopically. The pooled semen was divided into 2 aliquots. One aliquot was extended in OviPlus® and egg yolk. The second aliquot was extended with OviPlus® and coconut milk. Each of the aliquots was further subdivided into 2 parts and evaluated microscopically. One part was chilled at 4 oC and evaluated for the same parameters after extension at 24, 48 and 72 hours. Second part was loaded into 0.25 ml plastic straws for cryopreservation at -196 ℃ and analyzed after 24 hours. The post thaw spermatozoa motility, livability and morphological abnormalities were determined at 24, 48 and 72 hours. The motility and concentration of freshly collected Uda semen were 81.7 ± 1.7 % and 3.2 ± 0.3 ×109/ml respectively. After extension, the motility decreased significantly (P &lt; 0.05) from 82 % at 3 hours to 17 % at 72. The percentage live spermatozoa of the chilled semen did not differ significantly (P &gt; 0.05) between the two extenders. Post thaw spermatozoa motility and livability were significantly (P &lt; 0.05) reduced. There were significant differences (P &lt; 0.05) between the post-thaw proportions of morphological abnormalities, between semen preserved at 4 oC and the frozen-thawed semen. In conclusion, semen motility, livability, and morphological abnormalities of Uda ram are equally preserved in coconut milk and egg yolk-based extenders at 4 oC up to 24 hours post extension. However, semen motility and livability were significantly reduced in the Uda semen earlier cryopreserved at -196 oC after thawing.

Effects of sperm preparation techniques on sperm survivability and DNA fragmentation.

ObjectiveThis study was performed to determine the effect of swim-up (SU) and density gradient centrifugation (DGC) on sperm survival and DNA fragmentation.MethodsIndividual semen samples were analyzed before each was divided into two aliquots (half for SU and half for DGC) for calculation of sperm survival and the DNA fragmentation index (DFI). Sperm DNA fragmentation was determined using the sperm chromatin dispersion test.ResultsThe DFI of the 63 semen samples processed using both procedures was lower than that of the fresh semen samples. The DFI was significantly lower for samples processed using the SU than DGC method. In the sperm survival test, the SU technique was associated with increased sperm motility and vitality following preparation. After 24 hours, however, the concentration and percentage of surviving sperm were significantly lower in the SU than DGC group.ConclusionsBoth semen preparation techniques help to minimize sperm DNA fragmentation; however, when the DFI is <30%, the SU technique is more appropriate than DGC. While DGC may be superior for intrauterine insemination, the SU method may be preferable for in vitro fertilization or maturation.

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality, potentially affecting all treatment outcomes.

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality, potentially affecting all treatment outcomes.

Qualitative and Quantitative Estimation of Osteopontin in Bull Semen and its Effect on Sperm Quality Parameters and Fertility Rate In vivo

Background: Osteopontin plays a crucial role in fertilization and subsequent embryonic development. Methods: The fresh semen samples were collected from crossbred Jersey and Haryana breeding bulls. The proteins in the seminal plasma and sperm membrane were precipitated and analysed by SDS-PAGE followed by western blotting. Based on the concentration of osteopontin in the seminal plasma, the bulls were grouped as high (≥20 pg/ml), medium (10-20 pg/ml) and low (≤10 pg/ml) OPN groups. Result: Total 13 and 14 different proteins were identified in the seminal plasma and sperm membrane, respectively. The sperm velocity parameters (straight line velocity and straightness) and functional membrane integrity showed significant difference among high, medium and low OPN groups. Significant difference (P less than 0.05) was observed in malondialdehyde, SOD and glutathione reductase levels. Bulls which are having high osteopontin concentration in the semen had less sperm abnormality, less apoptotic cells, better resistance to cryopreservation, less damaged chromatin and higher conception rate (64.80±1.09) than bulls having low osteopontin concentration in their semen. Therefore, osteopontin may be a valuable protein marker to predict fertility in bulls.

The effects of cysteine and L-carnitine on the DNA integrity of post-thaw sperm of frozen buck semen.

The addition of antioxidants to semen extenders is fundamental to limit oxidative changes resulting from cryopreservation. This experiment was designed to clarify the effects of cysteine and L-carnitine (LC) on post-thaw sperm criteria. Semen samples were collected once weekly from five mature Zaraibi bucks for 10 successive weeks. Fresh semen samples were evaluated for basic semen characteristics, and the accepted samples were pooled and divided into seven aliquots: control (Tris egg yolk extender), cysteine (2.5, 5 and 10 mM) and LC (2.5, 5 and 7.5 mM). Aliquots were diluted and subjected to cryopreservation procedures. After thawing, sperm criteria were evaluated regarding motility, viability, plasma membrane (hypo-osmotic swelling test, HOST) and acrosome integrity, total antioxidant capacity (TAC), level of malondialdehyde (MDA), and DNA comet assay. Addition of both 10 mM cysteine and 7.5 mM LC significantly increased post-thaw sperm motility, viability, HOST, and acrosome integrity. Comet assay revealed significant decreases in DNA damage with best results at 2.5 mM cysteine and 7.5 mM LC. Besides, 10 mM cysteine and 7.5 mM LC exhibited significant diminish in MDA levels. Cysteine was positively correlated with sperm viability, acrosome integrity, and comet, but negatively correlated with MDA and tail length. Positive correlations were declared between LC and progressive motility, viability, HOST and TAC, while negative correlations were found with MDA, comet, tail DNA, tail moment, and olive tail moment. Supplementing cysteine and LC to frozen buck semen improved sperm criteria and decreased DNA damage, possibly due to their effectiveness in the suppression of MDA formation.

An In vitro Study on the Protective Effect of Melatonin on Human Sperm Parameters Treated by Cadmium.

ABSTRACTBackground:Male infertility account for nearly 50% of infertility cases. Cadmium is regarded as a well-known toxic metal for industrial applications; high amounts of cadmium in the human body can result in chronic toxicity. Melatonin as a free radical scavenger has anti-inflammatory, and even anti-cancer and antiapoptotic functions.Aim:In this work, we evaluated the protective effect of melatonin on human sperm parameters treated by cadmium.Study Setting and Design:This was an experimental study carried out from May to December 2019.Materials and Methods:A total of 41 fresh semen samples were collected from fertile men and were divided into 4 groups: (1) control, (2) sperm +25 Nm cd, (3) sperm +25 nM cd +0.1 mM melatonin,(4) sperm +0.1 mM melatonin treated for 60 min. In all groups, semen analysis was performed for motility, viability and DNA fragmentation index (DFI).Statistical Analysis:The groups were compared using the ANOVA test.Results:The group treated with cadmium showed a significant decrease in rapid and slow motility, and survival rate compared with the control group (P < 0.05). However, the degree of DFI and sperm with non-progressive motility in the group treated with cadmium had a significant increase compared to the control (P < 0.05). The use of melatonin significantly improved sperm parameters such as motility, survival rate and decreased sperm DFI with non-progressive motility.Conclusions:The use of melatonin reduces the amount of cadmium damage in human sperm in vitro.

Semen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bulls

Genomic selection in cattle breeding has gradually allowed younger bulls to be recruited for semen production. In this study, sperm quality parameters, seminal plasma and sperm metabolites, semen production capacity and fertility in young Norwegian Red bulls were analysed. For in vitro analyses of sperm quality and metabolites, ejaculates were collected from the same 25 bulls at both 14 and 17 months of age. Semen production and fertility data were collected for all Norwegian Red bulls in production from December 2017 throughout 2019. Bull fertility was measured as 56 days non-return rate (NR56), for both age groups. In both fresh and frozen-thawed semen samples, the proportion of hyperactive spermatozoa, average path velocity, curvilinear velocity and amplitude of lateral head displacement were higher in samples collected at 17 months of age compared to 14 months (P<0.05). In addition, several amino acids including arginine, glutamine, cysteine and proline, were affected by age (P<0.05). The concentrations of K and Ba increased significantly (P<0.05) with age in both seminal plasma and sperm cells. Sperm concentration and volume of the ejaculate increased significantly with increasing age, while the percentage of discarded batches decreased. The bulls’ NR56 decreased significantly with increasing age (75% vs. 74%). However, the difference of 1% is unlikely to be of biological importance. In conclusion, even small age differences in young bulls may significantly affect several sperm quality parameters, metabolite levels and semen production traits.

A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs

In order to accurately analyze the possible side effects of sperm cryopreservation, an in-depth screening of post-thaw sperm status is necessary. Thus, this study aimed to identify thorough effects of sperm cryopreservation, by evaluating the integrity of all specific structures of the canine spermatozoa. Thirteen (n = 13) mature dogs of different breeds were selected. Six dogs (n = 6) were subjected to sperm cryopreservation, whereas seven dogs (n = 7) were used as semen donors to validate a simultaneous assessment of sperm plasmatic, acrosomal, and mitochondrial membranes (triple stain) by fluorescent probes. Fresh and post-thaw semen samples were evaluated through a computer-assisted analysis of sperm motility, sperm morpho-functional evaluation, triple stain and sperm DNA integrity. Post-thaw semen samples had lower total and progressive motility, as well as higher percentage of minor and major defects. Moreover, post-thaw samples had higher percentage of sperm with plasma membrane and mitochondrial damage but intact acrosome, and also sperm with simultaneous damaged plasma, acrosomal and mitochondrial membranes. Furthermore, post-thaw sperm had higher protamination deficiency and DNA fragmentation. In conclusion, cryopreservation has a broad impact in sperm morphology and function, altering motility patterns, plasma, acrosome and mitochondrial membranes integrity, as well as sperm DNA.

Kualitas semen sapi pejantan berdasarkan umur, suhu, dan kelembaban di Taman Ternak Pendidikan Universitas Airlangga

This research was conducted at the Teaching Farm of the Faculty of Veterinary Medicine, Airlangga University, to know the effect of age, temperature, and humidity on the quality of semen of bulls which are turned into frozen semen. This research uses fresh semen samples of bulls of various ages grouped into 3 groups, young, middle age, and old groups and measures temperature and humidity during the storage process. Fresh semen is collected using an artificial vagina and then examined macroscopic and microscopic tests which is then checked for post thawing motility. Data analysis uses Analysis of Variants (ANOVA) followed by Duncan. The results showed that the percentage of spermatozoa motility of fresh semen and post thawing differed and age affected the quality of semen produced. There was no effect of temperature and humidity when processing of collecting semen. Bull’s semen at Teaching Farm Faculty of Veterinary Medicine Airlangga University has good quality and is suitable for Artificial Insemination.

A RAPID QC TESTING PLATFORM USING FROZEN SEMEN

The last twelve months have presented significant challenges for the ART laboratory. Mandatory shutdowns, lack of patient access, supply chain issues, and changing rules and recommendations brought on by COVID-19 have stretched laboratories to their limits in an attempt to maintain regular and required activities. One area of concern in the laboratory has been the lack of available fresh semen (FS) samples at the proper times for quality control (QC) and proficiency testing (PT). Cryopreserved semen (CS) would appear a reasonable alternative.

ASSESSMENT OF SEMEN CHARACTERISTICS IN CONSECUTIVE EJACULATES WITH SHORT ABSTINENCE USING SQA-V SPERM QUALITY ANALYZER

To determine the effect of consecutive ejaculates on seminal parameters after short abstinence using the SQA-V sperm quality analyzer tool. The prospective study was conducted from February 2020 to November 2020 in males undergoing treatment for primary infertility. Fifty-five fresh semen samples from infertile men were obtained by masturbation and analysed. Two semen samples were collected in succession after an abstinence period of 4 days followed by another sample after only 2 hrs. For both ejaculates, semen volume, sperm concentration, total motility & motility groups and detailed kinematic parameters were assessed using SQA-V sperm quality analyzer. The Statistical analysis was carried out by using the software, Statistical Package for Social Science, SPSS 20.0 (SPSS, Chicago, III., USA). The obtained results were analyzed statistically using student’s t-test & P-values <0.05 were considered significant. The second raw ejaculate demonstrated lower semen volume (1.39 ± 0.48 vs 3.0 ± 0.73, p<0.05) and sperm concentration (43.87 ± 14.69 vs 60.54 ± 14.50, p<0.05) than initial sample. While a significant increase in total motility and progressive motility in the consecutive sample than the initial sample (61.6 ± 8.21 vs 54.70 ± 8.38, p<0.05 and 49.83 ± 8.34 vs 43.45 ± 8.45, p<0.05). The findings of the present study indicate that pooling the spermatozoa from second ejaculate may allow for a simple method to select high quality spermatozoa and also could be an advantage for intrauterine insemination (where a large number of motile sperms are needed). Additionally, these spermatozoa also could prove advantage for conventional IVF or ICSI / fertility preservation where a large number of sperms are not required.

Paternal age over 50 years decreases assisted reproductive technology (ART) success: A single UK center retrospective analysis.

To study whether paternal age exerts an effect, independent of maternal age, on the outcomes of fresh in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) cycles. Semen quality deteriorates with increasing paternal age; however, there is conflicting evidence for any impact paternal age may have on the outcome of IVF/ICSI. Several retrospective and prospective cohort studies have shown that paternal age increases the miscarriage rate and reduces the live birth rate. Some studies have shown no effect of paternal age on live birth rate or miscarriage rate. Studies involving donor oocytes have tended to show no independent effect of paternal age on assisted reproductive technology (ART) outcomes. The age at which paternal age may exert a significant deleterious effect on outcome is not known and there is no limit to paternal age in IVF/ICSI treatment. A single-center retrospective cohort study was carried out at the Centre for Reproductive and Genetic Health, London, UK. Included in the analysis were all couples with primary or secondary infertility undergoing IVF/ICSI cycles in which the male partner produced a fresh semen sample and the cycle proceeded to fresh embryo transfer. All cycles of IVF/ICSI that used donor oocytes-donor sperm, frozen sperm, cycles leading to embryo storage and cycles including preimplantation genetic testing (PGT-A/PGT-M)-were excluded from analysis. The primary outcome was live birth rate and secondary outcomes were clinical pregnancy rate and miscarriage rate. Multivariate logistic regression analysis with live birth as a dependent variable and maternal and paternal age class as independent variables was performed. During the study period there were 4833 cycles, involving 4271men, eligible for analysis; 1974/4833 (40.8%, 95% confiene intervals [CI] 39.5-42.2%) cycles resulted in a live birth. A significantly lower proportion of men over 51years met World Health Organization semen analysis criteria (56/133, [42.1%, 95% CI 34.1-50.6]) compared with men under 51years of age (2530/4138 [61.1%, 95% CI 60.0-62.6]) (p=0.001). Both maternal and paternal age were retained in the multivariate model and for all maternal age subgroups the probability of live birth decreased with paternal age over 50years (odds ratio [OR] 0.674, 95% CI 0.482-0.943) (p=0.021). Paternal age over 50years was not an independent predictor of miscarriage (OR 0.678, 95% CI 0.369-1.250) (p=0.214). Paternal age over 50significantly affects the chance of achieving a live birth following ART. Paternal age does not independently affect the risk of miscarriage following ART. There should be a public health message for men not to delay fatherhood.

P–104 Assessment of sperm motility according to WHO classification using convolutional neural networks

Abstract Study question How does convolutional neural network (CNN)-predicted sperm motility correlate with manual assessment according to the WHO guidelines. Summary answer CNN predicts sperm motility comparable to reference laboratories in the ESHRE-SIGA External Quality Assessment Programme for Semen Analysis. What is known already Manual sperm motility assessment according to WHO guidelines is regarded as the gold standard. To obtain reliable and reproducible results, comprehensive training is essential as well as running internal and external quality control. Prediction based on artificial intelligence can potentially transfer human-level performance into models that perform the task faster and can avoid human assessor variations. CNNs have been groundbreaking in image processing. To develop AI models with high predictive power, the data set used should be of high quality and sperm motility assessment based on WHO guidelines. Study design, size, duration Videos of 65 fresh semen samples obtained from the ESHRE-SIGA External Quality Assessment Programme for Semen Analysis (from the period 2006–2018) were used in the development of the model. One video was captured for each semen sample. Sperm motility data was obtained from manual assessment of the videos according to WHO criteria by reference laboratories in the programme. Rapid progressive motility was also included. Ten-fold cross-validation was used to compensate for the relatively small dataset. Participants/materials, setting, methods The mean values of the reference laboratories were used. Sparse optical flow of the sperm videos was generated from each second of each video and fed into a ResNet50 convolutional neural network. For training, Adam was used to optimize the weights and mean squared error (MSE) to measure loss. For baseline, ZeroR (pseudo regression) was performed. Results are reported as MAE. For correlation analysis, Pearson’s r was used. Main results and the role of chance Predicting sperm motility based on the optical flow generated from the videos, achieved an average MAE of 0.05 across progressive (0.06), non-progressive (0.04) and immotile sperm (0.05). The ZeroR baseline was 0.09, indicating that the method is able to capture the movement of the spermatozoa and predict motility with low error. Pearson’s correlation between manually and AI-predicted motility showed r of 0.88, p &amp;lt; 0.001 for progressive, 0.59, p &amp;lt; 0.001 for non-progressive and 0.89, p &amp;lt; 0.001 for immotile sperm. When predicting rapid progressive motility, the average MAE was 0.07 across rapid progressive (0.11), slow progressive (0.09), non-progressive (0.04) and immotile sperm (0.05). Pearson’s correlation analysis between manually and AI-predicted motility showed r of 0.67, p &amp;lt; 0.001 for rapid progressive, 0.41, p &amp;lt; 0.001 for slow progressive, 0.51, p &amp;lt; 0.001 for non-progressive and 0.88, p &amp;lt; 0.001 for immotile sperm. The results show that differentiating between rapid progressive and slow progressive motility is difficult, but the model is still able to do this better than the ZeroR baseline, which was 0.15 for rapid progressive and 0.11 for slow progressive. This is interesting since rapid progressive motility has been regarded challenging to assess. The next step would be to compare the results of the algorithm to the human performance. Limitations, reasons for caution The sample size is small. The model is based on videos of high quality, and the performance may not transfer well to videos of lower quality. The performance for rapid progressive motility, which may have an important clinical value, has to be improved. Wider implications of the findings: This CNN model has a potential to assess sperm motility according to WHO guidelines for progressive motility and immotility. The error values for the automatic predictions are low, and the model shows a good performance taking into account that only videos were used to perform the prediction. Trial registration number Not applicable

The outcome of intra-cytoplasmic sperm injection (ICSI): do the sperm concentration and motility matter?

The male-factor subfertility was reported to be 30% globally; thus, the intracytoplasmic sperm injection (ICSI) procedure was implemented to improve the overall in vitro fertilization (IVF) rates. Nevertheless, at least 10% of fertilization failure still occurs. Concerning this issue, we explored the association of sperm concentration and motility with the quality of embryo development and pregnancy outcome in IVF-ICSI cycles. Retrospective analysis of 109 couples with male factor were done over 14months in a tertiary university hospital in Malaysia. The data were divided into four groups; Group I: normal sperm parameters, Group II: normal sperm concentration but reduced total sperm motility, Group III: reduced sperm concentration and motility, Group IV: reduced sperm concentration but normal sperm motility. Only fresh semen samples and fresh embryo transfers were included. The fertilization, cleavage rate, embryo quality and pregnancy outcome were assessed. Overall, group I had the highest oocytes yield and ICSI attempted; (10.12+6.50), whereas the lowest was in group IV; (7.00+2.82). Group II revealed the highest fertilization and cleavage rates; (54.14+25.36), (55.16+26.06), thus not surprisingly resulting in the highest number of good embryos and highest clinical pregnancy rates. The lowest cleavage and pregnancy rates were seen in group IV. However, all the outcomes were not statically significant (p>0.05). Similar fertilization rate and comparable pregnancy outcome was seen among couples with normal and reduced sperm concentration and motility.

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.

Sperm selection by rheotaxis improves sperm quality and early embryo development.

The objective of this work was to elucidate whether a sperm selection method that combines rheotaxis and microfluidics can improve the selection of spermatozoa over density gradient and swim-up. For this purpose human sperm selected by rheotaxis were compared against density gradient, swim-up and a control group of non-selected spermatozoa in split frozen-thawed (FT) and fresh (F) semen samples. Sperm quality was assessed in terms of motility, morphology, DNA fragmentation index (DFI), viability, acrosome integrity and membrane fluidity. Using a mouse model, we compared fertilisation and embryo development rates after performing ICSI with spermatozoa, sorted using rheotaxis or swim-up. Selection by rheotaxis yielded a sperm population with reduced DFI than the control (P < 0.05), improved normal morphology (P < 0.001) and higher total motility (TM; P < 0.001) than the other techniques studied in F and FT samples. Swim-up increased TM compared to density gradient and control in FT or F samples (P < 0.001), and yielded lower DFI than the control with F samples (P < 0.05). In FT samples, selection by rheotaxis yielded sperm with higher viability than control, density gradient and swim-up (P < 0.01) while acrosomal integrity and membrane fluidity were maintained. When mouse spermatozoa were selected for ICSI using rheotaxis compared to swim-up, there was an increase in fertilisation (P < 0.01), implantation (P < 0.001) and foetal development rates (P < 0.05). These results suggest that, in the absence of non-destructive DNA testing, the positive rheotaxis can be used to select a population of low DNA fragmentation spermatozoa with high motility, morphology and viability, leading to improved embryo developmental rates.