High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation

Study question Are there differences in fertilization, blastocyst development, or euploidy rates when comparing ICSI versus conventional insemination among patients with male and non-male factor infertility? Summary answer Fertilization rate was lower for male factor ICSI. There were no differences in blastocyst development. Euploidy rate was lower for non-male factor ICSI. What is known already ICSI was developed for male factor infertility due to its requirement for a very small number of viable sperm. Its use has expanded beyond male factor due to some studies suggesting enhanced fertilization, and lower risks of failed fertilization or inadvertent contamination of embryo biopsy specimens. There are potential risks associated with ICSI, including imprinting disorders, birth defects, and significantly increased cost. It is also unclear whether ICSI contributes to genetic abnormalities by bypassing the egg’s natural sperm selection and potentially disrupting the meiosis apparatus. Study design, size, duration This retrospective cohort study of 576 patients was conducted at a single clinic from January 2021 to December 2021. Patients were grouped into (1) ICSI with male factor (n = 201), (2) ICSI with non-male factor (n = 160), and (3) conventional insemination with non-male factor (n = 215). Primary outcome was ploidy status, determined by calculating the percentage of euploid, aneuploid, and mosaic (both low and high mosaic) among the three groups. Secondary outcomes included fertilization and blastocyst development rates. Participants/materials, setting, methods Patients undergoing autologous IVF at a single clinic, with PGT-A performed at a single lab were included. Fertilization rate was defined as the percentage of 2PNs per number of mature eggs injected/inseminated. Blastocyst rate was defined as the percentage of blastocysts per 2PNs. Ploidy rate was defined as the percentage of euploid/aneuploid/mosaic blastocysts per total biopsied blastocysts for which a result was obtained. Percentage data was compared using N-1 Chi-squared test. Main results and the role of chance There were no significant differences in patient age, number of oocytes retrieved, or number of mature oocytes among the three groups. Fertilization rate for male factor ICSI was significantly lower compared to both non-male factor ICSI (74.2% vs 77.8%, p = 0.005) and conventional insemination (74.2% vs 76.8%, p = 0.018). There was no significant difference in fertilization rate between non-male factor ICSI and conventional insemination. There were no significant differences between blastocyst rates on Day 5, 6, or 7, or total blastocyst rate among the three groups. The euploidy rate for conventional insemination was significantly higher than that for non-male factor ICSI (53.4% vs 46.8%; p = 0.008). Non-male factor ICSI yielded lower euploidy rate than male factor ICSI (46.8% vs 52.4%, p = 0.034). Aneuploidy rate for non-male factor ICSI was significantly higher than both male factor ICSI (36.1% vs 31.0%, p = 0.041) and conventional insemination (36.1% vs 31.0%, p = 0.030). Mosaicism levels were similar between the three groups. There were no significant differences in number of biopsied embryos with no results: 5 for ICSI male factor group (0.59%), 6 for ICSI non-male factor (0.96%), and 10 for conventional insemination (0.89%). Limitations, reasons for caution This was a retrospective analysis of patients from a single clinic, with a relatively small sample size so results may not be generalizable to all populations. Extensive demographic information and baseline characteristics (including reason for ICSI performed among non-male factor patients) were not analyzed so there may be confounding factors. Wider implications of the findings This is the first study that we are aware of to investigate the ploidy status of embryos created among non-male factor patients undergoing ICSI. Conservative use of ICSI can significantly decrease the cost of IVF. Larger studies are needed to further elucidate the role for ICSI among these patients. Trial registration number not applicable

Intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) is a powerful technique in the production of transgenic mice. However, it has not yet been exploited in cattle because of ICSI fertilization problems. The objective of this study was to evaluate fertilization rates, transgenesis, blastocysts rates, and enhanced green fluorescent protein (egfp) blastocyst quality parameters (mean cell number, Oct-4 expression, and day of formation) of ICSI-MGT derived bovine embryos treated with 5 different chemical activation protocols. COCs were aspirated from ovaries obtained from local slaughterhouses and IVM was performed using conventional protocols. Sperm samples were frozen/thawed by standard procedures. Coincubation of spermatozoa with DNA construct (pCX-EGFP) was carried out in 2.8% Na citrate, with 0.5 μg of plasmid per million spermatozoa for 5 min at 0°C. Then, spermatozoa were injected into metaphase II (MII) oocytes. Injected oocytes were treated with 5 μM ionomycin (Io) for 4 min, and after 3 h, incubated in 1.9 mM DMAP for 3 h (Io-DMAP); second Io followed by DMAP for 3 h (2Io-DMAP), second Io alone (2Io); 7% ethanol for 5 min (Io-EtOH); or 20 mM SrCl2 for 5 h (Io-SrCl2) immediately after first Io. Embryos were cultured in SOFaa medium. Fertilization efficiency (2 pronuclei and 2 polar bodies) at 16 h after ICSI and condensed sperm head presence at Day 4 were evaluated by Hoechst 33342 staining of ICSI-MGT embryos. Expression of EGFP was observed under the fluorescence microscope (488 nm). Oct-4 expression was detected by immunocytochemistry and confocal laser scanning microscopy. Each experiment was replicated 3 times. Differences between percentages (Fisher test) or means (ANOVA) were significant at P < 0.05. Fertilization rates did not differ between treatments. Transgenic and nontransgenic embryos were obtained by ICSI-MGT. All transgenic embryos were the product of ICSI fertilization (no condensed sperm head present at Day 4). On the other hand, at least 60% of nontransgenic embryos (>4 cells) were the result of parthenogenesis (condensed sperm present). Blastocyst rates after 2Io-DMAP (21/81; 25.9%) were not statistically different from Io-DMAP (15/80; 18.7%) or Io-EtOH (15/102; 14.7%) but they were higher than 2Io (8/85; 9.4%) or Io-SrCl2 (13/119; 10.9%). Transgenesis rates were higher for Io-DMAP (42/80; 52.3%), 2Io-DMAP (43/81; 53%) and Io-SrCl2 (51/119; 42.8%) than for 2Io (24/85; 28.2%) and Io-EtOH (30/102; 29.4%). Over 80% of the blastocysts were transgenic.An explant was derived from an egfp-blastocyst and it proliferated and expressed the transgene for 17 days. Mean cell numbers, Oct-4 expression, and day of formation (≤ Day 7 for all treatments except for 2Io) of the ICSI-MGT egfp-blastocysts were not statistically different neither between ICSI assistance treatments nor to IVF or parthenogenetic (Io-DMAP) controls, indicating similar embryo quality according to these parameters. All treatments proved to be highly efficient to assist ICSI-MGT and to produce transgenic bovine embryos. Moreover, ICSI-MGT with an early expressed marker can help to evaluate ICSI fertilization real efficiency.

Study question Does Microfluidic Sperm Selection (MSS) improve the blastocysts’ development rate and the euploidy rate compared to Swim-up sperm selection? Summary answer MSS doesn’t improve the rate of blastocyst development and euploidy compared to Swim-up. Furthermore, in women age ≥ 37 years D6 aneuploid blastocyst rate increases What is known already Sperm defects have been shown to have a negative impact on embryo quality and development. MSS was introduced as an alternative to traditional centrifugation-based sperm preparation techniques to efficiently isolate highly motile and healthy spermatozoa. Centrifugation has been suggested to compromise membrane integrity, motility, mitochondrial energy production, and DNA structure. Less DNA fragmentation and ROS formation was observed in spermatozoa isolated with MSS compared to standard techniques. Our study aims to analyze whether MSS also improves biological outcomes. Study design, size, duration In a prospective study of 51 ICSI/PGT-A cycles performed between September 2022 and November 2023, freshly ejaculated semen sample was divided into two aliquots and processed by Swim-up and MSS. Split sperm sample was used to inject siblings’ mature oocytes (249 vs 274 oocytes in Swim-up vs MSS groups). Inclusion criteria: retrieved MII oocytes ≥8, oligoteratozoospermic and normozoospermic semen. Results were analyzed according to women’s age <37 years (21 cycles) and ≥37 years (30 cycles). Participants/materials, setting, methods Spermatozoa were selected using either conventional Swim-up or a microfluidics device (ZyMōtTM). Post treatment sperm parameters (sperm count; total sperm motility; progressive motility and hyperactivated motility) were examined for both. Embryo culture was performed in Irvine Continuous Culture-NX media until day5 or day6 and biopsy was performed at the blastocyst stage by next generation sequencing. Primary endpoint was fertilization rate, blastocyst development and usable blastocysts rate. Chi-square analysis was performed and p < 0.05 was considered significant Main results and the role of chance There is no significant differences overall between Swim-up and MSS in fertilization rate (73% vs 75%), blastocyst development rate (47% vs 54%) and ploidy rate: aneuploid (47% vs 50%), usable blastocyst (i.e. euploid+mosaics) (53% vs 50%). When considering female age, no significant difference is observed in the primary endpoints in the <37 years group. However, in women aged ≥37 years, the rate of blastocyst development is similar in the two sperm preparation groups, but a significant difference is observed in the timing of blastocyst development: The percentage of blastocysts formed on day 6 in the MSS group is significantly higher than in the Swim-up group (43% vs. 19% p < 0.05). In addition, the aneuploidy rate of blastocysts on day 6 is also significantly higher than in the Swim-up group (79% vs. 50% respectively, p < 0.05). The concentration of sperm recovered post-treatment was similar in Swim-up vs MSS (23 x106 vs 19 x106); total motility and progressive motility were similar in Swim-up compared to MSS (92.7% vs. 95.3% and 85% vs. 87% respectively). Only hyperactivated motility assessed with the SCASCOPE CASA System (Microptic) was significantly higher in MSS vs Swim-up 41% vs 4% respectively (p < 0.0005). Limitations, reasons for caution Our study included couples with different infertility diagnosis and semen parameters. Further studies with an increased number of patients may provide more comprehensive data to better evaluate biological and clinical outcomes to eventually suggest the use of MSS in laboratory routines Wider implications of the findings Recently, some studies reported that microfluidic-sorted sperm showed significantly less ROS formation and DNA fragmentation compared to those treated with conventional Swim-up method. Nevertheless, our data does not currently show the expected increase in embryo euploidy rate to justify the routine use of these expensive devices in male infertility treatment Trial registration number non applicabile

Study question What is the efficacy of frozen thawed surgically retrieved spermatozoa in azoospermic men with AZFc microdeletion undergoing Introcytoplasmic sperm injection (ICSI)? Summary answer The comparison between frozen thaw to fresh testicular sperm did not show statistically significant difference in terms of fertilization, blastocyst development and pregnancy rates. What is known already In male infertility, Y chromosome microdeletion (YCM) may be the cause up to 10% of men with oligozoospermia or azoospermia. AZFc region deletion was mostly detected in YCM. In men with azoospermia and AZFc deletion spermatozoa can be retrieved via testicular sperm extraction (TESE). Today, couples suffering azoospermia due to AZFc deletion may have genetically own child by using testicular sperm with ICSI. Because of the rare incidence of AZFc deletion, the outcome of ICSI cycles in which frozen thawed spermatozoa utilized for assisted conception has not been widely known. Study design, size, duration The data of 611 men with non-obstructive azoospermia (NOA) who underwent microTESE surgery between 2014 and 2022 was retrospectively analyzed. Assisted reproduction treatment (ART) outcome of couples whose men with AZFc deletion was further analyzed according to the fresh or frozen thawed spermatozoa usage for ICSI. Participants/materials, setting, methods All of the patients were genetically screened with karyotype and Y chromosome microdeletion analysis and microTESE operation was performed for testicular sperm recovery. The patients who diagnosed AZFc deletion and had successful sperm recovery divided to two groups according to cryopreserved or fresh sperm utilization for ICSI. The spouse and men age, fertilization rate, blastocyst development, pregnancy and live birth rate was compared between the groups. Main results and the role of chance Twenty-seven (4.4%) men were diagnosed YMC deletion in total of 611 men with NOA who underwent microTESE. Spermatozoa was successfully retrieved in 14 men (51.8%) men who were diagnosed AZFc deletion. The mean age of women and men were 30.5 (3.5) and 32.8 (4.1) years, respectively. Oocyte retrieval and ICSI were accomplished in 18 cycles. In 7 (38.8%) cycles fresh testicular spermatozoa and in 11(61.2%) cycles frozen thawed testicular spermatozoa were used. In one cycle, embryo transfer was cancelled due to total fertilization failure. In total, fertilization rate as 2PN/M2 oocytes was 57% (119/206). In 5 cycles cleavage stage embryos were transferred whereas in remaining 13 cycles blastocysts were transferred. Blastocyst development rate as day 5 blastocyst/2PN was 45.2% (42/95). Comparison of fertilization and blastocyst development rates according to fresh TESE spermatozoa and frozen thawed TESE spermatozoa groups were 63.2% vs 54.4% and 50.0% vs 38.7% respectively (p > 0.5 for both). Clinical and live birth rates per embryo transfer cycle were 57.1% vs 40.0% and 42.8% vs 30.0% respectively (p > 0.5 for both). Limitations, reasons for caution Fresh and frozen thawed groups demonstrated no statistical difference in terms of ICSI outcome. However, relatively reduced fertilization and blastocyst development rates should be interpreted cautiously because beta type error. Further data is needed to clarify this difference. Wider implications of the findings The best of our knowledge our study is the largest in literature comparing frozen thawed and fresh sperm utilization for ICSI in men with AZFc deletion. This data may help for physicians and couples to give decision diagnostic microTESE surgery and cryopreservation of sperm further ICSI treatment. Trial registration number Not applicable

Study question Can the use of a microfluidic sperm selection device without centrifugation simplify the procedure without affecting fertilization rates and embryonic development after ICSI? Summary answer The microfluidic device can be used to select sperm in a simple procedure without reducing the fertilization or embryo development rate. What is known already In human ART, it is essential to process the semen and adjust the sperm sample according to the intended purpose, rather than using raw semen. Current sperm preparation methods at most IVF clinics include the density gradient centrifugation method, which utilizes the difference in density between the maturation stages of sperm, followed by washing using centrifugation. However, these methods require a lot of steps and the procedure is complicated. Recently, several non-centrifugal sperm processing devices have become available. One of them is a simple sperm conditioning method using a microfluidic device. Study design, size, duration This was a prospective study using sibling oocytes including 20 ART patients treated with 24 cycles and where there were 320 zygotes after ICSI. The duration of the study was 8 months (May 2020 to December 2020). Participants/materials, setting, methods For sperm preparation without centrifugation, a microfluidic device “ZyMōtⓇ Multi 850μL” was used. According to the labelled use, 850μL of semen was required with “ZyMōtⓇ” device (ZyMōt group), and the rest of the semen was processed according to routine laboratory procedure by monolayer density gradient centrifugation and washing by centrifugation (DGC group). Oocytes from the same patient were randomly divided into 2 batches and ICSI was performed using sperm treated with each method. Main results and the role of chance The 2PN formation rate in the ZyMōt group was 84.5% (142/168), which was not significantly different from 82.9% (126/152) in the DGC group. There was also no significant difference in 1PN formation rate (3.0% vs. 3.3%), multi PN formation rate (3.6% vs. 3.3%) and the non-fertilization rate (8.3% vs. 10.5%). The good quality embryo rate at Day 3 was 25.0% (31/124) in the ZyMōt group and 24.5% (27/110) in the DGC group, with no significant difference. The Day 5 blastocyst rate was 37.9% (47/124) in the ZyMōt group and 36.7% (40/109) in the DGC group, and the cumulative blastocyst rates by Day 7 were 54.0% (67/124) and 57.8% (63/109), respectively, with no significant difference. Limitations, reasons for caution This study was limited to samples with a motile sperm concentration of more over 1.0 × 106 in raw semen. Wider implications of the findings These results demonstrated that sperm processing using the microfluidic device without centrifugation does not affect the fertilization or blastocyst development rate after ICSI and that the sperm processing procedure can be simplified by using this device. Trial registration number not applicable

To investigate the effect of vascular endothelial growth factor (VEGF) on the early development and polyspermy rate of ovine embryos in vitro, 2 experiments were conducted with human recombinant VEGF165 supplemented to the media during maturation, fertilization, and culture in vitro, respectively. Ovaries were collected from ewes at a local slaughterhouse. All oocytes surrounded by a multilayer of cumulus cells were collected and rinsed 3 times in maturation medium (control medium and treatment medium, respectively). A total of 100 oocytes in each group were cultured in 4-well plates (Nunc) containing 800 μL of maturation medium at 38.5°C in an atmosphere of 5% CO2 with saturated humidity. Four replicates of each experiment were conducted. Statistical analyses were conducted by ANOVA with SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as means, and P < 0.05 was considered significant. In Experiment 1, to investigate the effect of VEGF on the early development of ovine embryos in vitro, VEGF was used at 5 ng mL–1 (treatment group A) and 10 ng mL–1 (treatment group B) in maturation medium (TCM-199 + BSA), HSOF fertilization medium, and SOF culture medium. The results showed that the maturation rate was increased significantly (P < 0.01), from 75.76% in the control treatment to 83.98 and 80.23% in treatment group A and treatment group B, respectively. The cleavage rate was increased from 75.85% in the control group to 79.39% in treatment group A (P > 0.05). The development rates of morulae (45.03%) and blastocysts (23.54%) in treatment group A were significantly higher (P < 0.01) than those in the control group (38.94 and 18.09%, respectively). In addition, the development rates of blastocysts in treatment group B (21.05%) were lower than those in treatment group A (P > 0.05) and higher than those in the control group (P > 0.05). In Experiment 2, to investigate the effect of VEGF on the polyspermy rate of ovine embryos in vitro, 5 ng mL–1 of VEGF was used in TCM-199 + BSA maturation medium in this experiment. The results showed that the fertilization rate after 18 h of IVF was increased significantly (P < 0.01), from 75.75% in the control group to 83.86% in the treatment group, and that the polyspermy rate was decreased significantly (P < 0.01), from 12.64% in the control group to 7.68% in the treatment group. These results indicate that VEGF significantly improved the maturation and fertilization rates of ovine oocytes and, consequently, the rate of embryo development in vitro, especially when the medium was supplemented with 5 ng mL–1 of VEGF. The VEGF obviously decreased the polyspermy rate and bated the phenomenon of polyspermy in the process of ovine oocyte IVF. The present study was supported by the National Natural Science Foundation of China (No. 30371035).

ABSTRACT: The main cause of low efficiency of in vitro produced porcine embryos is the high polyspermic penetration rates at fertilization, which is aggravated in low quality oocytes. Experiment 1 evaluated the embryo development in high and low quality oocytes. Experiment 2 evaluated the embryo development and quality of low quality oocytes fertilized with sperm pre-incubated during 0h (control), 0.5h, 1h and 1.5h. Experiment 3 investigated fertilization and monospermic rates of the same groups of Experiment 2. Experiment 4 evaluated embryo development, cell density, fertilization and monospermic rates of high quality oocytes using semen pre incubated during the best time observed in the previous experiments. Cleavage and blastocyst rates were analyzed by chi-square test, and remaining data by ANOVA and Tukey test (P≤0.05). The cleavage (74.8 vs 51.7%) and blastocyst (33.7 vs 9.8%) rates were greater in oocytes of high versus low quality, with no differences in cell density. Fertilization rates (65.6 to 79.5%) were not influenced by pre-incubation time. However, semen pre-incubation during 1.5h increased monospermic penetration (53.3%) and cleavage rates (92.5%) in low quality oocytes. Blastocyst rate was improved with 1.5h of semen pre incubation; however they were still lower than that observed with high quality control oocytes. Ultimately, pre-incubation did not influence fertilization, monospermic penetration, embryo development rates, nor cell density in oocytes of high quality. Low-quality porcine oocytes resulted in better rates of embryo development if in vitro fertilized with sperm pre-incubated for 1.5 hour.

Does the orientation of the first polar body during ICSI affect fertilization and embryo development rates?

Study question Are there any differences in the use of fresh or frozen-thawed oocytes in a PGT-A program? Summary answer While euploidy rates are comparable, the efficiency of a PGT program is adversely impacted when utilizing frozen-thawed oocytes. What is known already Oocyte vitrification has become one of the key assisted reproduction procedures. Some studies have reported rates of embryonic development, clinical pregnancy, and embryo implantation comparable between fresh and cryopreserved oocytes in egg donor patients. Vitrified oocyte accumulation before the blastocyst biopsy is a commonly used strategy in PGT programs to enhance the chances of obtaining a euploid blastocyst with the patient’s own oocytes. Further research is needed to assess the impact of frozen-thawed oocytes on aneuploidy and the efficiency of PGT programs. Study design, size, duration We conducted a retrospective comparison of 777 PGT-A cycles, using either fresh or frozen-thawed oocytes, regarding embryo development (fertilization rates, blastocyst formation, and rates of good-quality blastocysts), PGT outcomes (euploidy and mosaicism rates), and the overall efficiency of the PGT cycle, measured as the number of euploid blastocyst per available mature oocytes, between April 2018 and November 2023. The study groups were stratified based on the age of the patients (≤35 and >35 years old). Participants/materials, setting, methods PGT-A cycles were categorized into two groups: patients who utilized fresh oocytes (n = 641) and those who utilized frozen-thawed oocytes (n = 136). A total of 6,370 fresh oocytes and surviving oocytes from the initially vitrified ones (972 out of 1,153) were fertilized and cultured to the blastocyst stage, at which point biopsies were performed. The biopsied cells were subjected to NGS screening, and statistical data analysis was conducted using the Chi-square test, with significance set at P < 0.05. Main results and the role of chance In the group of patients aged ≤35 years old, significant differences were observed when comparing PGT cycles using fresh or frozen-thawed oocytes in terms of the fertilization rate (78.4% vs. 72.0%), blastocyst formation rate (47.4% vs. 37.7%), and the efficiency of the PGT cycle (24.0% vs. 16.0%), corresponding to a 33.3% reduction in efficiency. No differences were observed between fresh and frozen-thawed oocytes regarding euploidy rate (64.6% vs. 68.4%), mosaicism rate (4.9% vs. 5.1%), and the percentage of good-quality embryos (60.0% vs. 56.1%). Similarly, in patients aged >35 years old, fertilization (81.5% vs. 73.5%), blastocyst formation (40.5% vs. 33.4%), and the efficiency of the PGT cycle (11.7% vs. 7.2%), which correspond to a 38.5% reduction in efficiency, were improved when using fresh oocytes. No differences were observed regarding euploidy (35.6% vs. 35.3%) or mosaicism rate (3.8% vs. 5.3%), and the percentage of good-quality embryos (55.2% vs. 54.7%). The oocyte survival rate was 84.3%. Limitations, reasons for caution Our study is limited by its sample size and retrospective design, as well as the inclusion of a non-selected and general infertile population, which could amplify the impact of confounding variables. Wider implications of the findings We found that the utilization of frozen-thawed oocytes reduces the PGT program efficiency by decreasing fertilization and blastocyst formation rates. This suggests that the oocyte accumulation strategy should be approached with caution. However, vitrification did not lead to an increase in the rates of aneuploidy, mosaicism, or poor-quality blastocysts. Trial registration number NOT APPLICABLE

Study question Does the administration of micronutrients and probiotics supplement during an oocyte donation program improve fertilization rate, blastocyst development, and pregnancy outcomes? Summary answer Micronutrients and probiotics supplementation in oocyte donors does not influence fertilization or blastocyst rates, but significantly improved pregnancy outcomes of recipients that used vitrified oocytes. What is known already Micronutrient supplementation could have a positive impact on IVF clinical results. Evidence about its role in improving oocytes’ quality and reproductive outcomes in normoresponders patients are still lacking. The OvoVid double-blind, randomized, controlled trial Phase 1 suggested that a micronutrients-probiotics supplementation enhances oocyte yield and quality in normoresponder oocyte donors. A significantly higher number of total oocytes retrieved and mature MII were achieved in the supplement group, which also showed a better follicular response to controlled ovarian hyperstimualtion (COH) than placebo in term of more follicles at the end of COH, despite a similar initial antral follicle count. Study design, size, duration Multicentric, double-blind, randomized controlled trial involving 130 oocyte donors and 107 recipients during a two-year period involving three Fertility Clinics in Spain. Oocyte donors were randomized in supplement or placebo group, taking one tablet a day at least for 30 days before and during COH. The micronutrients-probiotc supplement was a combination of Myoinositol+d-chiro-inositol (ratio 40:1), folic acid, melatonin, coenzyme Q10, vitamin D3, B6, B12, E, iron, zinc, Lactobacillus Gasseri and Crispatus. Participants/materials, setting, methods The Ovovid Phase 2 study analyzed the reproductive outcomes of 107 oocyte donation treatments in recipients, classified in supplement (n = 62) or placebo (n = 45) group depending on the oocytes’ origin, realized with donors’ oocytes of Ovovid Phase 1. Primary outcomes included fertilization and blastocyst development rates, while secondary outcomes encompassed pregnancy rates and oocyte survival after thawing. Statistical analysis was performed using Fisher’s exact test and Mann-Whitney U test, with significance set at p<0.05. Main results and the role of chance No significant differences were observed in fertilization rate (76,7% vs. 78%, p > 0,05) comparing supplement and placebo group, as well as in blastulation development (43,4% vs. 45,4%, p = 0,61), despite similar number of inseminated oocytes (6,7 ± 1,3 vs. 6,3 ± 1,2, p = 0,13). When vitrified oocytes were used, similar survival rate after thawing were described in the two groups (94,3% vs. 93,8%, p = 0,3). Pregnancy rate was similar regardless of multivitamin-probiotics supplement use (44,4% vs. 68,2%, p = 0,07). However, in the sub-analysis for type of oocyte, pregnancy rates with vitrified oocytes (Supplement n = 24; Placebo n = 18) were significantly higher in the supplement group compared to placebo (50% vs. 0%, p = 0,02). No significant differences were found for fresh oocytes (n = 65; Supplement n = 38; Placebo n = 27) outcomes. Limitations, reasons for caution The study was limited by the relatively small sample size for subgroup analyses and the lack of long-term follow-up for pregnancy outcomes. Further studies are warranted to confirm these observations and optimize supplementation strategies in oocyte donation programs and assisted reproductive technologies. Wider implications of the findings Micronutrients and probiotics supplementation during oocyte donors’ COH did not seem to improve overall fertilization or blastulation rates. However, it may be a valuable adjunct in improving recipients’ pregnancy outcomes with vitrified oocytes, suggesting a role in enhancing oocyte competence during vitrification, potentially optimizing outcomes for specific protocols. Trial registration number No

Study question Does vitrification of oocytes in oocyte donation program alter the morphokinetic pattern of human embryos? Summary answer Both the extrusion of the second polar body (tPB2) and the appearance of the pronuclei (tPNa) occur earlier in vitrified oocytes without affecting further development. What is known already Vitrification of oocytes has been a breakthrough in oocyte cryopreservation and significantly altered practices in IVF labs. Oocyte vitrification/warming has become an integral part of daily routine and thorough assessment of its efficiency is crucial. Though, studies of vitrified-warmed oocytes have shown similar fertilization, cleavage, blastulation, pregnancy and live birth rates to fresh oocytes, the morphokinetic pattern of embryos originating from vitrified oocytes has not been thoroughly studied. According to some studies vitrification may be related to delayed blastocyst formation while others failed to see any alteration in the morphokinetic pattern between embryos derived from fresh or frozen oocytes. Study design, size, duration This retrospective observational study was performed at Embryolab Fertility Clinic, in Thessaloniki, Greece between April 2020 and September 2021 and included 590 vitrified oocytes from 68 oocyte donation cycles. Control group consisted of 31 good prognosis cases (oocyte donation cycles or homologous oocyte cycles of women under 35 years old). Participants/materials, setting, methods 68 vitrified oocyte donation cycles and 31 fresh control cycles were analyzed. All oocytes after ICSI were cultured in Embryoscope time-lapse incubator up to blastocyst stage. Key time parameters and dynamic events were analyzed using generalized estimating equations (GEE) regression analysis for the non-independent nature of the data. Moreover, the following comparisons were performed between groups: fertilization, cleavage, top cleavage, blastocyst, top blastocyst and pregnancy rates. Main results and the role of chance There was no significant difference in fertilization rates between control and frozen oocytes (77.79% ± 15.73 vs 71.26% ± 21.50 respectively, p = 0.15). Cleavage rate and top cleavage rate (more than 6 cells, equal size, with no or minor fragmentation), were higher in frozen oocytes with this difference being significant (p = 0.02 for cleavage rate and p = 0.015 for top cleavage rate). Blastocyst rate and top blastocyst (2AA, 3AA, 3AB, 4AA, 4AB according to Gardner criteria) rate, though, showed similar rates with no statistically significant difference (Blastocyst rate: 74,15% ± 17.19-control vs 76.42% ± 30.75-frozen oocytes, p = 0.6 & Top blastocyst rate: 45.54% ± 24.04-control, 45.30% ± 32.04-frozen oocytes, p = 0.82). Pregnancy rates were not significantly different (control:85.19%, frozen oocytes:75.38%, p = 0.29). The comparison for key time parameters and dynamic events that were analyzed (tPB2, tPNa, tPNf, t2, t3,t4, t5, t6, t7, t8, t9+, tSC, tM, tSB, tB, tEB, S2, S3, S5, CC1, CC2, CC3,Compaction and Blastulation) showed significant difference in 2 time parameters: tPB2 (4.62 hours-control, 3.67 hours-frozen, Coef: -1.09, p = 0.015) and tPNa (9.07 hours-control, 8.25 hours-frozen oocytes, Coef: -1.06, p = 0.043). Limitations, reasons for caution The fact that the oocytes under comparison were not sibling as well as its retrospective nature, constitute the main limitations of the present study. Moreover, the number of cases included in this study was limited and the available information about ongoing pregnancy and live births is still pending. Wider implications of the findings tPB2 and tPNa seem to occur earlier in vitrified oocytes without affecting further development, implying a need for reviewing timings between OPU/vitrification/warming/ICSI. Moreover, vitrified oocytes displayed higher cleavage and top cleavage rates with unaffected blastocyst or pregnancy rates. Overall, vitrification of oocytes seems to be safe and reliable. Trial registration number N/A

The objectives of this study were 1) to compare the efficiency of intracytoplasmic sperm injection (ICSI) with and without additional artificial stimulation using frozen-thawed sperm and in vitro-matured porcine oocytes and 2) to determine the nuclear anomalies of ICSI oocytes that failed to fertilize or develop. In experiments 1 and 2, we evaluated the effects of additional activation treatments, e.g., electrical stimulus, Ca ionophore (A23187), and/or cycloheximide, on fertilization and development of ICSI porcine oocytes. Significantly higher fertilization, cleavage, and blastocyst rates were obtained for oocytes treated with a combination of ICSI and electrical activation (EA) (P < 0.05) than for those treated with ICSI alone. However, different combinations of electrical and chemical activation treatments did not further improve the rates of fertilization, cleavage, and blastocyst development for ICSI embryos. To elucidate the association between sperm head decondensation and oocyte activation and to investigate the cause of embryonic development failure, in experiment 3 we evaluated the nuclear morphology of oocytes 16-20 h after ICSI. Nearly 100% of oocytes showed female pronucleus formation after ICSI regardless of activation treatment. However, failure of male pronucleus formation with intact or swelling sperm heads was observed in some ICSI embryos, suggesting that these embryos underwent cell division with the female pronucleus only. Artificial activation (EA and A23187) had a beneficial effect on embryonic development, sperm decondensation was independent of the resumption of meiosis, and the failure of formation of a male pronucleus was the major cause for fertilization failure in porcine ICSI embryos.

Does the application of three different artificial activating stimuli lead to a difference in pre- and post-implantation embryo development in the wobbler mouse, a mouse model with oocyte activation deficient round-headed sperm cells similar to human globozoospermia? No gross differences were found between strontium chloride, electrical pulses or ionomycin with respect to the pre- and post-implantation development in the wobbler mouse. Fertilization failure following intra-cytoplasmic sperm injection (ICSI) occurs in 1-3% of the ICSI cycles in human assisted reproduction technology (ART) and has been successfully overcome by different artificial activating stimuli. No comparison has been made yet in terms of their efficiency and safety. Calcium release and embryo development were compared between oocytes fertilized by wobbler and wild-type (WT) sperm following ICSI with or without three different artificial activating agents. Preimplantation development was assessed on 70 injected oocytes on average per group. On average, 10 foster mothers were used per activating group to compare post-implantation development. We used the wobbler mouse model that possesses oocyte activation deficient round-headed sperm cells. First, the calcium release following ICSI using wobbler sperm was compared with that of WT sperm. Outcome measures were the percentage of oocytes that showed calcium release and their mean amount of calcium rises. Secondly, the pre- and post-implantation development was assessed following ICSI with wobbler sperm plus artificial oocyte activation using either: (i) strontium chloride (Wob-Sr), (ii) electrical pulses (Wob-E) or (iii) ionomycin (Wob-I). Outcome measures were the activation, cleavage and blastocyst rates and the assessment of blastocyst quality by differential staining. Following mouse embryo transfer, pregnancy and birth rates as well as mean litter sizes were examined. Finally, pups were followed up until 8 weeks of age and then mated with fertile controls to assess their fertility. The percentage of oocytes showing calcium rises as well as the number of calcium rises per oscillating oocyte were significantly lower in the wobbler group when compared with the WT group (9.3 versus 96% and 2.1 calcium rises versus 31 calcium rises) (P < 0.001). The fertilization rate was significantly lower in the wobbler group (11.4%) when compared with the WT group (92.1%) and the artificial activation groups (strontium chloride: 99%, electrical pulses: 99% and ionomycin: 81%, respectively) (P < 0.001). Post-implantation development did not differ significantly between the WT and artificial activation groups, with pregnancy rates in favor of strontium chloride and electrical pulses. The weight of the male pups did not differ between the study groups, whereas the weight of the female pups originating from Wob-Sr embryos was significantly lower at weeks 2, 3 and 4 when compared with female pups originating from WT embryos. However, the latter difference was not observed at later time points, nor in the other artificial activating groups. All offspring mated successfully with fertile controls. Results in animal models should be extrapolated with caution to a subfertile human population. Also, ionomycin is currently the most widely used artificial oocyte activating agent in human ART. The low frequency of observed calcium rises and the low activation rate make the wobbler mouse a highly suitable model to study oocyte activation deficiency. Strontium chloride and electrical pulses were more efficient means to restore fertilization rates and to support pre- and post-implantation embryonic development than ionomycin. This work was supported by the Flemish foundation of Scientific Research (FWO-Vlaanderen) (aspirant clinical research mandate to F.V.M., fundamental clinical research mandate to P.D.S.); and Ghent University grant (KAN-BOF E/01321/01 to B.H.). The authors have no competing interests to declare.

The aim was to study whether a limited exposure of embryos outside the incubator has an effect on embryo development, blastocyst quality and euploid outcomes. This retrospective study was performed at ART Fertility Clinics, Abu Dhabi, United Arab Emirates (UAE) between March 2018 and April 2020 and included 796 mature sibling oocytes that were split randomly between two incubators after intracytoplasmic sperm injection (ICSI): an EmbryoScope™ (ES) incubator and a benchtop incubator, G185 K-SYSTEMS (KS). The fertilization, cleavage, embryo/blastocyst qualities, useable blastocyst and euploid rates were assessed to evaluate the incubator performance. In total, 503 (63.2%) mature oocytes were cultured in the EmbryoScope and 293 (36.8%) in the K-SYSTEMS. No differences were observed in fertilization rate (79.3% vs 78.8%, P = 0.932), cleavage rate (98.5% vs 99.1%, P = 0.676) and embryo quality on Day 3 (P = 0.543) between both incubators, respectively. Embryos cultured in the EmbryoScope, had a significantly higher chance of being biopsied (64.8% vs 49.6%, P < 0.001). Moreover, a significantly higher blastocyst biopsy rate was observed on Day 5 in the EmbryoScope (67.8% vs 57.0%, P = 0.037), with a highly significant increased euploid rate (63.5% vs 37.4%, P = 0.001) and improved blastocyst quality (P = 0.008). We found that exposure of embryos outside the incubator may negatively affect the in vitro blastocyst development and euploid rate on Day 5.

Study question What effect does Annexin-V MACS method have on sperm selection by pLCZ1 expression and blastocyst rate in male DNA fragmentation? Summary answer AnnexinV-MACS method could be a good choice for sperm selection with high pLCZ1 expression and high blastocyst rate in high DNA fragmentation male factor. What is known already Sperm selection based on morphology and motility in ART techniques, is not enough for choosing the best sperm especially in male factor patients. In Annexin-V magnetic activated cell sorting (MACS) technique, apoptotic sperm are separated from non-apoptotic one by negative selection. So, this method can improved quality of compaction rate in embryo. PLCζ is oocyte activating factors that it starts oscillations of calcium ca2+ in oocyte and it has a significant effect on fertilization and implantation. Study design, size, duration Semen samples from 30 male factor infertile couples with high DFI (DFI&amp;gt;30%) were selected and divided into two group (control and experimental) in each patient. Participants/materials, setting, methods Control was washed with DGC and experimental one was selected by MACS-DGC. Retrieved eggs in each patient, were divided in 2. Both group were injected by DGC and MACS respectively. Semen parameters and DFI (SCD test) were analyzed before and after processing. After ICSI, rate of fertilization, embryo development and blastocyst formation were evaluated. Real time PCR evaluate expression of PLCζ. Comparison between results of two groups was assessed by SPSS analysis. Main results and the role of chance Results showed that, sperm motility and morphology after MACS method (45%, 1.7%) was significantly higher than DGC method (40%, 1.1%) and before washing (35%, 0.9%). Percent of DFI in MACS group (36%) was significantly decreased compared to DGC (45%) and primitive group (55%). The number of oocytes were injected in DGC group was 93 and in MACS group was 111. Fertilization rate in both groups was almost the same (72.07% in MACS vs 73.11 in DGC). Rate of day 3 embryo with good grade in MACS group (72. 5%) was significantly higher than DGC (51.47%) (P &amp;lt; 0.05). The blastocyst rate in MACS-DGC group (69.69%) was significantly increased compared to DGC group (48%). PLCζ gene expression in MACS-DGC was significantly higher than DGC group (p-value=0.046). Limitations, reasons for caution This experiment was performed invivo. Wider implications of the findings: Sperm selection by MACS-DGC method can improve sperm motility, morphology and reduce sperm DFI. No significant difference was observed in fertilization rate, but percent of high-quality embryo on days 3 and 5 was significantly higher by this method,also it can be suggested as a good choice for patients with highDF. Trial registration number Not applicable