P–081 Microfluidic sorting does not improve clinical outcomes compared to magnetic activated cell sorting (MACS) in Assisted Reproduction

Study question Does microfluidic sorting improve clinical outcomes over magnetic activated cell sorting (MACS) in ovum donation cycles? Summary answer Performing microfluidic sorting does not seem to improve clinical outcomes compared to MACS in ovum donation cycles. What is known already Novel sperm selection techniques, such as magnetic activated cell sorting (MACS), have been described as useful procedures to increase reproductive outcomes when male factor is present. Because of centrifugation steps associated to swim-up or density-gradient can induce sperm DNA fragmentation, microfluidic sperm sorters are being used to isolate motile human spermatozoa based on fluid dynamics and avoiding sample manipulation. This new technology has been shown to reduce the level of sperm DNA damage, especially double strand breaks, but an improvement of clinical outcome by using this technique remain unclear. Study design, size, duration Prospective and observational study to evaluate the efficacy of a sperm sorting technique based on microfluidic technology versus a technique based on the removal of apoptotic spermatozoa by MACS. The study was performed between May 2019 to January 2021 in IVI Madrid and IVI Sevilla. All men attending for an ovum donation cycle during the aforementioned study period were included. The exclusion criteria were sperm concentration <5 mill/mL and <15% of progressive motile spermatozoa. Participants/materials, setting, methods Seminal samples from couples participating in the study were divided into two aliquots; each of them was processed according to one of the study methods. Subsequently, each of the processed sperm samples was used to microinject half of the oocytes obtained during oocyte retrieval. In all case, a single-embryo transfer was performed. Variables were expressed as mean values and standard deviations. Statistical analysis was performed by ANOVA and Chi-squared where applicable; significance established under 0.05. Main results and the role of chance We included 48 couples in the study; of these, n = 31 transferred an embryo derived from a MACS processed sperm sample, while n = 17 received an embryo from the microfluidic one. Groups were homogeneous in terms of number of transferred embryos and frozen embryos, usable blastocyst rate and fertilization rate; results were as follows for MACS and microfluidic processing respectively: number of transferred embryos (1.0±0.0 vs. 1.0±0.0, p = 0.978); number of frozen embryos (1.6±0.5 vs. 1.4±0.7 p = 0.168); usable blastocyst rate (40.7% vs. 43.1%, p = 0.384); and fertilization rate (80.4% vs. 75.3%, p = 0.075). However, according clinical outcomes, we observed significant differences in implantation rate (74.2% vs. 58.8%, p < 0.001) and in clinical pregnancy rate (74.2% vs. 58.8%, p < 0.001); finally, the miscarriage rate was similar between the two groups of study (6.4% vs. 5.8%, p = 0.876). Limitations, reasons for caution This study has not considered the indication in male factor couples due to a high degree of double-strand DNA fragmentation. Therefore, more specific studies are required to determine in which patients, microfluidics sorter selection would significantly improve clinical outcomes. Wider implications of the findings: In an unselected population, magnetic activated cell sorting significantly improves clinical outcomes compared to a microfluidic technique, so this latter method should not be recommended without a male factor indication associated with sperm DNA damage. The proposed microfluidic technology does not seem to offer a flow-free approach to select spermatozoa. Trial registration number NCT04061486

From passive to active sorting in microfluidics: A review

Achieving high-efficiency sorting of microfluidics (such as cells, particles, droplets, etc.) has great significance in the fields of biology, chemistry, medical treatment, material synthesis, and drug development. This paper introduces the microfluidics sorting methods in recent years. The current research status and progress can be divided into the active sorting system and passive sorting system according to whether there is an external field. They can control the microfluidics by promoting more selective separation, so as to obtain higher resolution and selection rate. In this paper, the above methods are analyzed and discussed, and the future microfluidic sorting is prospected.

61 Swim-up and microfluidic techniques improve the kinetic parameters of selected bovine spermatozoa for invitro fertilization: Preliminary results

For invitro embryo production, spermatozoa with fertilizing capacity must possess optimal kinetic, morphometric, vitality, and DNA integrity characteristics. The objective of this study was to evaluate the effect of 3 sperm selection methods on sperm quality and invitro embryonic development in bovine. Frozen commercial semen (0.5 mL/straws) from one bull with known fertility was thawed at 37°C for 20s and was divided for 3 sperm selection techniques: density gradient, swim-up, and microfluidic sperm sorting. The sperm kinetic parameters (VCL=curvilinear velocity, VSL=straight line velocity, VAP=average path velocity, ALH=lateral displacement of sperm head, BCF=beat frequency cross, STR=path straightness) were assessed using computer-assisted sperm analysis (CASA). Sperm morphometric parameters were evaluated using Diff-Quick staining followed by automated analysis. To assess vitality, the sperm were stained with propidium iodide and acridine orange, then analysed under a fluorescence microscope. In addition, DNA fragmentation was assessed using sperm chromatin dispersion method. Last, the fertilizing capacity of the selected sperm was tested by fertilizing cumulus–oocyte complexes (2×106 sperm mL−1) obtained from slaughterhouse ovaries and matured invitro for 24h. A standardized invitro embryo production protocol was used with commercial medium from Vitrogen. The cleavage rate and blastocyst yield were measured on Day 2 and 7, respectively (fertilization=Day 0). The results were calculated with analysis of variance and Tukey’s test (P<0.05). The values of sperm kinetic parameters obtained with swim-up (VCL 132.5µm/s; VSL 73.5µm/s) and microfluidic technique (VCL 129.5µm/s; VSL 64.4µm/s) were significantly higher (P<0.05) than those obtained by density gradient (VCL 98.3µm/s; VSL 45.01µm/s). However, the total and progressive motility by density gradient method was slightly higher (89% and 57%) compared with that assessed by swim-up (64% and 43%) or microfluidic technique (74% and 54%) respectively. Microfluidic sorting (11.3%) showed lower (P<0.05) DNA fragmentation levels compared with density gradient method (16.6%), whereas the swim-up technique (12.5%) was similar between both groups. No significant difference was detected between the 3 groups for sperm morphometric and vitality parameters. Moreover, cleavage rates were similar (P>0.05) between the 3 sperm selection techniques: density gradient (84.0%), swim-up (75.2%), and microfluidic sorting (67.3%). However, blastocyst yield was significantly higher (P<0.05) using sperm selected by density gradient (28.1%) and swim-up (21.9%) compared with microfluidic sorting (15.3%). In conclusion, sperm selection using microfluidic and swim-up techniques improved kinetic parameters with lower levels of DNA fragmentation, without affecting sperm morphometry. However, both the density gradient and swim-up techniques are efficient systems for producing invitro bovine embryo.

High-throughput screening of high lactic acid-producing Bacillus coagulans by droplet microfluidic based flow cytometry with fluorescence activated cell sorting

A high-throughput screening system based on droplet microfluidic sorting was developed and employed for screening of high lactic acid-producing Bacillus coagulans.

Hybrid microfluidic sorting of rare cells based on high throughput inertial focusing and high accuracy acoustic manipulation

The first example of integration of sized-based inertial sorting and surface biomarker-based acoustic sorting to achieve >2500-fold enrichment of rare cell populations.