Sperm Sorting Research Articles

Prediction of sperm motion behavior in microfluidic channel using sperm swimming model

Several investigations have recently been conducted using microfluidic channels to sort highly motile sperm and thereby increase the probability of fertilization. To further enhance the efficiency of sperm sorting, predicting sperm movement in microfluidic channels through simulation techniques could be beneficial. In this study, we constructed a sperm swimming model based on the concept of an agent-based model. This model allows analysis at the same spatio–temporal scale similar to microfluidic channels. Sperm movement was simplistically modeled as a random walk, utilizing the distribution of sperm velocity and deflection angle obtained from experimental data. We have developed a thigmotaxis model to describe the phenomenon where sperm near the wall exhibit a reduced tendency to move away from it. Additionally, we created a rheotaxis model, in which sperm reorient in the direction opposite to the flow depending on the shear rate. Using these models, we investigated sperm behaviors within a microchannel featuring a tapered area. The results reveal that sperm accumulate within the tapered area, leading to a significant increase in sperm concentration for specific flow velocity ranges in the microchannel. This model provides valuable information for predicting the effects of sperm sorting in various microfluidic channels.

Effect of Two Different Sperm Selection Methods on Boar Sperm Parameters and In Vitro Fertilisation Outcomes.

Porcine in vitro embryo production (IVP) protocols have conventionally used density gradient selection (DGS) by centrifugation to prepare sperm samples and achieve successful fertilisation. However, the possible toxicity of the solutions used and the potential damage caused by the centrifugation step may have a negative effect on the quality of the sample. Microfluidic chip-based sperm (MCS) sorting has been proposed as an alternative technique for the selection of high-quality sperm with the purpose of improving reproductive outcomes in IVF. This device does not require centrifugation or any toxic solution to prepare the sample for fertilisation. The sample is not subjected to unnecessary stress, and the process is less operator-dependent. In this study, we compared the sperm parameters of unselected extender-diluted boar semen samples with selected samples using DGS and MCS methods. The results show an expected reduction in sperm concentration after both methods. All the groups were significantly different from one another, with MCS being the group with the lowest concentration. Though the three groups had a similar overall motility, significant differences were found in progressive motility when comparing the unselected group (control, 19.5 ± 1.4%) with DGS and MCS. Progressive motility in DGS was also significantly higher than in MCS (65.2 ± 4.9% and 45.7% ± 5.3, respectively). However, MCS selection resulted in enriched sperm samples with a significantly lower proportion of morphologically abnormal sperm compared to DGS. After fertilisation, no statistical differences were found between the two methods for embryological parameters such as cleavage rates, blastulation rates, and embryo quality. The number of cells in blastocysts derived from MCS was significantly greater than those derived from DGS sperm. Thus, we demonstrate that MCS is at least as good as the standard DGS for most measures. As a more gentle and reproducible approach for sperm selection, however, it could improve consistency and improve IVP outcomes as mediated by a greater proportion of morphologically normal sperm and manifested by a higher cell count in blastocysts.

Live Motile Sperm Sorting Device Improves Embryo Aneuploidy: A Retrospective Cohort Study

Background: Conventional sperm selection methods, involving centrifugation, exert a detrimental effect on sperm DNA integrity due to mechanical stress. The recent noninvasive sperm selection device, CA0, based on live sperm sorting technology, facilitates the retrieval of highly motile sperm and minimizes sperm DNA fragmentation (SDF). This study was to investigate the impact of various sperm separation methods, with and without centrifugation, on embryo ploidy status. Methods: The retrospective study comprised 82 intracytoplasmic sperm injection (ICSI) cycles involving preimplantation genetic testing for aneuploidy (PGT-A) cases, with a focus on recruiting egg donation cycles to thoroughly investigate the impact of male factors. Two populations are classified based on semen quality: normozoospermic ([Formula: see text] = 33) and non-normozoospermic ([Formula: see text] = 49). Subjects were allocated to either swim-up (SU) or CA0. Preimplantation genetic testing results were recorded. Results: When comparing male characteristics between subgroups, no significant differences were observed except for a lower normal morphology rate in the CA0 group compared to SU (SU: 3 [3–4] vs. CA0: 2 [2–2.8], [Formula: see text] < 0.0001) in the non-normozoospermic cohort. There were no differences in female factors such as age and mature oocyte count (MII) number between subgroups, indicating that this model is ideal for assessing the impact of male factors on clinical outcomes. In the normozoospermic cohort, euploidy rates were similar between SU and CA0 (SU: 71.9% vs. CA0: 64.2%). However, in the non-normozoospermic cohort, CA0 showed a significantly higher euploidy rate compared to SU (SU: 53.6% vs. CA0: 74.2%) and a lower aneuploidy rate (SU: 37.1% vs. CA0: 25.8%). Additionally, CA0 minimized the incidence of mosaic embryos, whereas a mosaicism rate of 9.3% was observed with SU. This trend highlights CA0’s distinct advantage in optimizing outcomes for non-normozoospermic cases. Conclusions: CA0 is a reliable intervention to optimize paternal genetic quality before assisted insemination, thereafter effectively reducing the incidence of embryo aneuploidy associated with male factors.

P-033 Microfluific sperm sorting does not impact the primary sex ratio in IVF-ICSI treatments

Abstract Study question Does sperm selection through microfluidics alter the primary sex ratio (PSR) when compared to density gradients? Summary answer The PSR is not modified when employing a microfluidic sperm sorting when comparing to the conventional density gradient technique for sperm selection. What is known already The PSR refers to gender after fertilization, this is often expressed as the male births per female births. Little is known about the sex ratio in natural and assisted reproduction treatments (ART) since it is difficult to establish in ART as it requires preimplantation genetic testing (PGT). Previous studies have been estimating the variation in the PSR based on the fertilization technique used or the day of embryo transfer. Even the influence of sperm selection techniques has not received much attention, using traditional sperm selection methods such as density gradients or swim-up, a PSR of 1.07 has been estimated. Study design, size, duration This is a retrospective study that includes 1764 blastocysts stage embryos biopsied from 522 autologous preimplantation genetic testing for aneuploidies cycles (PGT-A) performed between January of 2020 and December of 2022 and analysed through Next Generation Sequencing (NGS). All the embryos were cultured under the same conditions with one-step media in a time-lapse incubator and the PGT-A was performed in day 5 or day 6 of culture to determine the embryo ploidy status. Participants/materials, setting, methods Two groups were established based on the sperm selection method before ICSI. The study group using a microfluidic system (MS; ZyMōtICSI® or ZyMōtMulti®) included 256 blastocysts from 98 cycles, and a control group using density gradients (DG), with 1058 blastocysts from 424 cycles were compared. PGT-A determined the embryonic sex and for the PSR estimation, number abnormalities in the sexual chromosomes were excluded (i.e. XXY, X0, XYY). Chi-square test was performed to compare the groups. Main results and the role of chance Our data show that there was no statistical significance in the overall ratio of XY:XX embryos biopsied between both groups (p = 0.09). The PSR was 0.89 (47:53) in the study group (MS) and 1.12 (53:47) in the control group (DG). Although not statistically significant, the rate of XX embryos was 6% higher than XY embryos in the MS group. In contrast, in the DG group, the same increase in the rate of XY embryos was observed. If we consider only the euploid embryos which are the potentially born babies, the PSR acquires values of 0.96 for the DG group and 0.92 for the MS group (p = 0.86), showing an increase in euploid female embryos transferred compared to those euploid male embryos. However, it was not statistically significant. Limitations, reasons for caution The performance of PGT-A on blastocysts does not provide an entirely accurate representation of the PSR since it is carried out 5/6 days after the moment of fertilization; nevertheless, it is the technique that comes closest. Also, the low number of blastocysts in the study group represents a limitation. Wider implications of the findings The sperm selection for ICSI with microfluidic technology does not significantly alter the PSR. Trial registration number Not applicable

P-009 Impact of sperm preparation methods on embryo development rate and embryo ploidy status: ICSI egg donation as a model

Abstract Study question Do different sperm separation techniques result in distinguishable contributions to embryo development rates and ploidy status? Summary answer CA0 sperm separation device, devoid of centrifugation, yielded a higher euploidy rate than conventional swim-up (50% vs. 74%, p = 0.02), notably observed in abnormozoospermic cases. What is known already Sperm DNA fragmentation (SDF) emerges as a key paternal risk factor associated with embryo aneuploidy, increasing the risk of ART failures. Factors like advanced male age, prolonged ejaculatory abstinence, and the detrimental effects of centrifugation during sperm separation jeopardize sperm DNA integrity. A recent centrifugation-free sperm separation device (CA0), employing live sperm sorting technology, exhibits efficiency in producing highly motile sperm with minimized SDF. This retrospective cohort study solely focusing on ICSI egg donation cycles to assess the influence of sperm separation methods (conventional swim-up vs. CA0) on early embryo development, and embryo ploidy. Study design, size, duration This retrospective study included 83 couples who underwent ICSI egg donation cycles at Chachava Clinic, Tbilisi, Georgia. Thirty-three semen specimens were processed using conventional swim-up (SU) and 427 Mii were inseminated. Forty-four semen samples were separated by CA0 and 519 Mii were inseminated. Clinical outcome measures included fertilization rate (number of 2PN over number of inseminated oocyte), good embryo rate (embryo number with more than 8 cells over inseminated oocyte), and euploidy rate using PGT-A. Participants/materials, setting, methods Male age and basic semen parameters via CASA (CEROS II, Hamilton-Thorne) were recorded. SU involved layering 1 mL neat semen to 1.2 mL HTF medium, inclining the tube at 45° for 60 min at 37 °C, collecting 1 mL uppermost, adding 2 mL HTF for centrifugation at 500×g for 5 min, and resuspending sperm pellet with 0.5 mL HTF. Semen samples were sorted using LensHooke® CA0 device following the manufacturer’s instructions. Main results and the role of chance In comparing demographic characteristics between SU and CA0 arms, CA0 exhibited higher median total motility (SU: 40% vs. CA0: 54%, p = 0.02), with no significant differences in female age and Mii oocyte count. This study assessed the impact of sperm separation methods on early embryo development, finding no significant differences in fertilization rate (SU: 79% vs. CA0: 77%) and good embryo rate (SU: 64% vs. CA0: 62%). However, CA0 demonstrated a significantly higher euploidy rate than SU (SU: 59% vs. CA0: 69%, p = 0.03). Further analysis within subpopulations, including normozoospermic and asthenozoospermic (total motility <40%), revealed equivalent euploidy rates between SU and CA0 for normospermic specimens (SU: 66% vs. CA0: 68%). However, a significant differentiation in euploidy rate emerged for asthenozoospermic cases (SU: 59% vs. CA0: 74%, p = 0.02). Specifically, comparing outcomes in normozoospermic, a significant reduction was observed in asthenozoospermic cases using SU (66% vs. 50%, p = 0.02), whereas CA0 provided improved euploidy rates regardless of semen quality (68% for normozoospermic and 74% for asthenozoospermic). In conclusion, this study suggests that the non-invasive live sperm sorting device enhances embryo euploidy rates, serving as a reliable sperm preparation method, ensuring selection consistency despite sample variations. Limitations, reasons for caution This study’s limitations include its retrospective nature and single-center trial design. To more effectively assess CA0 efficiency, a randomized controlled trial targeting diverse patient eligibility criteria is warranted. Wider implications of the findings Attributed to the improved embryo euploidy with CA0, we anticipate enhanced ART outcomes, such as increased embryo transfer rate and prevention of ART failures related to embryo aneuploidy. Additionally, CA0 offers practical benefits by streamlining and standardizing the sperm separation process with only three steps involved. Trial registration number not applicable

P-053 Comparative analysis between microfluidic sperm sorting and Swim-up technique to select spermatozoa with better characteristics in hyper viscous samples

Abstract Study question What is the best technique to select spermatozoa in hyper viscous semen samples? Summary answer Microfluidics is the best technique that allows to select spermatozoa with better seminal characteristics and hyperactivated motility, and less sperm DNA fragmentation. What is known already Density gradient centrifugation and Swim-up (SU) are the two traditional selection methods used during assisted reproduction techniques (ART) to collect the highly motile sperm fraction. Many studies reported that centrifugation increase sperm DNA fragmentation and oxidative status which could affect reproductive outcomes. To overcome the limitations of centrifugation, an alternative approach by using microfluidic-based devices was recently designed. In microfluidic protocol, sample is applied to the device’s inlet port, connected to a lower chamber. This chamber consists of a microporous filter, where the most motile spermatozoa migrate upward through the filter. Study design, size, duration An observational study was conducted on 29 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from January 2023 to January 2024. In these samples we compare two different methods for sperm selection: indirect SU technique and microfluidic devices (MSS). Participants/materials, setting, methods We evaluated seminal parameters (sperm progressive and total motility, viability, and concentration), sperm kinematic parameters by Computer-Assisted Sperm Analysis (C.A.S.A. system), sperm DNA fragmentation by TUNEL/PI assay and sperm oxidative status by CellROX® Orange (which we have recently shown to identify a fraction of viable oxidized spermatozoa with better characteristics (Traini G., et al. 2022)) in 29 normozoospermic and 18 hyper viscous semen samples selected by SU and MSS. Main results and the role of chance In normozoospermic samples, sperm selection with both indirect SU technique and microfluidic devices, ameliorated sperm characteristics compared to not selected samples. Spermatozoa selected by MSS showed statistically significant higher progressive and total motility, viability and concentration compared to those selected by indirect SU. Moreover, an increase in most of kinematic semen parameters (like VAP, VCL and VSL) and hyperactivated motility was observed in MSS selected samples. Also, sperm DNA fragmentation was significantly reduced after MSS selection compared to indirect SU. We evaluated the percentage of viable oxidized spermatozoa by using CellROX® Orange. A significant increase of the percentage of CellROX® Orange positive spermatozoa was observed in most of MSS selected samples. In all 18 hyper viscous samples, the results obtained in normozoospermic samples were confirmed. Indeed, an overall improvement of sperm progressive and total motility, viability and concentration together with an increase in most of kinematic semen parameters and in hyperactivated motility were evidenced in MSS selected spermatozoa respect to those selected by indirect SU. Again, a significant decrease of sperm DNA fragmentation and an increase of the percentage of CellROX® Orange positive spermatozoa, were observed after MSS selection. Limitations, reasons for caution The number of semen samples included in the study is not very elevated. Results obtained in hyper viscous semen samples should be confirmed in a greater number of samples. Wider implications of the findings Our data indicate that MSS selects the sperm fraction with better characteristics and less DNA fragmentation. These results suggest that MSS could improve ART outcomes, particularly for specific categories of patients, including hyper viscous semen samples, for which the selection by SU does not always guarantee good yields. Trial registration number not applicable

Quality and fertilizing ability of frozen-thawed porcine sperm separated using a migration sedimentation method.

We evaluated the quality and fertilizing ability of frozen-thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen-thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen-thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen-thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize invitro-matured oocytes and cultured invitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high-quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation.

Development of a thermotaxis and rheotaxis microfluidic device for motile spermatozoa sorting

Male infertility is a pervasive global reproductive challenge, primarily attributed to a decline in semen quality. Addressing this concern, there has been a growing focus on spermatozoa sorting in assisted reproductive technology. This study introduces a groundbreaking development in the form of a thermotaxis and rheotaxis microfluidic (TRMC) device designed for efficient motile spermatozoa sorting within a short 15-min timeframe. The TRMC device mimics the natural sperm sorting mechanism of the oviduct, selecting spermatozoa with superior motility and DNA integrity. The experimental outcomes demonstrate a remarkable enhancement in the percentage of progressive spermatozoa following sorting, soaring from 3.90% to an impressive 96.11% when subjected to a temperature decrease from 38 °C to 35 °C. Notably, sperm motility exhibited a substantial 69% improvement. The TRMC device exhibited a commendable recovery rate of 60.93%, surpassing current clinical requirements. Furthermore, the sorted spermatozoa displayed a notable reduction in the DNA fragmentation index to 6.94%, signifying a substantial 90% enhancement in DNA integrity. This remarkable advancement positions the TRMC device as highly suitable for applications in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), offering a promising solution to male infertility challenges.

Microfluidics as an emerging paradigm for assisted reproductive technology: A sperm separation perspective.

Millions of people are subject to infertility worldwide and one in every six people, regardless of gender, experiences infertility at some period in their life, according to the World Health Organization. Assisted reproductive technologies are defined as a set of procedures that can address the infertility issue among couples, culminating in the alleviation of the condition. However, the costly conventional procedures of assisted reproduction and the inherent vagaries of the processes involved represent a setback for its successful implementation. Microfluidics, an emerging tool for processing low-volume samples, have recently started to play a role in infertility diagnosis and treatment. Given its host of benefits, including manipulating cells at the microscale, repeatability, automation, and superior biocompatibility, microfluidics have been adopted for various procedures in assisted reproduction, ranging from sperm sorting and analysis to more advanced processes such as IVF-on-a-chip. In this review, we try to adopt a more holistic approach and cover different uses of microfluidics for a variety of applications, specifically aimed at sperm separation and analysis. We present various sperm separation microfluidic techniques, categorized as natural and non-natural methods. A few of the recent developments in on-chip fertilization are also discussed.

USING MICROFLUIDIC SPERM SORTING CHIPS IN PATIENTS WITH INFERTILITY

The purpose of the work is to analyze the effectiveness of the FERTILE microfluidic sperm sorting chip.
 Materials and Methods. The technique under consideration is currently experimental. It was used in patients after obtaining voluntary informed consent. A retrospective analysis of medical records of 4 groups of patients was carried out. The patients were divided into 2 experimental and 2 control groups. In total the authors analyzed 54 medical records. The microfluidic sperm sorting chip (FERTILE/FERTILE PLUS CHIP) are intended for the selection of normal mobile spermatozoids, which can be used for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The indicators of preimplantation embryo development in vitro were assessed.
 Results. Embryological parameters of infertility treatment using ART were analyzed. Key indicators of preimplantation embryo development (fertilization rate, blastocyst development, embryo cryopreservation) were higher in both experimental groups compared to control ones (standard semen processing). Embryos were obtained by fertilization with sperm which undergone microfluidic sorting. Embryo culture suggests a more physiological nature of gamete separation. During standard ejaculate processing centifugation is used to obtain a fraction of active sperm. However, it negatively affects the cytophysiology of male gametes and causes breaks in the DNA chain in the spermatozoid head.
 Conclusion. The authors observed the improvement in embryological parameters of in vitro embryos, which were obtained by means of microfluidic chip-based sperm selection. This method allows us to select cytophysiologically competent sperm and improve the embryo quality without increasing DNA fragmentation rate.

Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. The study is funded by the Iran University of Medical Sciences and no competing interest exists. N/A.

Sperm Preparation with Microfluidic Sperm Sorting Chip May Improve Intracytoplasmic Sperm Injection Outcomes Compared to Density Gradient Centrifugation.

Does sperm preparation using the FERTILE PLUS™ Sperm Sorting Chip improve fertilization rates, blastocyst formation, utilization, and euploidy rates in patients undergoing intracytoplasmic sperm injection (ICSI), compared with density gradient centrifugation (DGC)? A single-cohort, retrospective data review including data from 53 couples who underwent ICSI cycles within a 12-month period. For each couple, the two closest, consecutive cycles were identified, where one used the standard technique of sperm preparation (DGC) and the subsequent used FERTILE PLUS™, therefore, couples acted as their own controls. Paired samples t-test was used to compare means for the outcomes (fertilization, blastocyst formation, utilization, and euploidy rates). Binary logistic regression analysis assessed the relationship between female age, the presence of male factor infertility, and euploidy rates. Blastocyst, utilization, and euploidy rates were significantly higher for cycles using FERTILE PLUS™ compared to DGC (76% vs 56%, p = 0.002; 60% vs 41%, p = 0.005, and 40% vs 20%, p = 0.001, respectively). Although there was an increase in fertilization rates for cycles using FERTILE PLUS™, this was not significant (72% vs 68%, p = 0.449). The euploidy rates of females ≤ 35 years were significantly increased when the FERTILE PLUS™ sperm preparation method was used, compared to the older age group (OR 2.31, p = 0.007). No significant association was found between the presence or absence of male factor infertility and euploidy rates between the two cycles. This study provides tentative evidence that the FERTILE PLUS™ microfluidic sorting device for sperm selection can improve blastocyst formation, utilization, and euploidy rates following ICSI in comparison to the DGC method.

New insights into semen separation techniques in buffaloes.

Male infertility is frequently caused by idiopathic or unexplained reasons, resulting in an increase in demand for assisted reproductive technologies. In buffaloes, more than in other animals due to reproductive hardiness, successful fertilization needs spermatozoa to effectively transit the female reproductive system to reach the oocyte. This mechanism naturally picks high-quality sperm cells for conception, but when artificial reproductive technologies such as in vitro fertilization, intracytoplasmic sperm injection, or intrauterine insemination are utilized, alternative techniques of sperm selection are necessary. Currently, technology allows for sperm sorting based on motility, maturity, the lack of apoptotic components, proper morphology, and even sex. This study provides current knowledge on all known techniques of sperm cell sorting in buffaloes, evaluates their efficiency, and discusses the benefits and drawbacks of each approach.

Comparative Bioinformatic Analysis of the Proteomes of Rabbit and Human Sex Chromosomes.

Studying proteins associated with sex chromosomes can provide insights into sex-specific proteins. Membrane proteins accessible through the cell surface may serve as excellent targets for diagnostic, therapeutic, or even technological purposes, such as sperm sexing technologies. In this context, proteins encoded by sex chromosomes have the potential to become targets for X- or Y-chromosome-bearing spermatozoa. Due to the limited availability of proteomic studies on rabbit spermatozoa and poorly annotated databases for rabbits compared to humans, a bioinformatic analysis of the available rabbit X chromosome proteome (RX), as well as the human X (HX) and Y (HY) chromosomes proteome, was conducted to identify potential targets that could be accessible from the cell surface and predict which of the potential targets identified in humans might also exist in rabbits. We identified 100, 211, and 3 proteins associated with the plasma membrane or cell surface for RX, HX, and HY, respectively, of which 61, 132, and 3 proteins exhibit potential as targets as they were predicted to be accessible from the cell surface. Cross-referencing the potential HX targets with the rabbit proteome revealed an additional 60 proteins with the potential to be RX targets, resulting in a total of 121 potential RX targets. In addition, at least 53 possible common HX and RX targets have been previously identified in human spermatozoa, emphasizing their potential as targets of X-chromosome-bearing spermatozoa. Further proteomic studies on rabbit sperm will be essential to identify and validate the usefulness of these proteins for application in rabbit sperm sorting techniques as targets of X-chromosome-bearing spermatozoa.

Advances in microfluidic technology for sperm screening and in vitro fertilization.

About 18% of reproductive-age adults worldwide are affected by infertility. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are widely used assisted reproductive technologies (ARTs) aimed at improving clinical outcomes. Efficient and noninvasive selection and isolation of highly motile sperm with intact DNA are essential for the success of IVF and ICSI and can potentially impact the therapeutic efficacy and the health of the offspring. Compared to traditional methods, microfluidic technology offers significant advantages such as low sample consumption, high efficiency, minimal damage, high integration, similar microenvironment, and high automation, providing a new platform for ARTs. Here, we review the current situation of microfluidic technology in the field of sperm motility screening and evaluation and IVF research. First, we focus on the working principle, structural design, and screening results of sperm selection microfluidic platforms. We then highlight how the multiple steps of the IVF process can be facilitated and integrated into a microfluidic chip, including oocyte capture, sperm collection and isolation, sperm sorting, fertilization, and embryo culture. Ultimately, we summarize how microfluidics can complement and optimize current sperm sorting and IVF protocols, and challenges and possible solutions are discussed.

Embryonic development through in vitro fertilization using high-quality bovine sperm separated in a biomimetic cervix environment.

This study is the first to identify bovine blastocysts through in vitro fertilization (IVF) of matured oocytes with a large quantity of high-quality sperm separated from a biomimetic cervix environment. We obtained high-quality sperm in large quantities using an IVF sperm sorting chip (SSC), which could mimic the viscous environment of the bovine cervix during ovulation and facilitates isolation of progressively motile sperm from semen. The viscous environment-on-a-chip was realized by formulating and implementing polyvinylpyrrolidone (PVP)-based solutions for the SSC medium. Sperm separated from the IVF-SSC containing PVP 1.5% showed high motility, normal morphology and high DNA integrity. As a result of IVF, a higher rate of hatching blastocysts, which is the pre-implantation stage, were observed, compared to the conventional swim-up method. Our results may significantly contribute to improving livestock with superior male and female genetic traits, thus overcoming the limitation of artificial insemination based on the superior genetic traits of existing males.

#89 : Higher Euploidy Rate in Embryos Generated by ICSI Using Sperm Obtained After Microfluidics Sperm Sorting (MFSS). A Randomised Controlled Trial

Background and Aims: MFSS is a state-of-the-art technology that has the ability to sort a subpopulation of sperm cells with lower DNA fragmentation index (DFI), higher motility and higher normal forms without the need for centrifugation. Methods: A randomised controlled trial that included 925 couples that were planned for ICSI with day 5/6 biopsy, PGT-A with Next Generation Sequencing (NGS) and blastocyst vitrification. The embryo transfer was planned for future cycles. The inclusion criteria for women were age (18–45), BMI (18–30), AMH level more than 1.1 ng/ml, regular menstrual cycle and normal uterine ultrasound. Whereas the inclusion criteria for men were age (18–50), fresh semen sample with count more than 10 million/ml and total motility more than 30%. Semen samples were randomly assigned with 1:1 ratio to either preparation by routine density gradient (DG) or (MFSS). The primary outcome of the study is PGT-A aneuploidy results. The secondary outcome is the fertilisation rate, and blastocyst development rate. Results: The study was performed between January 2020 and July 2021. 463 semen samples were prepared with density gradient and 462 semen samples were sorted with microfluidic sperm sorting. All of the prepared semen samples were used for ICSI. The euploid embryo rate for the embryos resulted from density gradient preparation was 53.1% versus 59.3% for the embryos resulted from microfluidics sperm sorting (p=0.0034). The fertilisation rate was 77.9% for density gradient group versus 78.3% microfluidics group (p=0.063) and blastocyst rate was 51.6% for the density gradient group versus 59.2% for the microfluidics group (p=0.058). Conclusion: Semen samples prepared with MFSS for ICSI generated higher euploidy embryos following PGT-A with NGS.

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.

Revelation of β-galactosidase variation in senile sperms and organs via a silicon xanthene near-infrared probe

β-galactosidase has become the most widely used bio-marker in cellular senescence, however, it is unknown whether there are same changes in β-galactosidase activity during sperm senescence. Understanding the changes in such indicator during sperm senescence is important for sperm sorting and in vitro fertilization. Herein, we developed a NIR probe Si-βGal-P by using silicon xanthene as fluorophore. Si-βGal-P showed a good affinity to β-galactosidase together with high specificity and strong anti-interference ability. And Si-βGal-P was proved to be able to assess β-galactosidase in three different replicate senescence models. Besides, Si-βGal-P could easily pass blood-brain barrier and successfully imaged the up-regulated β-galactosidase activity in 34-week-old mouse brain. In addition, Si-βGal-P was successfully applied to hydrogen peroxide-induced premature sperm and visualized the up-regulated β-galactosidase activity for the first time, providing sperm senescence with a valid labeling tool.

Technologies for Separation of ‘X’ and ‘Y’ Spermatozoa in Bovines: An Overview

Several approaches to sperm sexing such as Albumin gradient, Identification of H-Y antigen, Free-flow electrophoresis, Detection of sex-specific proteins, Centrifugal counter current distribution, Volumetric differences, Percoll density gradient, Flow-cytometry, Laser ablation, etc were put forward by the scientists. However, the alteration in DNA content between X- and Y-sperm remains the key and only discriminating feature for sperm sorting based on which only Flow cytometry and Laser ablation techniques are being used commercially. Flow cytometry is based on precise staining of the DNA of sperm with the nucleic acid-specific dye, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized highspeed sorter. Laser ablation (LA) is a system and method for sorting a mixture of stained particles in a fluid flow path. The system and method includes an electromagnetic radiation source for exciting fluorescence emissions from the stained particles, a photodetector for detecting the fluorescence emissions, a processor for classifying the stained particles, and a photo-damaging laser for damaging selected particles in the flow path.