Blastocyst Stage Research Articles

P-544 Viability assessment using fluorescent markers, ultrastructure and implantation of human embryos following blastocyst biopsy using three different laser systems

Abstract Study question Are there any differences in viability, ultrastructure and implantation of human blastocysts biopsied using three different laser systems? Summary answer No statistically significant differences are observed in viability, ultrastructure and implantation of human blastocysts biopsied using three different laser systems What is known already Most studies in human embryos concentrate on assessing the success of biopsy/vitrification on survival rates and clinical outcomes following transfer. Our previous published studies examined in addition to the survival rates, the cytoskeleton, ultrastructure and viability of cleavage and blastocyst stage biopsied embryos, using a specific laser system, emphasizing differences between the two methods and alerting on effective strategies to minimise potential laser induced damage. This is the first study to investigate the safety of 3 different laser systems for blastocyst biopsy by comparing implantation rates and by analysing cell viability using Fluorescence Microscopy and ultrastructure by Transmission Electron Microscopy(TEM). Study design, size, duration 405 human blastocysts were biopsied on day 5 for PGT-A or PGT-SR or PGT-M using the Saturn laser (n = 145-58 cycles) or the Octax laser (n = 132-55 cycles) or the Lykos laser (n = 128-46 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer due to being diagnosed as abnormal were processed following vitrification/warming for viability analysis (n = 30 Saturn laser, n = 30 Octax laser, n = 30 Lykos laser) or TEM (n = 20 Saturn laser, n = 20 Octax laser, n = 20 Lykos laser). Participants/materials, setting, methods The study was conducted in an academic hospital with an IVF/PGT laboratory, 2 private IVF Units and an IVF Unit of a Naval hospital. Blastocyst viability after biopsy/vitrification/warming was assessed using two fluorescent markers: carboxyfluorescein-diacetate succinimidylester (CFSE-viable cells) and propidium iodide (PI–damaged cells) in combination with DAPI to visualise DNA. TEM analysis was carried out following embryo fixation in gluteraldehyde, incubation in osmium, aqueous uranyl acetate, dehydration through ethanol series, and immersion in Epon. Main results and the role of chance The implantation rates (+ve hCG / ET) were not statistically different among the 3 laser systems (64.4% -Saturn, 63.4%- Octax, 64.7%- Lykos p > 0.05). Viability staining with CFSE/PI following vitrification/warming revealed that all biopsied blastocysts had damaged cells at the position of cutting (range 4-8cells) and additional PI stained cells in other parts (range 2-15), possibly due to damage sustained during vitrification/warming. The majority of embryos across the 3 groups had ≤7% PI stained damaged cells (73.3% Saturn, 73.3% Octax, 76.7% Lykos), indicating no statistically significant difference in blastocyst viability among the 3 laser systems (p > 0.05). No difference was also observed in the incidence of lysed biopsied TE cells or the PGT results. TEM analysis revealed similar ultrastracture among the 3 groups, as documented by the presence of normal organelles’ structures including smooth/rough endoplasmic reticulum, Golgi apparatus, mature mitochondria with cisternae, zona and microvilli. Multivesicular bodies, residual bodies, lipofuscins and autophagic vacuoles were also evident and indicative of elimination and rescue process. Excessive accumulation of glycogen granules and distension of mitochondria were more prominent in Grade B blastocysts compared to Grade A blastocysts, which may indicate a ‘block’ of glycogenolysis secondary to the mitochondrial alterations, suggesting changes in normal embryo metabolism. Limitations, reasons for caution The biopsied blastocysts used for viability and ultrastructural analysis in this study were all diagnosed with either chromosomal abnormalities or single gene defects following PGT-A or PGT-SR or PGT-M. Wider implications of the findings The similarities observed in implantation rates, the minimal levels of cell damage and the embryo quality dependent limited ultrastuctural anomalies identified confirm the safety of all three laser systems and emphasize the blastocysts’ ability to initiate elimination/rescue processes in their attempt to fully recover from ‘traumas’ sustained during biopsy/vitrification. Trial registration number not applicable

P-025 Mitochondrial activity in sperm is associated to blastocyst development: a double-donation study

Abstract Study question Do oxidative parameters in sperm affect oocyte fertilization or blastocyst development in double-donation ICSI cycles? Summary answer While intracellular oxidative stress in sperm does not suppose an impact on fertilization or embryo development, mitochondrial activity is related to increased blastocyst development rates What is known already Reactive oxygen species(ROS) are molecules causing a detrimental effect on sperm quality and function, especially when they are imbalanced with respect to the sperm and seminal plasma antioxidant capacity. Previous research have extensively demonstrated that sperm affected by oxidative stress(OS) show reduced fertilizing capacity, and lead to embryos with impaired development after conventional IVF. The role of OS in sperm in the case of ICSI is, however, controversial because many factors may cause a bias. Not only could this explain the inconsistent results reported thus far, but would also support the need of testing healthy donor gametes to reduce variability. Study design, size, duration This research was devised in the context of a collaboration between a sperm bank and a public university, and was set as a blinded, retrospective study that included samples from 45 sperm donors used in a total of 55 oocyte donation cycles. Intracellular levels of total ROS and of superoxides (O2•-), oxidative stress and mitochondrial activity (MA) were evaluated in frozen-thawed sperm donor samples, and related to the embryo outcomes obtained from the same samples. Participants/materials, setting, methods Samples from selected sperm donors were used to fertilize oocytes through ICSI in oocyte donation programs, and fertilization and blastocyst rates were recorded. Sperm viability and intracellular levels of total ROS and superoxides were determined through a combined assay (2’,7’-Dichlorofluorescin diacetate/Hydroethidine/Sytox Red); oxidative stress and sperm viability were assessed through co-staining with CellRox Far Red and propidium iodide (PI); and MA (mitochondrial membrane potential) and sperm viability were evaluated after staining with DiOC6(3) and PI. Main results and the role of chance The average and standard deviation for fertilization and blastocyst rates were 76.8%±17.3% and 67.5%±22.8%, respectively. Taking the viable sperm population into account, percentages of sperm with high ROS and low O2•- were 17.6%±15.9%, those with high ROS and high O2•- were 0.5%±0.4%, percentages of sperm with low ROS and low O2•- were 25.9%±9.2%, and those with low ROS and high O2•- were 54.5%±21.1%. The evaluation of oxidative stress with CellRox revealed that the percentage of viable sperm with high OS was 63.1%±16.5%. Neither intracellular levels of ROS and superoxides, nor oxidative stress in viable sperm were found to be correlated to fertilization or blastocyst rates (P>0.05). In spite of this, the percentage of viable sperm with high MA, which was 20.7%±10.5%, was observed to be positively correlated to blastocyst rates (Rs=0.276; P=0.04). While sperm mitochondria do not participate in further embryo development, a greater mitochondrial activity in post-thawed donor sperm could indicate a healthier sperm function. Mitochondrial activity could, therefore, be used as a predictive biomarker for embryo development. Limitations, reasons for caution The sample size is the main limitation of this study, despite the efforts made to include a high number of sperm donors. Wider implications of the findings Results obtained in the present study show that ICSI evades the detrimental effects of sperm oxidative stress on fertilization and embryo development. Yet, sperm mitochondrial activity could be used as a biomarker to test the ability of a sperm donor to generate healthy embryos that develop up to blastocyst stage. Trial registration number Not applicable

P-182 High-resolution synthetic embryo images generated by AI

Abstract Study question How effectively can a latent diffusion model generate high-fidelity embryo images tailored to the specific contextual needs of researchers, based on the user’s text input? Summary answer The latent diffusion model successfully generated high-resolution embryo images, providing a novel approach to address data scarcity in embryo imaging. What is known already The use of AI in generating synthetic data has been increasingly explored to address the scarcity of real-world datasets in various fields. Particularly in medical imaging, AI-generated synthetic data offers a potential solution to overcome the limitations imposed by data privacy concerns and ethical considerations. Previous studies have shown that AI can replicate complex patterns in data, but its application in generating embryo images remains less explored. Study design, size, duration Single static images of 5,133 Day 5 blastocysts and 2,093 Day 3 cleavages were retrospectively collected from seven in vitro fertilization clinics between June 2011 and May 2022. The images were analyzed along with relevant metadata including clinical information and embryo grades. Day 3 embryo grading was based on the number of blastomeres, evenness, and fragmentation percentage. Day 5 grading criteria included the inner cell mass, trophectoderm, and blastocyst stage evaluations. Participants/materials, setting, methods An AI model using latent diffusion was developed to generate 10,051 Day 5 and 10,088 Day 3 synthetic embryo images. The authenticity was assessed through visual Turing tests, where embryologists discerned real from synthetic images. For the evaluation, 200 real (100 Day 5, 100 Day 3) and 200 synthetic (100 Day 5, 100 Day 3) images were randomly chosen from each dataset, ensuring a comprehensive test of the generated images’ realism. Main results and the role of chance The AI model’s efficacy in generating synthetic embryo images was assessed through visual Turing tests on Day 5 and Day 3 embryos, yielding accuracies of 0.59 and 0.57, respectively. These accuracies fall within the 99% confidence interval of near-random performance (0.41 to 0.59), highlighting the challenge in distinguishing synthetic from real images. For Day 5 embryos, the sensitivity and specificity were recorded at 0.52 and 0.66, indicating a moderate challenge in identifying synthetic images and a relatively higher ease in recognizing real ones. Day 3 embryos presented a lower sensitivity of 0.41, suggesting greater difficulty in detecting synthetic images, while the specificity increased to 0.73, indicating a stronger ability to identify real images. Collectively, with an overall accuracy of 0.58, sensitivity of 0.47, and specificity of 0.70, these findings confirm the synthetic images’ remarkable realism, closely emulating actual embryo features to the extent that differentiation by experts proved challenging. This level of realism underscores the synthetic images’ potential to significantly enrich embryological research datasets, promising advancements in the field. Limitations, reasons for caution The study’s limitation lies in the generated synthetic embryos being based on three embryo grade categories, limiting feature diversity. Additionally, there was no quantitative assessment method for these images, relying instead on expert evaluation, which required deploying a large number of embryologists. Wider implications of the findings The model’s generation of highly realistic embryo images counters data scarcity in embryological research, potentially elevating AI’s utility, enriching educational content, advancing reproductive medicine, and ensuring ethical data usage. Trial registration number not applicable

P-191 Pronuclear transfer overcomes embryonic developmental arrest in Padi6-deficient mice

Abstract Study question Can pronuclear transfer (PNT) overcome embryonic developmental arrest (EDA) observed in the created Padi6-/- mouse model? Summary answer Applying PNT significantly improved the blastocyst rate of Padi6-/- zygotes, indicating its potential in overcoming female-related infertility. What is known already Various components of the subcortical maternal complex (SCMC), including peptidyl arginine deiminase 6 (PADI6), play a crucial role in early embryonic development and female fertility. Recent studies have revealed that mutations in PADI6 can result in EDA and thus female infertility, which is hard to overcome by current available treatment methods. The PNT technique, designed to replace compromised cytoplasm with a healthy counterpart, has been proposed to overcome specific female-related infertility indications. The Padi6-/- mouse displaying EDA, offers a valuable model to investigate the efficacy of PNT to overcome female-related infertility. Study design, size, duration From April 2022 to December 2023, an experimental study was conducted with CRISPR/Cas9 created Padi6 knockout mice. For fertility evaluation, Padi6-/- and Padi6+/- females were paired with wildtype males. Next, 30 wildtype and 25 Padi6-/- oocytes were collected for parthenogenetic activation. A total of 81, 54 and 145 zygotes were collected from wildtype, Padi6+/- and Padi6-/- females, respectively, for evaluating embryonic development and the effectiveness of PNT in overcoming EDA. Participants/materials, setting, methods The C57BL/6J Padi6-/- mouse model was established by introducing Padi6 c.1577_1578insT with CRISPR/Cas9. Female mice aged 6-12 weeks underwent ovarian hyperstimulation and mated with wildtype males. Oocytes were parthenogenetically activated with SrCl2 in presence of Cytochalasin D, and checked for 2-cell and blastocyst rates. PNT treatment was applied by transferring the 2PN from Padi6-/- to wildtype enucleated zygotes using electrofusion, and the efficiency was evaluated in terms of embryonic developmental potential. Main results and the role of chance After mating Padi6-/- female mice with wild-type males, no offspring could be obtained. In contrast, Padi6+/- females exhibited normal fertility, yielding an average of 7.3 offspring per litter. In addition, Padi6-/- males are also fertile. Twenty-eight wildtype oocytes (n = 30) survived parthenogenetic activation, with 85.71% (24/28) reaching the 2-cell stage, and 75% (18/24) subsequently forming blastocysts. In contrast, all Padi6-/- oocytes (n = 25) survived after activation, but only 20% (5/25) reached the 2-cell stage (P < 0.0001), with none progressing to blastocyst (P = 0.0039). Regarding zygote development, 82.14% (46/56) of wildtype zygotes and 98.14% (53/54) of Padi6+/-embryos reached the 2-cell stage, showing blastocyst rates of 95.65% (44/46) and 100% (53/53) respectively. For zygotes obtained from Padi6-/- females, 72.50% (87/120) of them progressed to the 2-cell stage (P = 0.1908, vs wildtype), but only 4.60% (4/87) reached blastocysts (P< 0.0001, vs wildtype), exhibiting the EDA phenotype. Following PNT to wildtype cytoplasm (n = 25), 23 reconstructed PNT zygotes were obtained. Of these, 91.30% (21/23) reached the 2-cell stage (P = 0.4924, vs wildtype), and 85,71% (18/21) developed to the blastocyst stage (P = 0.3151, vs wildtype), which is equivalent to the wildtype zygotes. Limitations, reasons for caution This study is limited by the small sample size of the PNT, which will still be extended. Next to the blastocyst rate, additional assessment methods such as multi-omics analyses will be employed to assess the efficiency and safety of PNT treatment. Wider implications of the findings This study provides further insight into the potential of the nuclear transfer technology for female-related infertility issues due to cytoplasmic incompetence. For couples experiencing recurrent EDA after ICSI due to inferior oocyte quality, PNT still offers them the possibility of having genetically related children. Trial registration number Not applicable

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-511 Euploidy potential of day 7 embryos and the relevance in Assisted Human Reproduction treatments

Abstract Study question Is it worth waiting embryos with late blastulation (Day 7) for preimplantation genetic analysis? Summary answer Embryo biopsy on Day 7 (D7) benefits cases of ovarian insufficiency and severe male factor with a euploidy rate of 15.1%. What is known already New technologies and improvements in IVF laboratories has allowed the extended cultivation of blastocysts to increase the pool of embryos per IVF cycle, resulting in greater chances, especially in cases of low blastulation rate, blastocysts with low quality or with few cells at Day 5(D5) and Day 6(D6). The variation in the time of the compaction stage depends on many factors, which may take more time, leading some embryos to reach the blastocyst stage only on D7 of development. These embryos have a lower euploidy rate, but there is embryonic competence and these embryos can be eligible for embryo transfer. Study design, size, duration Retrospective study that evaluated the chromosomal status (by Next Generation Screening - NGS) of 33 blastocysts biopsied on day 7 of development, between 2016 and 2023. Subsequently, it was compared the rate of euploidy and mosaicism with 295 blastocysts biopsied at D5 and D6, biopsied from January to October 2023. D7 embryos were originated from IVF cycles of couples with female age between 26 and 44 years, and males age between 29 and 56 years. Participants/materials, setting, methods D7 blastocysts from fresh and own oocytes fertilized by ICSI. The euploidy and mosaicism rates was compared, as well as the morphology of the ICM and trophectoderm (A-C; by Gardner Blastocyst Grade), the day which biopsy was performed and the respective infertility factors, in the groups: Group 1: D7 euploid and Group 2: D7 aneuploid. Subsequently, the rate of euploidy and mosaicism was compared between D7 embryos (Group 3) and D5/D6 embryos (Group 4). Main results and the role of chance There was no difference (p > 0.05) between maternal age for group D7 euploid (38.4 years) vs group D7 aneuploid (37.9 years) and group 3 (38.4 years) vs group 4 (38.2 years). Embryonic morphological evaluation of D7 embryos showed that ICM was classified as B (18.2%) or C (81.8%) and trophectoderm as A (6.1%), B (24.2%) or C (69.7%). Comparisons of ICM and trophectoderm classifications between euploid (group 1) and aneuploid (group 2) in D7 blastocysts showed no difference (p > 0.05). There was a predominance of 5CC embryos in both groups (Group 1: 40.0% vs Group 2: 42.4%), with no difference between them (p > 0.05). In most cases, the biopsy was performed on two days (D5+D7 – 39.3%; D6+D7 – 35.7%), while in 10.7% of cases only on Day 7, when the infertility factor was 100% ovarian insufficiency. In cases of biopsy within 3 days (D5+D6+D7), we observed that 100% of cases were related to severe male factor. When comparing the euploidy rate of D7 embryos – group 3 (15.1%) with that of D5/D6 – group 4 (34.2%), we observed a statistical difference (p < 0.05), while, for in mosaicism rates, D7 embryos represented 6.1% and D5/D6 embryos 9.1%, with no statistical difference (p > 0.05). Limitations, reasons for caution The retrospective nature of our study may lead to bias derived from clinical practices and variability in relation to patient age, embryonic quality and infertility factor. Wider implications of the findings Extended culture and biopsy on D7 benefits cases of ovarian insufficiency and severe male factor by optimizing the IVF cycle. Although the euploidy rate in D7 embryos is lower than D5/D6 embryos, 15% of D7 embryos are euploid, demonstrating that the slower development is not caused exclusively by aneuploidy Trial registration number NA

O-322 What is the number of IVF cycles needed to produce at least one euploid blastocyst in patients of all ages with varied AMH levels

Abstract Study question How many cycles are needed to get at least one blastocyst in patients of different AMH levels and varied ages? Summary answer Age and AMH determine the number of cycles, which ranges from 1 cycle in young patients up to 10 cycles in over-44-year-old patients. What is known already AMH secretion by ovarian granulosa cells initiates during the 36th week of gestation in pre-antral and antral follicles. This persists throughout a woman’s reproductive lifespan. AMH serves as a biomarker for assessing ovarian reserve and forecasting the ovarian response to stimulation. AMH is strongly associated with the quantity of antral follicles and the production of oocytes. AMH can be utilised to determine the initial dosage of FSH in an IVF cycle, as well as serving as a quantitative indicator of the number of oocytes produced. Study design, size, duration In this retrospective cohort study, data was collected from 16,417 ICSI cycles with Preimplantation Testing for Aneuploidy (PGT-A) conducted at three referral fertility centers from Jan 2019 to Dec 2023. We excluded cycles without serum AMH, endocrinopathies or recurrent pregnancy loss, males with total sperm count of less than 5 million and total motility less than 30%, and those with parental chromosomal abnormalities. Participants/materials, setting, methods Patients were stratified into three different categories of AMH (ng/ml), low (< 1.1 ng/mL), intermediate (>1.1 to < 3.0 ng/mL) and high (> 3.0 ng/mL). Each category was subdivided into 6 age sub-categories as follow (< 35), (35 to < 38), (38 to < 40), (40 to < 42), (42 to < 44), (> 44). All of the cycles underwent blastocyst stage biopsy and PGT-A utilising next generation sequencing (NGS). Main results and the role of chance Euploidy rate was statistically insignificant for all AMH levels in the same age group. However, euploid embryo numbers vary significantly. Euploid embryos numbers are shown in low, medium, and high AMH by age group. In the (<35) years was 1.9±1.5, 2.9±2.3, and 4.7±3.5, p < 0.0001. In the (35 to > 38) years was 1.3±0.8, 2.1±1.8, and 3.2±2.6, p < 0.0001. In the (38 to > 40) years was 0.9±0.8, 1.7±1.4, and 2.6±1.7, p < 0.0001. In the (40 to > 42) years was 0.6±0.5, 0.9±0.7, and 1.5±1.4, p < 0.0001. In the (42 to > 44) year was 0.26±0.2, 0.49±0.38, and 0.6±53, p < 0.0001. In the (≥44) years was 0.10±0.10, 0.23±0.21, and 0.40±0.37, p = 0.0170. We calculated the number of cycles needed to produce at least one grade BB or higher euploid blastocyst from the mean number of euploid embryos per cycle: The number of cycles is presented in low, medium, and high AMH. In the (<35) and (35 to > 38), 1 cycle is needed. In (38 to > 40) years, 2 cycles, 1 cycle and 1 cycle. In the (40-42) group 2 cycles, 2 cycles and 1 cycle. In the (42 to < 44) group 4 cycles, 3 cycles and 2 cycles. In the (≥44) group 10 cycles, 5 cycles and 3 cycles. Limitations, reasons for caution The retrospective nature of the study is the main limitation. Wider implications of the findings The study offers valuable insights for patient counselling regarding the required number of cycles to achieve euploid embryos, as well as for financial budgeting for both patients and insurance companies. Trial registration number Not applicable

P-136 Non-invasive PGT (niPGT) using spent culture medium: Relationship between time lapse embryo dynamics and accuracy of niPGT analysis

Abstract Study question Do differences in the morphological characteristics of embryos or the dynamics of the developmental process affect the results of niPGT analysis? Summary answer Embryos with blastomeres that were excluded at compaction showed reduced accuracy of niPGT analysis, but other measured morphological characteristics had no impact. What is known already The PGT-A test, is invasive and requires training to be competent at the technique, aswell as the need for specialized equipment and consumables. In recent years, non-invasive PGT-A (niPGT-A), which analyzes Cell-Free DNA released from cultured embryos, has been attracting attention. Regarding the analysis accuracy of niPGT-A, there are reports that it is equivalent to or even superior to conventional PGT-A. However, at present, it is not clear how methodological differences or embryo dynamics impact results. Study design, size, duration From August 2021 to February 2022, 284 pronuclear-stage frozen embryos (with consent for research use), were thawed and cultured. They were individually cultured for at least 40 hours after Day 4. For the 150 embryos that reached the blastocyst stage, culture medium and the blastocysts were collected and subjected to chromosome aneuploidy analysis by NGS. For the 138 blastocyst with a result, the concordance rate was calculated with that obtained from the culture medium samples. Participants/materials, setting, methods The embryos were individually cultured in a single step medium using a 10 μl droplet from Day 4. The entire culture period was recorded in a time-lapse incubator. The concordance rate for all chromosomes between culture medium sample and the blastocyst was compared to recorded embryo dynamics, e.g. presence or absence of cumulus cell attachment, sperm attachment, fragmentation, blastomeres that excluded at compaction, shrinkage, and hatching. Chi-square test was used to compare the concordance rates. Main results and the role of chance The overall concordance rate for all chromosomes between the culture medium and the blastocysts samples was 46.4%(64/138). In the blastocysts where there were cumulus cells attached or not it was 43.6% (17/39) vs 47.5% (47/99) respectively, cases where there was still a sperm attached or not it was 53.1% (17/32) vs 44.3% (47/106) resepctively and in blastocysts where there had been fragmentation or no fragmenattion it was 46.9%(38/81) vs 45.6％(26/57) respectively. In cases where shrinkage of the blastocyst had been observed vs no shrinkage the respective concordance rates were 44.9% (48/107) vs 51.6% (16/31), whilst in cases where the blastocyst had hatched or not it was 40.6% (13/32) vs 48.1% (51/106) respectively.For the blastocysts where the blastomeres had been excluded at compaction or not it was 35.6％(31/87) vs 64.7% (33/51) respectively.Significant differences were only identified in cases with blastomere exclusion or no exclusion at compaction (P < 0.01). Limitations, reasons for caution In this study, the culture was continued to day 6 regardless of morphology of the embryo on day 5, which differs from the usual clinical culture process. Wider implications of the findings These results are useful in that they may aid in recognising which morphological features of embryos may impact the results obtained from niPGT and hence its reliability for clinical application. The blastomeres that excluded at compaction are clearly important for improving the accuracy of the niPGT analysis in the future. Trial registration number None

P-012 Euploid and good-quality blastocysts using testicular sperm from men with high sperm DNA fragmentation (SDF), oligozoospermia, and previous failed ICSI with ejaculated sperm

Abstract Study question Are euploid rate and blastocyst quality improved when testicular sperm is used in patients with elevated SDF and poor semen parameters or/and previous ICSI failure? Summary answer The use of testicular spermatozoa in non-azoospermic males does preserve blastocyst ploidy while favoring the proportion of good-quality embryos available for the couple. What is known already Sperm extracted from the testicle has been proposed as a strategy to improve reproductive outcomes in couples with elevated DNA fragmentation in the ejaculate and with severe male infertility or previous ICSI failure. Different studies and meta-analyses show to increase pregnancy and live birth rates, and one of the reasons could be the lower genetic damage of spermatozoa retrieved from the testicle. However, there is little information on the effect on the quality of the embryos generated and their ploidy since the use of testicular sperm tends to have a higher rate of aneuploidy. Study design, size, duration This is a prospective, blinded, randomized, and controlled trial conducted in a private university-affiliated IVF center between October 2020 and June 2023. In each patient (n = 6), oocytes were randomly microinjected by sperm from the testis (T-ICSI) or ejaculate (E-ICSI) assigned in a 1:1 ratio. All males had an SDF>30% (measured by Sperm Chromatin dispersion (SCD) test), severe oligozoospermia, and/or a previous ICSI failure. Participants/materials, setting, methods Males underwent testicular sperm extraction (TESE) to obtain spermatozoa, which were frozen along with an ejaculated semen sample obtained previously on the same day. Women were <38 years old with good ovarian reserve (antral follicle count>10). SDF level was compared between both sources. The primary outcome was the euploid rate (among a total number of biopsied embryos). Secondary outcomes were fertilization rate, blastocyst rate, good-quality blastocyst rate, and ongoing pregnancy rate per embryo transfer (ET). Main results and the role of chance Mean ejaculated SDF was 44.83±16.6 (IC95% 27.5 -62.2), while mean women age was 31.8±2.9 (28.76-34.9). An average of 14.5±5.8 metaphase II oocytes were microinjected per patient. A total of 87 oocytes were randomized to the T-ICSI group (n = 45) or E-ICSI group (n = 42). There was a 14% difference between the SDF of testicular (44.7% (31.3-58.0) and ejaculated sperm (58.5% (37.6-79.4) (p = 0.2). The fertilization rate was slightly lower in the T-ICSI group (53.3% (38.1-68.1) compared to the E-ICSI group (61.9 (61.8-76.0) (p = 0.6). A total of 21 embryos reached the blastocyst stage, being the blastocyst arrival rate per correctly fertilized oocyte 41.7% (10/24) (22.8-63.1) for T-ICSI and 42.3% (11/26) (24.0-62.8) for E-ICSI (p = 1.0). Of them, a higher proportion of good-quality blastocysts was found in the T-ICSI (29.2% (13.4-51.6)) group than in the E-ICSI (19.2% (7.3-40.0)) group, although the difference was not statistically significant. In addition, the euploidy rate was comparable between the T-ICSI (60% (27.4-86.3)) and E-ICSI (63.6% (31.6-87.6)) groups (p = 1.0). Seven embryos were transferred in 6 ET, three in each study group. Only one of the ET in the E-ICSI group resulted in pregnancy (33.3% (1.8-87.5), while the three transfers in the T-ICSI group resulted in an ongoing pregnancy (100% (31.0-100)) (p = 1.0). Limitations, reasons for caution Despite being a randomized controlled trial, the number of subjects recruited is small; it is necessary to increase the number of cases to obtain results with greater statistical power. TESE is a technique that is not risk-free, so its performance should be evaluated in terms of its cost-benefit for patients. Wider implications of the findings The use of less genome-damaged testicular spermatozoa in non-azoospermic males seems to be a reliable strategy for couples with high ejaculated SDF and severe oligospermia and/or previous ICSI failures. However, although we have found an improvement, remains to be statistically confirmed with more cases. Trial registration number NCT04795440

P-548 An investigation into the mechanisms of active enhancers regulating the initial lineage differentiation in mammals

Abstract Study question What role do active enhancers play in determining the fate of early embryonic cells? Are there differences in active enhancers between mouse and human blastocysts? Summary answer Tissue-specific active enhancers in inner cell mass (ICM) and trophoblast (TE) are essential for blastocyst formation, regulating gene expression through interactive loops with promoters. What is known already At the two-cell stage of nuclear transfer embryos, the absence of interaction between Zscan4d promoter and distal active enhancer leads to reduced Zscan4d expression, resulting in lower developmental rates. Overexpressing the demethylase Kdm4d alleviates reprogramming-resistant H3K9me3 modification, restoring the structural loop between the Zscan4d promoter and active enhancer and enhancing developmental efficiency. These findings strongly suggest a close association between the formation of regulatory chromatin interactions in early embryonic development and histone modifications. Indicating active enhancers marked with H3K27ac may regulate differentiation gene expression through distal interactions in blastocyst. Study design, size, duration Analyze variations in histone H3K27ac modification, chromatin accessibility, higher-order structures, and transcriptomes during mouse embryo differentiation from 8-cell stage to ICM and TE formation. Identify tissue-specific active enhancers in ICM and TE, validate their gene expression regulation, explore their role in early differentiation-associated genes in mice. Compare and clarify similarities and differences in enhancer regulation mechanisms during early embryo development in humans and mice. Participants/materials, setting, methods The 8-cell, morula and blastocyst stage embryos were collected from the oviducts and uterus of super-ovulated female ICR mice mated with male mice. The clinical human blastocysts were collected and separated the ICM and TE. Low-input multi-omics sequencing, including Cut-Tag, ATAC-seq, Hi-C, and RNA-seq, was performed on the embryonic samples. After analyzing and identifying active enhancers, tissue-specific ones were selected and validated for their impact on gene expression via CRISPR-Cas9 knockout in embryos. Main results and the role of chance Inhibiting ep300 expression in mouse embryos through siRNA interference reduces blastocyst formation from 90% to approximately 40%. Introducing the small molecule inhibitor A485 at the 8-cell stage, blocking the active center of CBP/p300 enzymes, completely arrests embryos at the morula stage, preventing their development into blastocysts. Emphasizing the crucial role of correctly establishing active enhancers marked by H3K27ac modification in embryonic differentiation. Multi-omics data demonstrates high repeatability and quality. PCA clustering analysis reveals a clear differentiation path in the distribution of H3K27ac-modified histones, chromatin accessibility sites, and the transcriptome from the 8-cell stage to the blastocyst. Differences in H3K27ac histone modification distribution between the ICM and TE indicate the presence of tissue-specific active enhancers in blastocyst. The disparities in H3K27ac modification distribution between blastocyst ICM and TE primarily occur in distal gene regions rather than proximal ones. Moreover, these distal enhancers exhibit a positive correlation with the expression of lineage-specific genes, suggesting a probable regulatory role of distal enhancers in gene modulation during embryonic differentiation. Tissue-specific enhancer-promoter (EP) interaction loops emerge during embryonic differentiation, allowing direct interaction between tissue-specific active enhancers and promoters, thereby activating the expression of tissue-specific genes. Limitations, reasons for caution While DNA sequences with H3K27ac modification are currently considered markers for active enhancers, H3K4me modification is another histone marker for enhancers. However, enhancers associated with H3K4me modification may not necessarily be in an activated state. Future research can simultaneously incorporate appropriate detection methods for H3K4me modification. Wider implications of the findings After elucidating the mechanisms through which active enhancers regulate mouse differentiation, comparing the differences in active enhancers during the first lineage differentiation process between mice and humans can help identify factors that enhance human blastocyst quality. This can potentially improve clinical outcomes in assisted reproduction. Trial registration number not applicable

O-004 Oocyte image assessment by novel artificial intelligence (AI) model predicts blastocyst PGT-A results, outperforming age as a predictor

Abstract Study question Can a non-invasive, image analysis artificial intelligence (AI) model be built to predict the likelihood that a mature oocyte will develop into a euploid blastocyst? Summary answer A novel AI model was developed to assess images of mature oocytes, predicting chromosomal ploidy status of the developed blastocyst with an AUC of 0.71. What is known already The genetic status of blastocysts greatly impacts implantation and cycle success; therefore, PGT-A has become widely adopted as routine to aid in blastocyst selection for transfer and aims to decrease the time to pregnancy. Most chromosomal abnormalities arising in a blastocyst embryo are of maternal meiotic origin–indicating that the oocyte may have valuable information to be harnessed at the earliest stage in the process. Gaining insights into the potential genetic status of a blastocyst at the oocyte stage in a non-invasive manner would provide critical information to the overall fertility cycle. Study design, size, duration This study included a labeled dataset—9,260 images of mature denuded oocytes, immediately post-ICSI—from 3 clinics (Canada, United States (US), Spain) with euploid or aneuploid trophectoderm biopsy results (excluding mosaic/inconclusive embryos, n = 980). The labeled dataset was split into training (60%), validation (20%), and test (20%). An unlabeled dataset was also included—34,709 oocyte images (2 clinics; Canada, US)—and utilized in training. An external dataset (n = 473) from a separate US clinic was assessed for model generalization. Participants/materials, setting, methods Fully-supervised and semi-supervised machine learning models were experimented to predict blastocyst PGT-A from oocyte images. The labeled data were utilized in both methods. The semi-supervised method integrates unlabeled data using the well-known Mixmatch algorithm—learning important features from augmented images (with pseudo labels) that were then utilized in the supervised phase to predict outcomes. Age was incorporated into the models, therefore, a model only using age to predict ploidy was developed to act as a comparison. Main results and the role of chance Following various experiments, the fully-supervised model displayed an AUC 0.61, sensitivity 0.41, specificity 0.76, and accuracy 0.62 on the test set. The semi-supervised model displayed a performance of AUC 0.71, sensitivity 0.63, specificity 0.67, and accuracy 0.65–notably higher AUC and balanced performance on the positive/negative classes. The semi-supervised method benefits from both labeled and augmented unlabeled data for enhanced learning efficacy. The model utilizing age only to predict ploidy was extremely imbalanced in performance on the test set with AUC 0.63, sensitivity 1, specificity 0, and accuracy 0.48, confirming the importance of image features. Performance of the semi-supervised model was assessed by clinic. On Clinic 1 (n = 364; 40% euploid) it displayed AUC 0.61, sensitivity 0.60, specificity 0.56, and accuracy 0.58; on Clinic 2 (n = 675; 52% euploid) it displayed AUC 0.64, sensitivity 0.56, specificity 0.65, and accuracy of 0.60; on Clinic 3 (n = 466; 50% euploid) it displayed significantly better performance with AUC 0.85, sensitivity 0.76, specificity 0.79, and accuracy 0.77–warranting further investigation into model generalizability. On the external dataset (n = 473, 58% euploid), the semi-supervised model displayed improved performance (AUC 0.62, sensitivity 0.61, specificity 0.61, accuracy 0.60) in comparison to the age only model (AUC 0.59, sensitivity 1, specificity 0, accuracy 0.58). Limitations, reasons for caution This dataset includes oocytes that developed into blastocysts and underwent PGT-A. Datasets including oocytes that did not reach the blastocyst stage must also be assessed to validate the model’s clinical utility. Despite utilizing diverse data, advancing the generalizability of the model requires additional investigations to ensure balanced performance across clinics. Wider implications of the findings Utilizing images of mature oocytes, an AI model predicts the ploidy status of resulting blastocysts, providing a powerful tool for assessing oocyte quality and genetic integrity. These preliminary findings are promising for those undergoing oocyte cryopreservation by striving to elucidate genetic potential at the earliest phase of the IVF process. Trial registration number Not applicable.

P-248 Egg vitrification related to female age: survival and laboratory outcome of 1233 vitrified-thawed oocytes in non-medical autologous use

Abstract Study question Is the female age at retrieval a determinant factor for the viability and potential of the thawed eggs? Summary answer In patients over 40, thawed oocytes display more abnormal fertilization, develop less in extended culture and yield less top quality blastocyst. What is known already Oocyte cryopreservation is a well-established procedure especially since the advent of the vitrification technique. The successful outcomes achieved with vitrification encourage its application in fertility preservation (FP) for medical and non-medical reasons. Besides, the age-related decline in women’s fertility and the oocyte quality negatively affect the outcome of assisted reproduction. Some studies have reported that age at oocyte retrieval strongly influences reproductive prognosis after FP. However, the appropriate age for non-medical FP is still debated. Although oocyte freezing at a younger age is recommended, there should be a balance between a favourable outcome and the cost-effectiveness of the strategy. Study design, size, duration Retrospective study on 166 patients with frozen-thawed oocytes who underwent ICSI during 2021-2023 in a single hospital. Oocyte vitrification had different purposes: banking for poor responders, to limit the number of embryos, and to solve unexpected situations where partner’s semen sample was unavailable. Patients were divided into three groups according to their age at retrieval (Group A ≤ 37 n = 77, Group B 37-40 n = 44, Group C ≥ 40 n = 45). Post-warming survival, fertilization, and embryological data were studied. Participants/materials, setting, methods 1233 oocytes were thawed. The intact oocytes were microinjected 1-2 hours after warming. Embryos were cultured in Sage 1-StepTM to blastocyst stage, and rated on daily basis. Embryo transfer was performed on day 3 or day 5 and remaining blastocysts were frozen on Day 5 or Day 6. Post warming survival, lysis after ICSI, fertilization, embryo development, blastulation rate (BR) and top blastulation rate (TBR), and morphokinetic features were studied in the 3 groups. Main results and the role of chance 84.6% of the 1233 oocytes (vitrified in a high-security closed system) survived the warming process. 1043 eggs were injected and the post-ICSI lysis rate was 11.2%. No significant differences were seen in thawing survival rate in groups A, B and C (85.6% vs. 83.5% vs. 83.8%, p=0.635) nor in the post-ICSI lysis rate (13.1% vs. 7.4% vs. 11.2%, p=0.072). 2PN fertilization rate was similar between the groups (67.8% vs. 61.3% vs. 67.7%, p=0.178) as well as the total number of embryos (303 vs 146 vs 188 p=0.605). However, triploid fertilization rate (3PN) statistically grew with age (2.8% vs. 5.8% vs. 6.5%, p=0.034). Significant differences were also reported in the global abnormal fertilisation rate (1PN+3PN) (6.4% vs 10.3% vs. 13.3%, p= 0.005). BR and TBR were statistically lower after the age of 40 (BR: 61.5% vs. 62.7% vs 40% p<0.001, TBR: 43.3% vs. 47.9% vs. 24.3% p<0.001). Morphokinetic features in the 3 groups were studied (abnormal cleavage pattern, tSB and tB). Overall results were similar between group A and group B but oocyte potential decreases drastically from the age of 40. Limitations, reasons for caution The major limitation of the study is its retrospective nature and the relative small size of each group. As most of patients were transferred during 2023, the pregnancies have not delivered yet so the live birth rate is not mentioned. Wider implications of the findings Studies have reported the impact of age at oocyte retrieval in case of medical FP in cancer and endometriosis. Here we report a negative effect on outcome after the age of 40 in non-medical FP. Patients should be informed that FP is not a certification of a successful outcome. Trial registration number not applicable

P-258 Not too big, not too small: euploid blastocysts that lead to live birth have an optimal surface area, diameter and optimal pace of development

Abstract Study question Does embryo diameter and surface area at tsB and tB as determined by AI predict live birth (LB) potential amongst euploid blastocysts? Summary answer Euploid blastocysts with optimal diameter (tsB:120-129; tB:136-140μm) and surface area (tsB:11771-13577; tB:15000-15643μm2) are more likely to lead to LB than larger or smaller blastocysts. What is known already Current embryology consensus favours larger blastocysts based on Gardner grading, associating expansion with higher quality. Human assessment of embryos is subjective, and it is unknown whether this dogma holds when assessing pre-expansion blastocyst stages. The impact of blastocyst surface area and diameter at tSB and tB; and pace of development on LB remains unclear. This may help clarify whether large blastocysts are associated with accelerated embryo growth and abnormal metabolic processes. The study’s objective was to use AI to quantitatively determine the optimal embryo surface area and diameter of euploid blastocysts that lead to live birth. Study design, size, duration Retrospective study including 97 time-lapse videos of euploid embryos with known live birth outcomes from January to December 2021 Participants/materials, setting, methods Embryo diameter and surface area were measured at start of blastulation (tSB) and at time of blastulation (tB) by an AI model (CHLOE-EQ, Fairtility Ltd). Live birth (LB) outcomes were manually annotated by embryologists. An Optimal embryo surface area and diameter were determined by the highest live birth rate and the suboptimal range was determine by a decreased Live birth rate. A comparison between the optimal and suboptimal live birth rate was measured using chi-square. Main results and the role of chance Moderately sized embryos were found to have higher LB outcomes. At tSB, optimal surface area was 11771-13577 um2, while optimal embryo diameter was 120-129μm. LB rate for optimal embryo surface area was higher than for suboptimal [Optimal 80% (20/25) vs Suboptimal 55% (40/72), p < 0.001]. In addition, optimal embryo diameter had higher LB rates than embryos with suboptimal diameter [80% (16/20) vs 52% (31/60), p < 0.05). At tB, optimal embryo area was 15000-15643μm2 and optimal embryo diameter 136-140μm. LB rate for optimal embryo surface area was higher than for suboptimal 82% (14/17) vs 49% (36/73), p < 0.05]. Optimal embryo diameter had higher LB rates than embryos with suboptimal diameter (77% 20/26 vs 47% 30/64, p < 0.05). Embryos with an optimal surface area and diameter were slower than bigger suboptimal embryos: t2 (26±2 vs 25±2), t8 (59±8 vs 53±6), t9 + (71±6 vs 67±6), tM (82±7 vs 77±8), tSB (99±6 vs 93±6), tB (107±7 vs 100±6), tEB (137±2 vs 122±12); t8-t5 (12±10 vs 6±5), t8-t2 (33±8 vs 28±5), tEB-tSB (34±2 vs 23±9), p < 0.05. Optimal vs small suboptimal embryos were faster in t9 + (71±6 vs 76±4), tEB (121±11 vs 137±2, tEB-tSB (20±10 vs 34±1). But were slower in t8-t5 (12±10 vs 5±6), t8-t2 (33±8 vs 28±5), p < 0.05. Limitations, reasons for caution This is the first time a quantitative embryo surface area and diameter has been studied in its correlation with live birth. Generalisation requires the replication across diverse centers with varying demographic profiles to validate findings and establish broader applicability within the context of assisted reproductive technologies. Wider implications of the findings This data-driven approach can support the embryo selection process, correlating quantitative parameters with live birth outcomes, thereby refining the precision of the decision-making in IVF. This level of quantitative data of embryo surface area and diameter is only accessible through AI tools. Trial registration number IRB-007CITY-FL21.08.2023

P-504 Examining the success rates following a second embryo biopsy for inconclusive results in preimplantation genetic testing for monogenic diseases (PGT-M): a matched case-control study

Abstract Study question Can an embryo re-biopsy for a previous inconclusive result influence clinical pregnancy compared to a successful PGT-M diagnosis from the first biopsy? Summary answer The embryo re-biopsy intervention for PGT-M seems to reduce the implantation potential of a blastocyst. What is known already PGT-M allows embryo genetic profiling prior to transfer in couples with hereditary genetic disorders, as part of in vitro fertilization process. The need of a trophectoderm biopsy for the retrieval of a fragment of about 8-10 blastomeres is generally sufficient for optimal DNA amplification and molecular analysis. However, a proportion of embryos (approximately 10-12%) fails to be properly diagnosed after PGT-M testing due to inconclusive results. Therefore, as an attempt to obtain a genetic diagnosis, the embryo biopsy can be repeated on the same embryos although retesting involves a second round of biopsy and a second round of vitrification. Study design, size, duration Twenty-seven embryos from women undergoing PGT-M at an infertility center and transferred after two biopsies for genetic analysis were matched for age, fertility status, and study period, to embryos transferred after a conclusive PGT-M result after the first biopsy. The main outcome was the clinical pregnancy rate following embryo transfers in cases in whom embryos had a single biopsy, and when a re-biopsy was performed. The secondary outcome was the live birth rate. Participants/materials, setting, methods Ovarian stimulation, oocyte collection and fertilization were performed according to standard protocols. Embryos were cultured to blastocyst stage in groups of up to five, using one-step media. Blastocysts were biopsied on day 5, 6, or 7, employing either the pulling or flicking method based on their appearance. Biopsied cells were stored at -20 °C for genetic analysis. Vitrification was performed using Kitazato protocol. If results were inconclusive, a second biopsy was performed in surviving blastocysts. Main results and the role of chance The median [interquartile range] number of retrieved and injected oocytes was similar between the two groups, as well as fertilization rate and number of blastocysts obtained. No difference between the two groups was also observed pertaining to the day of embryo culture in which the first biopsy was performed. Conversely, the number of non-diagnosticated and transferable blastocysts was lower in patients whose blastocysts were re-biopsied. In 90% of the re-biopsies an informative result was obtained. Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst. Limitations, reasons for caution The matching design, which aligns women’s age and fertility status in the two groups, allowed the assessment of pregnancy rates while avoiding potential confounding factors associated with retrospective analysis. Given that it is the first study with this design, a larger sample size is necessary to further elucidate these findings. Wider implications of the findings Despite the limited sample size, these results are of extreme clinical relevance. They offer valuable insights for guiding decisions about embryos with unclear results, contributing to the improvement of strategies for selecting those with the highest probability of pregnancy. Trial registration number not applicable

P-155 Reproductive Potential of Metaphase-I (M-I) Intra Cytoplasmic Sperm Injection (ICSI)

Abstract Study question To evaluate the reproductive potential of performing ICSI with metaphase-I (M-I) oocytes derived from In Vitro Fertilization (IVF)-ICSI cycles Summary answer M-I ICSI oocytes can fertilize and develop into blastocysts, but with low rates and limited efficiency in producing more embryos, highlighting challenges in reproductive outcomes. What is known already ICSI for initially immature oocytes undergoing in vitro maturation in IVF cycles is a topic of controversy with varying practices. While it has the potential to increase usable embryos, it may be time-intensive and yield uncertain results. Assessing the maturation status of M-I oocytes multiple times on collection day is challenging. If viable embryos can be obtained after ICSI with M-I oocytes, it simplifies workload management in busy embryology labs for patients with a limited number of mature oocytes. Despite a few reported cases of healthy live births resulting from ICSI with M-I oocytes, their reproductive potential remains inadequately explored. Study design, size, duration The study included IVF cycles (n = 929) with both immature and mature oocytes from Jan, 2022-Dec, 2023. Fertilization and blastocyst were assessed based on oocyte maturity. Participants/materials, setting, methods The study included IVF-ICSI cycles obtaining at least one immature (Germinal vesicle (GV), M-I) and one mature (M-II) oocyte at oocyte denudation. Analyzing 9,793 inseminated oocytes from 929 women (mean age: 34.0), we divided them into groups: M-II (Group 1: n = 7678), M-I at ICSI (Group 2; n = 835), M-IàM-II at ICSI (Group 3; n = 643), M-IàM-II on day 1 (Group 4; n = 129), and GVàM-II on day 1 (Group 5: n = 508). Fertilization and blastocyst development were then examined. Main results and the role of chance Overall, embryos originating from initially immature oocytes exhibited lower cleavage and blastulation rates compared to those from initially mature oocytes (P < 0.05). In Group 2, where ICSI was performed with M-I oocytes, fertilization and blastocyst rates were 23.6% and 16.1%, respectively. However, these rates were significantly lower than in other groups (P < 0.05). In Group 3, where ICSI was performed with M-II oocytes matured from M-I within 2 hours after oocyte denudation, the fertilization rate per oocyte (55.2%) was lower than in Group 4 (75.7%) (P < 0.05). However, the blastocyst rate per 2PN embryo was higher (30.7%) in Group 3 than that of Group 4 (18.0%) (P < 0.05). The blastocyst rate from the inseminated oocytes was directly associated with oocyte maturation: 29.3% (2250/7678) for in vivo M-II, 14.9% (96/643) for Group 3, 11.6% (15/129) for Group 4, 8.9% (45/508) for Group 5, and 2.9% (24/835) for M-I (Group 2). There were few pregnancies resulting from fresh transfers with only embryos produced from day 1 M-II oocytes (3/18=16.7%). While there were pregnancies after transferring only embryos generated from M-II matured from M-I on day 0 (4/10=40%), no patients transferred only embryos produced from M-I oocytes. Limitations, reasons for caution Despite the study possessing a substantial sample size, limited availability of embryos for fresh transfers arises from the deprioritization of those originating from immature oocytes. Consequently, it is crucial to conduct further research involving frozen embryo transfers and chromosome analysis to determine the efficacy of M-I ICSI. Wider implications of the findings Immature M-I oocytes exhibit a blastocyst formation rate of less than 3%. Consequently, these immature M-I oocytes demonstrate very limited developmental potential in progressing to the blastocyst stage, and ICSI for the M-I stage should be re-evaluated. Trial registration number Bnoon20240101

Vitamin D levels in couples undergoing in vitro fertilization treatment: lack of association with embryo quality or pregnancy rates

ObjectiveTo investigate the potential impact of vitamin D serum levels of couples going through in vitro fertilization in terms of embryo quality and pregnancy rates. DesignRetrospective cohort study SettingFertipraxis, private human reproduction center on Rio de Janeiro, Brazil. Subjects267 couples who underwent intracytoplasmic sperm injection between January 2017 and March 2019. ExposureThe couples were categorized into four groups based on 25OH vitamin D levels measured at the beginning of the stimulation protocol: Group 1 with levels ≥ 30 ng/mL for both women and men; Group 2 with levels < 30 ng/mL for both; Group 3 with women < 30 ng/mL and men ≥ 30 ng/mL; and Group 4 with women ≥ 30 ng/mL and men < 30 ng/mL. Main Outcome MeasuresWe consider quantity and quality of embryos during the cleavage and blastocyst stages as primary outcomes. Correspondingly, clinical pregnancy rate was regarded as a secondary outcome. ResultsOur findings revealed no significant correlations between the studied VD groups and the evaluated outcomes. This includes quantity and quality of embryos during the cleavage and blastocyst stages, as well as clinical pregnancy rate. Primary analysis revealed a small but statistically significant difference in the duration of controlled ovarian stimulation between group 1 and group 2 (p=0.035; CI=0.07 – 3.04) and between group 1 and group 3 (p=0.040; CI=0.05 – 3.23). ConclusionThe present study found no correlation between the studied VD levels and quantity and quality of cleavage or blastocyst stage embryos, nor did it show any impact on clinical pregnancy rates. Further well designed, prospective studies are warranted to determine whether and how vitamin D affects reproductive outcomes.

Utilisation of Oocyte Diameter as a Non-invasive Indicator of Oocyte and Embryo Quality: Investigating Protein Kinase B Protein Expression in Cumulus–Granulosa Cells and Embryo Morphokinetics

Background: Oocyte diameter is crucial for initiating meiosis and supporting the maturation capability, a prerequisite for successful fertilisation and high-quality embryo development. While numerous studies have explored the correlation between oocyte diameter and embryo development capacity in animal models, limited research appears concerning human oocytes. Aim: This study aims to ascertain whether oocyte diameter can serve as a reliable predictor of oocyte and embryo quality, achieved through its correlation with protein kinase B (Akt) protein levels on cumulus–granulosa cells and blastocyst morphokinetics. Setting and Design: Prospective cohort study included cumulus–granulosa cells collected from 228 mature oocytes of 32 infertile women undergoing in vitro fertilisation treatment. The diameter of the oocytes was measured using the Image J software, and the samples were categorised into three groups based on their diameter: &lt;105.95 µm, 105.96–118.96 µm and &gt;118.97 µm. Quantification of pAkt and Akt protein total of cumulus–granulosa cells exploited the ELISA method. Embryo morphokinetics and anomaly development events were observed at the blastocyst stage to evaluate embryo quality relative to oocyte diameter. Statistical Analysis Used: The Kruskal–Wallis test and Chi-square test were used. P &lt;0.05 was considered statistically significant. Results: The study revealed no notable difference in pAkt and Akt protein total in cumulus–granulosa cells among the three groups (P &gt; 0.05). Likewise, there were no significant differences in morphokinetics and anomaly development except for the S2 parameter and reverse cleavage. Conclusion: Reflecting on the molecular profile including pAkt, total Akt protein, morphokinetics and anomaly development, it was observed that oocyte diameter does not correlate with its competency and the quality of the resulting embryo. In addition, oocytes with a diameter below 105.95 µm may have an equal potential to develop into top-quality embryos.

Obtaining cattle embryos using ready-made media of the scientific and production enterprise "Paneco"

The development and widespread practical application of in vitro embryo production technology will make it possible to replenish the herd population in a short time, preserve and replicate valuable genotypes, as well as create new genotypes using the method of genome editing. Livestock breeding is tasked with providing agriculture with a high–value herd in a fairly short time, while farmers are charged with replenishing the herd in a short time, which is possible only with the active introduction of assisted reproductive technologies, such as in vitro embryo production (IVP technology). The use of domestically produced cultural media in technology will ensure greater accessibility, reduced economic costs and the rapid development of breeding workin Russia. The purpose of the work was to evaluate the effectiveness of the IVP technology when using culture media from the domestic manufacturer SPE “PanEco” (Russia) to obtain cattle embryos in vitro. Our own protocol for obtaining cattle embryos was based on the protocols of culture media manufacturers SPE “PanEco” and IVF Bioscience (UK). Thus, for the first time using the line of media from the domestic manufacturer “PanEco” to obtain cattle embryos in vitro under incubation conditions at temperatures from plus 38.6 ◦C to plus 38.9 ◦C, CO2 content 6.2-6.8 % and uncontrolled humidity, on 6-8 days after a period of time from 140 to 182 hours from the moment of fertilization, 30.4 % of embryos were obtained at the blastocyst stage in relation to the total number of fertilized oocytes that reached the stage of 2 – 4 blastomeres.

CRISPR-mediated editing of β-lactoglobulin (BLG) gene in buffalo

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (−/−) heterozygous, bi-allelic (−/−) homozygous, and mono-allelic (−/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (−/−) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

Early Embryonic Development in Agriculturally Important Species.

The fertilization of oocytes ovulated by pigs, sheep, cows, and horses is not considered a limiting factor in successful establishment of pregnancy. Pig, sheep, and cow embryos undergo cleavage to the blastocyst stage, hatch from the zona pellucida, and undergo central-type implantation. Hatched blastocysts of pigs, sheep, and cows transition from tubular to long filamentous forms to establish surface area for exchange of nutrients and gases with the uterus. The equine blastocyst, surrounded by external membranes, does not elongate but migrates throughout the uterine lumen before attaching to the uterine luminal epithelium (LE) to begin implantation. Pregnancy recognition signaling in pigs requires the trophectoderm to express interleukin 1 beta, estrogens, prostaglandin E2, and interferon gamma. Sheep and cow conceptus trophectoderm expresses interferon tau that induces interferon regulatory factor 2 that inhibits transcription of estrogen and oxytocin receptors by uterine epithelia. This prevents oxytocin-induced luteolytic pulses of prostaglandin F2-alpha from regressing the corpora lutea, as well as ensuring the secretion of progesterone required for maintenance of pregnancy. The pregnancy recognition signal produced by equine blastocysts is not known. Implantation in these species requires interactions between extracellular matrix (ECM) proteins and integrins as the conceptus undergoes apposition and firm attachment to the uterine LE. This review provides details with respect to early embryonic development and the transition from spherical to filamentous conceptuses in pigs, sheep, and cows, as well as pre-implantation development of equine blastocysts and implantation of the conceptuses.