Sperm Selection and Embryo Development: A Comparison of the Density Gradient Centrifugation and Microfluidic Chip Sperm Preparation Methods in Patients with Astheno-Teratozoospermia

In recent years, microfluidic chip-based sperm sorting has emerged as an alternative tool to centrifugation-based conventional techniques for in vitro fertilization. This prospective study aims to compare the effects of density gradient centrifugation and microfluidic chip sperm preparation methods on embryo development in patient populations with astheno-teratozoospermia. In the study, the semen samples of the patients were divided into two groups for preparation with either the microfluidic or density gradient methods. Selected spermatozoa were then used to fertilize mature sibling oocytes and the semen parameters and embryo development on days 3 and 5 were assessed. While the density gradient group was associated with a higher sperm concentration, motility (progressive and total) was significantly higher in the microfluidic chip group. No significant differences were observed in the fertilization rates or grade 1 (G1) and grade 2 (G2) proportions of the third-day embryos. Furthermore, while the proportions of the poor, fair and good blastocysts on day 5 did not differ significantly, excellent blastocysts (indicating high-quality embryos) were observed in a significantly higher proportion of the microfluidic chip group. When compared to the classical density gradient method, the microfluidic chip sperm preparation yielded sperm with higher motility and higher quality blastocysts at day 5; in patients with astheno-teratozoospermia.

P–221 Prospective randomized sibling study on gamete preparation, insemination and subsequent culture of human oocytes in a time-lapse system using media systems with and without antioxidants

Study question Does the addition of antioxidants for gamete preparation, insemination and embryo culture lead to differences in embryo development and clinical outcome Summary answer Using an antioxidant-containing media system for sperm preparation, insemination and embryo culture imparts significantly higher good-quality blastocyst rates and improved clinical outcome in elderly patients. What is known already A previous study showed that adding combined antioxidants for sequential embryo culture in conventional incubators (interrupted culture) improves embryo viability and clinical outcome, especially for elderly patients. Here we investigated the combined effect of three antioxidants Acetyl-L-Carnitine (10 µM), N-Acetyl-L-Cysteine (10 µM), and α-Lipoic Acid (5 µM) during sperm preparation, insemination, and time-lapse culture in a single step medium on human embryo development and clinical outcome. Study design, size, duration Prospective randomized single center study including 143 couples for IVF/ICSI between August 2018 and December 2019. Inclusion required at least eight cumulus-oocyte-complexes (COCs) after retrieval. Cycles involving PGT, split IVF/ICSI, and surgically retrieved sperm were excluded. Immediately after retrieval oocytes were randomly distributed to a study or control media system with or without antioxidants (Vitrolife). Similarly, ejaculates were split and prepared with and without antioxidants. Participants/materials, setting, methods Sibling oocytes were inseminated in the respective group with accordingly prepared sperm. Single step embryo culture was conducted in medium with (Gx-TL) and without (G-TL) antioxidants in the EmbryoScope+. Embryo quality and clinical outcome were assessed in relation to maternal age (<35/>35 years). Good-quality embryos on day 3 were defined as 8- to 10-cells with even cells and low fragmentation; good-quality blastocysts as > 3BB. Clinical outcome was assessed after single vitrified blastocyst transfer (SVBT). Main results and the role of chance From 143 participants (female age, 34.7±3.2 years), a total of 2424 COCs were collected; 1180 COCs/916 metaphase-II (MII) oocytes were allocated to Gx-TL media and 1244 COCs/981 MII oocytes to G-TL media. Age-related analysis in Gx-TL compared with G-TL in relation to allocated MII oocytes revealed a trend for higher fertilization rates in Gx-TL for both age groups (<35: 72.1% vs. 66.9%; >35: 70.7% vs. 64.9%, P < 0.1). Good-quality day 3 embryo development/MII oocytes was higher, albeit not significant, in the elderly patients in Gx-TL (<35: 35.9% vs. 34.4%; >35: 31.1% vs. 27.9%). Overall day 5/6 blastocyst rate was similar for both media (<35: 48.2% vs. 49.9%; >35: 42.3% vs. 39.5%). Day 5/6 GQB rate was comparable for younger patients (<35: 23.8% for Gx-TL vs. 26.0% for G-TL) but significantly higher in Gx-TL in elderly patients (>35: 20.7% vs. 14.4%; P < 0.05). A total of 200 SVBT were performed; 99 in the Gx-TL- and 101 in the G-TL-arm. We noted almost similar implantation and ongoing pregnancy rates between Gx-TL vs G-TL in the younger (<35) age group (50.0% vs. 55.4%; 50.0% vs. 55.6%) but higher albeit not significant rates for Gx-TL in older (>35) patients (44.1% vs. 33.3%; 44.1% vs. 33.3%). Limitations, reasons for caution In almost 95% of the cycles, oocytes were inseminated by ICSI; thus results may not equally apply for cycles with IVF. The use of a closed time-lapse system may have prevented from some environmental oxidative stress. Therefore results may come out different with a similar study using standard incubation. Wider implications of the findings: Supplementation of antioxidants to media for gamete isolation and preparation, as well as subsequent single step time-lapse culture may improve GQE/B rates and clinical outcomes in certain age groups, plausibly through the reduction of oxidative stress. Further studies in selected sub-groups (severe OAT syndrome / testicular cases) may be indicated. Trial registration number UMIN000034482

P–007 Effect of swim-up on sperm morphology

Study question To evaluate the effect of swim-up on the percentage of certain morphological defects in the semen population Summary answer Swim-up preparation led to significantly lower percentage of spermatozoa with cytoplasmic droplets, thick neck and also multiple defects. What is known already Swim-up is routinely used sperm preparation technique in ART practice. It is widely known that swim-up enhances sperm quality in terms of motility and sperm morphology. However, the effect of swim-up on the frequency of occurrence of the specific sperm morphological abnormalities is still missing. Study design, size, duration This observational study involved 30 teratozoospermic patients of Nadezhda Women’s Health hospital between December 2020 and January 2021. Sperm morphology was evaluated before and after swim-up preparation. Participants/materials, setting, methods Native semen was liquefied and was subjected to swim up. Semen analysis performed according to WHO 2010. Native semen and swim up samples from the same men were subjected to Kruger strict morphological evaluation. The analyzed sperm morphological defects included: head defects (large, small, tapered, pyriform, round, amorphous and double heads); midpiece defects (bent, asymmetrical, thin, thick, presence of cytoplasmic droplet); tail defects (short, hairpin, bent, coiled tail and terminal droplet) and multiple defects. Main results and the role of chance Wilcoxon paired test showed that the percentage of morphologically normal spermatozoa was significantly higher in the swim-up samples in comparison to the native semen (8.5±4.2% vs 4.9±3.2%, p < 0.05). In addition, the percentage of spermatozoa bearing multiple defects was found to be significantly lower in the swim-up samples than in the native semen (25.8±11.6% vs 37.0±15.0%, p < 0.05). Two specific sperm morphological defects were found to be significantly lower after swim-up preparation: the presence of cytoplasmic droplets (6.0±1.0% vs 8.6±1.5%, p < 0.05) and the thick neck (9.7±5.5% vs 12.8±5.8%, p < 0.05). No significant different were observed in the other morphological defects between swim up samples and native semen (p > 0.05). Limitations, reasons for caution Results obtained from this study need to be confirmed by larger group of samples. Wider implications of the findings: Our study showed a significant reduction of certain midpiece defects after swim-up. The observed selection of spermatozoa without thick necks and cytoplasmic droplets explains the effectiveness of swim-up on ART. In addition, the obtained results can serve as a guide for future validation of new sperm preparation techniques. Trial registration number Not applicable

P–012 A new sperm preparation technique for ICSI

Study question Is there any difference in this technique(swim-in) according to clinical results in ICSI cycles? Summary answer Fertilization rates are significantly better with this new technique(swim-in). There is no difference in pregnancy and ongoing pregnancy rates between swim-in and DGS techniques. What is known already Sperm separation is crucial in assisted reproductive technologies, based on different principles like migration, filtration, or density gradient centrifugation techniques. However, there are some studies that centrifugation steps and using gradient solutions may increase sperm DNA fragmentation. On the other hand, multiple contacts to different plastic surfaces such as pipettes, plastic tubes, etc., and also pipetting the ejaculate in sperm preparation may have detrimental effects. The ideal sperm separation technique should be non-invasive, not time-consuming, easy, and cost-effective, not cause sperm damage or non-physiological alterations of the separated sperm cells, eliminate dead spermatozoa and other cells, including leukocytes. Study design, size, duration This is a prospective randomized study between 01/02/2019...01/01/2021. Sperms were prepared either with this new technique that we call “swim-in”(n = 359) or with density gradient centrifugation(n = 404) before microinjection (ICSI). Fertilization rates, clinical pregnancy rates, and on-going pregnancy rates were compared between groups. Sperm motility is less than%20 patients are not included in the study. t-test and Chi-square test used. p < 0.05 significant. Participants/materials, setting, methods For this new centrifugation free technique(swim-in), a Braun injector, 21G green needle, and sperm washing medium are used. First, we pulled a 0.5 ml sperm wash medium in a Braun injector. After sperm liquefaction, we put Braun injector with the needle in the sperm cap perpendicularly and waited 30 minutes at 37-degree Celcius. Sperms swam through the needle to the injector used for ICSI. For DGC sperms were prepared according to the 2015 WHO manual. Main results and the role of chance: We try to make a centrifugation-free, gentle, cost-effective, and easy sperm preparation technique and called it a swim-in technique. Groups were comparable according to mean age, collected oocyte numbers, and oocyte maturation ratios. Fertilization rates are%73,2(1786/2438) vs%66,9(2167/3237) respectively for swim-in and DGC groups. Clinical pregnancy rates are%45,1 vs%42,3 respectively for swim-in and DGC groups. On-going pregnancy rates are%41,5 vs%39,6 for swim-in and DGC respectively. There is a significant difference between the two techniques according to fertilization rates but no difference according to clinical pregnancy and ongoing pregnancy rates. Limitations, reasons for caution Sperms can move by their own motility from ejaculate to the sperm washing medium for that reason with severe sperm problem patients are not suitable for this technique. Wider implications of the findings: This technique is centrifugation free so there are no detrimental g-forces. The needle used may mimic the physiological environment like Uterine Tube. Alternative sperm washing mediums like follicular fluid or chemical attractor progesterone rich medium can be used.DNA fragmentation rates and live birth rates should be established for future aspect. Trial registration number Not applicable

P–103 Novel sperm preparation techniques compared with conventional preparation method

Study question Which sperm preparation technique separate the best quality sperm? Summary answer Microfluidic method improved the sperm parameters and decreased sperm DNA damage. What is known already About 40% of infertility issues are due to male factor. One of the known causes of male infertility is associated with low sperm parameters and high level DNA fragmentation. Sperm preparation techniques in ICSI procedures is used in order to obtain the best-quality sperm. Study design, size, duration The present study was designed to compare Microfluidic, Zeta potential, Magnetic Activated Cell Sorting (MACS) and Swim-up methods for sperm preparation and the effect of these methods on semen parameters and sperm DNA integrity in infertile men (n = 25) with a mean age of 38. Participants/materials, setting, methods In this study, each sample was divided into 4 groups, one part for preparing by Microfluidic method, one of them for preparing by Swim -up method, the other one was prepared by MACS and the last one was prepared by zeta potential. Then sperm count, viability, motility and morphology were assessed according to WHO 2010. DNA damage were assessed by Sperm DNA Fragmentation assay and sperm chromatin packaging assessed by CMA3 staining test Main results and the role of chance Sperm parameters including viability, motility, and morphology in the Microfluidic method were significantly improved and sperm DNA damage were significantly lower than three other methods (P-value <0.05). The sperm parameters and sperm DNA damage after preparation by MACS and Zeta potential methods were not significantly different however in the Swim-up method sperm parameters were lower than three other methods (P-value <0.05). Limitations, reasons for caution The fertilization and pregnancy rate of the resulting embryos are not available. Wider implications of the findings: Our results showed that Microfluidic can be an effective way to improve sperm quality of infertile male compared to conventional preparation methods. We also found instead of the MACS method, we can use the Zeta potential method according to their costs, for sperm preparation during ART cycle. Trial registration number *

P–069 microfluidic sperm sorting vs density gradient to yield sperm with reduced DFI for patients undergoing IVF-ICSI

Study question To compare the effect of sperm preparation methods on the DFI of semen sample for patients undergoing ICSI. Summary answer On comparing the results, microfluidic sperm sorting yielded sperms with significantly less DFI as compared to density gradient method of sperm preparation. What is known already The DNA integrity of the sperm plays an important role to ensure formation of good quality embryos with increased potential of fertilization, growth and ultimately implantation.. Centrifugation has shown to add stress to the sperm and leading to DNA damage, therefore there is a need to develop techniques of sperm preparation which help in retrieving as many sperms with intact DNA from the unprocessed sample as possible. Microfludic is fluid dynamic based technique of sperm preparation. in this study, we evaluated if microfluidic sperm sorter can recover motile sperm with better DNA integrity compared to density gradient preparation method. Study design, size, duration Prospective randomized study conducted in 80 patients undergoing IVF-ICSI with normal semen parameters (based WHO criteria 2010). DFI was done using Sperm Chromatin Dispersion (SCD) test in split semen samples prepared by microfluidic sperm sorter and density gradient method. Sperm morphology and motility were also recorded and evaluated based on the WHO 2010 criteria. Participants/materials, setting, methods Semen parameters of the sample were assessed by microscopic examination. DFI of each unprocessed sample was carried out using SCD test, following that the sample was split and sperm preparation was done using microfluidic sperm sorter and density gradient. the recovered sperm were tested for DFI and the results were compared. Main results and the role of chance Mean DFI in unprocessed semen samples was 23%. the analysis of split semen samples post preparation showed that the DFI was significantly reduced with the use of microfluidic sperm sorter (mean DFI 0.6%) as compared to density gradient (mean DFI 9%). Limitations, reasons for caution A major limitation of the microfluidic sperm sorter is the use sperm concentration and motility of the semen sample. In oligospermic and asthenospermic samples, density gradient is the preferred method of preparation. Lack of data showing improvement in clinical outcomes with reduced DFI is also a major limitation. Wider implications of the findings: Microfluidics has shown to significantly reduce the DFI of the semen sample, it requires no extra equipment and cost and is relatively easy to pick up. Density gradient method of sperm preparation continues to be the preferred method due to its versatility and recovery of good quality sperm. Trial registration number Not applicable