Is the size of TE biopsy specimens related to the chromosomal status of the blastocysts in PGT-A? Smaller biopsy specimens are associated with higher proportion and altered composition of mosaic blastocysts in PGT-A. Concordance between TE biopsy and inner cell mass or whole embryos is influenced by mosaicism type, degree, biopsy location, number of cells biopsied, and cell loading. Particularly, mosaic embryos and those with structural rearrangements have a higher level of inconsistency. Recent studies have demonstrated the potential reclassification of chaotic embryos as euploid, leading to successful live births. This study aimed to investigate the potential influence of TE biopsy specimen size on the chromosomal status of blastocysts undergoing PGT-A. This retrospective study, conducted from October, 2023, to January, 2024, included 246 blastocysts from patients undergoing TE biopsy and PGT-A at the blastocyst stage on days 5, 6, or 7. Next-generation sequencing (NGS) was used to obtain PGT-A results, categorizing blastocysts into euploid, aneuploid, and mosaic based on their genetic composition. The biopsy specimen area was measured using the Image J program. Mosaic blastocysts were categorized into either a chaotic mosaic group or another group, which included blastocysts with fewer than three chromosomal aneuploidies and segmental mosaicism. Further analysis was conducted by classifying blastocysts into high-level mosaic (>30% aneuploidy) and low-level mosaic (≤30% aneuploidy) groups. Group differences were analyzed using t-tests and chi-square, with a significance threshold set at p<0.05. In the analysis of expanded mosaic blastocysts, the chaotic mosaic group had smaller biopsy specimens than those in the comparison group (p<0.05). The high-level mosaic group also had significantly smaller biopsy specimens than the low-level mosaic group (p<0.05). When categorizing the biopsied sample size into two groups—below 1350 µM2 (n = 98) and above 1500 µM2 (n = 97)—the rates of mosaic embryos were 37% (36/98) and 30% (29/97), respectively, showing a numerical difference without statistical significance. These findings suggest a correlation between the size of biopsy specimens and the chromosomal status of mosaic blastocysts, particularly indicating that a smaller sample size is associated with an elevated chance of mosaic embryos showing chaotic or high-level mosaic characteristics. The biopsy specimens were photographed, and their areas were measured using the Image J program, lacking a comprehensive representation of the three-dimensional sample morphology. This constraint could potentially affect the accuracy and in-depth understanding of the spatial characteristics of the biopsy specimens. The study emphasizes the crucial role of optimizing biopsy specimen size in PGT-A. The identified correlation with the chromosomal status in mosaic embryos, especially the association with chaotic or high-level mosaic embryo outcomes in smaller samples, highlights the need for careful size considerations. not applicable

This study aimed to investigate the influence of cytoplasmic strings (CSs) on the development and quality of the embryo. Blastocysts with CSs exhibited accelerated development from t7 to the tB stage and a higher rate of good-quality on day 5. Time-lapse (TL) culture during in vitro fertilization is an established method for evaluating the morphological and morphokinetic features of embryos. CSs, identified through TL culture, are among the characteristics of developing embryos. Although the impact of CSs on embryo quality is recognized, their specific functions in human embryos remain unclear. This retrospective study was conducted from January 2022 to December 2022. A total of 1050 injected oocytes were cultured in the EmbryoScope® (Vitrolife),with images captured every 20 minutes for 5-6 days. CSs are observed the straight lines connecting the inner cell mass to the mural trophectoderm. On days 5-6, the presence of CSs was assessed in 610 blastocysts. The evaluation of differences in development rate and quality related to CSs was conducted using morphological or morphokinetic parameters: the time to second polar body extrusion (tPB2), time to pronuclei appearance and fading (tPNa, tPNf), time to reach 2–8 cells (t2, t3, t4, t5, t6, t7, t8, t9), time to the start of blastulation and blastocyst formation (tSB, tB). Statistical analysis employed Student’s t-test for developmental time and the Chi-square test for quality comparisons. Blastocysts were categorized into Group 1 if they had CSs and Group 2 if they lacked CSs. No differences in developmental rates between the groups were observed until the t6 stage. However, from t7, the pre-cleavage stage, to the tB stage, the Group 1 exhibited a significantly faster developmental compared to the Group 2 (t7: 56.97h vs. 58.82h, p = 0.013; t8: 61.68h vs. 63.39h, p = 0.049; t9: 72.05h vs. 74.63h, p = 0.001; tSC: 82.32h vs. 85.56h, p < 0.001; tM: 92.60h vs. 95.48h, p < 0.001; tSB: 105.11h vs. 107.57h, p = 0.004 and tB: 114.73h vs.117.97h, p = 0.001). In examining of good-quality embryos (GQE) on day 5(≥ grade BB), there was a notable association in Group 1, demonstrating 71.6% compared to 28.4% in the Group 2 (p < 0.001).These findings indicate a positive correlation between the presence of CSs and embryo developmental progress and quality. The determination of CSs relies on visual evaluation, containing the potential for subjective bias. Future research requires an automated time course monitoring system for more objective methods for identifying CSs to improve accuracy. This research has advanced our comprehension of the impact of CSs on embryonic development. Moreover, the evaluation of CSs features suggests the possibility of their introduction as novel morphological parameter in future embryonic grading systems. Not Applicable

Abstract Study question Do high-level mosaic embryos exhibit differences in developmental speed compared to euploid or low-level mosaic embryos? Summary answer High-level mosaic embryos exhibit slower development than euploid and low-level mosaic embryos during the blastulation and blastocyst formation stages. What is known already Time-lapse technology (TLT) has facilitated the real-time observation of human embryo development without disrupting culture conditions, generating a wealth of morphokinetic data linked to outcomes such as implantation potential, blastocyst development, and ploidy status. However, the developmental patterns of mosaic embryos based on their aneuploidy level using TLT remain not well understood. Consequently, we aimed to assess the clinical significance of morphokinetic parameters in human embryo development, with a specific focus on embryos categorized by their mosaic status. Study design, size, duration This retrospective study examined 385 blastocysts undergoing preimplantation genetic testing for aneuploidy (PGT-A) between January and December 2022. Out of the total, 191 expansion blastocysts underwent PGT-A biopsy on day 5. The PGT-A results, which categorized embryos into euploid, aneuploid, and mosaic based on their genetic composition, were determined through next-generation sequencing (NGS) by an analysis company. Mosaic embryos were further categorized into two groups: high-level mosaicism (>30% aneuploidy cells) and low-level mosaicism (≤30%). Participants/materials, setting, methods Differences in development rates were assessed through the examination of morphological and morphokinetic parameters. These parameters included the time to second polar body extrusion (tPB2), time to pronuclei appearance and fading (tPNa, tPNf), time to reach 2–8 cells (t2, t3, t4, t5, t6, t7, t8, t9), and time to the onset of blastulation and blastocyst formation (tSB, tB). Statistical analyses, incorporating ANOVA and post-hoc tests, were employed to compare developmental timings across different groups. Main results and the role of chance When categorizing mosaic embryos based on their level, high-level mosaic embryos showed a significantly slower developmental rate than low-level mosaic embryos, evident at both the blastulation stage (tsB, high-level vs. low-level: 101.80h vs. 97.3h, p=0.003) and blastocyst stage (tB, high-level vs. low-level: 109.98h vs. 105.92h, p=0.005). In comparison to euploid embryos, low-level mosaic embryos demonstrated comparable outcomes. Conversely, high-level embryos exhibited a significant difference in both blastulation (tsB, euploidy vs. high-level: 98.85h vs. 101.80h, p=0.026) and blastocyst (tB, euploidy vs. high-level: 106.73h vs. 109.98h, p=0.011) stages. These findings suggested that the mosaic level of embryos might influence developmental kinetics, proposing this approach as an additional tool for the selection of mosaic embryos in the context of embryo transfer. Limitations, reasons for caution This study focused on morphokinetic parameters captured using time-lapse imaging, which may not fully represent the complex cellular dynamics within embryos. Furthermore, the categorization of mosaic embryos into high-level and low-level groups based on the percentage of mosaicism (>30% and ≤30%, respectively) might not capture all embryo developmental potential. Wider implications of the findings The findings suggest valuable predictive potential in assessing the development of aneuploid and mosaic embryos. Further research into the development of euploid, aneuploid, and mosaic embryos, as well as high-level and low-level mosaic embryos, could provide valuable insights for ART practices. This could significantly impact ART decision-making and patient outcomes. Trial registration number not applicable

Abstract Study question Does putrescine, a type of polyamine, affect embryonic development in patients with recurrent implantation failure (RIF)? Summary answer Putrescine supplementation on cumulus-oocyte complexes (COCs) was associated with better embryologic development from fertilization to blastulation in patients with RIF. What is known already Putrescine, a type of polyamine, plays a role in cell growth and proliferation. Several animal studies suggest the potential impacts of putrescine on embryo development, but its effects on human IVF have yet to be extensively studied. Study design, size, duration This retrospective study analyzed 442 patients who have undergone IVF cycles three or more times from January to December 2023. The average IVF cycle number of RIF patients was 7.4 times. A total of 1356 oocytes from the putrescine group and 1391 oocytes from the control group underwent aspiration and ICSI. Further analysis was conducted by classifying RIF patients into two groups: patients under 40 years of age and patients over 40 years of age. Participants/materials, setting, methods COCs were cultured for 4 hours in a culture medium supplemented with 0.5mM putrescine. 2 hours after oocyte collection, cumulus cells were denuded, and ICSI was performed. Embryonic development was evaluated based on fertilized oocytes (2PN), good quality embryos (GQE) on day 3, and blastulation rates on day 5/6. Group differences were analyzed using t-tests and chi-square tests (p < 0.05). Main results and the role of chance When maternal age was ≥40, the fertilization rate in the putrescine group was significantly higher than that in the control group (86.6% vs. 80.5%, p=0.002). The putrescine group had a numerically higher proportion of embryos with top-grade morphology on day 3 (47.8%, compared with 53.3% for control; p=0.057), without statistical significance. Additionally, the rate of blastulation on day 5 indicated a significant difference between the two groups (14.5% vs. 6.1%, p=0.0001). Further analysis was conducted in younger patients (<40 years old), and it also showed a significantly higher fertilization rate (89.7% vs. 71.1 p=0.0001) and blastocyst development rates on the day 5 in the putrescine group (22.3% vs. 11.1%, p=0.0001), respectively. This study suggests that putrescine supplementation can be utilized to improve embryo quality and facilitate blastocyst formation in IVF procedures for patients with RIF, irrespective of age. Limitations, reasons for caution Putrescine supplementation in IVF showed improved fertilization and blastulation rates, but caution is needed due to the small sample size used. Therefore, a larger sample size will help improve the robustness of the conclusions drawn with respect to the embryologic development in patients with RIF. Wider implications of the findings The study’s findings suggest that putrescine supplementation in IVF may enhance outcomes for patients with RIF, potentially leading to personalized treatments. Trial registration number not applicable

Abstract Study question Does embryo compaction positively correlate with blastocyst development and further embryonic ploidy status? Summary answer Fully compacted embryos develop into good-quality blastocysts, have shorter developmental times, and are related to ploidy status. What is known already To succeed in assisted reproduction technique (ART), it is necessary to select embryos that have the highest potential. So, numerous studies make an effort to establish parameters for selecting embryos. A Time-lapse system (TLS) allows embryologists to understand dynamic embryo change through continuous monitoring. In development, embryos undergo dynamic functional changes during compaction, which play a crucial role in blastocyst formation. It is also known that embryonic genome activation can be seen with compaction. Incomplete compaction leads to blastocyst developmental failure. Nevertheless, the details about the compaction of human embryos have not been paid sufficient attention, so still rarely known. Study design, size, duration This was a retrospective cohort study including couples that underwent an IVF cycle at the CHA Fertility Center, Gangnam, between January 2019 to October 2022. A total of 371 reached the blastocyst from 113 patients cultured in the TLS were analyzed. Among these, 94 blastocysts were analyzed by preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis was performed by prism9 using a t-test and chi-square test. P values <0.05 were regarded as statistically significant. Participants/materials, setting, methods Embryos were classified into two categories by compaction pattern: fully compacted (Group1, N = 194) and partially compacted (Group2, N = 177). Blastocyst quality was determined by morphology and divided into three groups (Good, Average, and Poor). The developmental time ranging from morula to blastocyst was annotated based on the embryo scope image. The surface of the blastocyst was measured every hour starting at blastocyst formation (tB) by using the ellipse tool of the Embryo Viewer software. Main results and the role of chance Good and average quality blastocysts are significantly higher in Group 1 than in Group 2 (21.6% vs. 3.4%, p < 0.01; 47.9% vs. 26.6%, p < 0.01, respectively). In contrast, poor-quality blastocysts are lower in Group 1 than in Group 2 (30.4% vs. 70.1%, p < 0.01). The beginning and completion of compaction, and blastocyst formation times of embryos from Group 1 were significantly shorter than those of embryos that Group 2(78.6h vs. 82.4, p < 0.01; 87.0h vs. 92.2h, p < 0.01; 100.2h vs. 103.7, p < 0.01, respectively). Also, there is a significant difference in longitudinal surface area at 3, 6, and 12h from the time of tB between the two groups. Consequently, the average expansion rate in Group 1 was significantly faster than in Group 2 (653.6 μm2/hour vs. 499.2μm2/hour, p < 0.05). According to the result of PGT-A, Group 1 had statistically significantly higher euploid and lower aneuploidy rates compared to Group 2 (47.2% vs. 36.2%, p < 0.001; 52.8% vs. 63.8%, p < 0.001, respectively). However, in the PGT-A group, there was no significant difference in developmental time between the two groups, regardless of fully or partial compaction. Meanwhile, the average expansion rate in euploidy blastocyst was significantly faster than in aneuploidy blastocyst (747.8 μm2/hour vs. 564.3 μm2/hour, p < 0.05). Limitations, reasons for caution The main limitation is the single-center retrospective approach. Therefore future prospective research is needed to verify and extend our findings. Another one, in the PGT-A there was no difference in developmental time regardless of fully or partially compaction. The reason is that we selected blastocysts of sufficient quality for biopsy. Wider implications of the findings Using the TLS, we have observed the dynamics of compaction in detail. We found a positive relationship between compaction and blastocyst quality and its association with embryo ploidy. The assessment of compaction is a significant parameter for predicting competent embryos and should be given priority when selecting blastocysts. Trial registration number Not applicable

Mitochondria have a central role for cell viability and in early mammalian embryogenesis. Asymmetric distribution of mitochondria at pronuclear stage has been associated with asymmetric segregation into different blastomeres might be the reason of abnormal embryonic development. We investigated the possible causes of low blastocyst development of vitrified/warmed oocytes by evaluating the changes of high-polarized mitochondria redistribution. Animal study. Immature GV mouse oocytes were vitrified in EG+ DMSO with an EM grid and slush-nitrogen. We used a milrinone, as phosphodietsterase 3 inhibitor, to block spontaneous germinal vesicle breakdown during oocyte collection and vitrification. The thawed/warmed oocyte were matured in vitro or subjected to test high-polarized mitochondria distribution. In vitro matured MII oocyte were fertilized and cultured in KSOM for 5 days to analyze embryonic development. As high-polarized mitochondria indicator, JC-1was used and monitored with CLSM with Z-stack. There is no difference in in vitro maturation rate and fertilization rate between fresh and vitrified/warmed immature oocyte. However, the development rate to blastocyst in thawed/warmed oocyte was significantly lower than those in fresh oocyte (P<0.05). It was recovered if these oocytes were incubated in milrinone for more than 3hr for recovery of mitochondria redistribution before oocyte maturation. Most of thawed/warmed oocyte showed low-polarized mitochondrial inner membrane potential, on the other hand, for 3hr incubated oocyte in milrinone recovered to high polarized mitochondria. Alteration of high polarized mitochondria distribution in vitrified oocyte may affect on mitochondria activity during fertilization and further embryo development.

Multi-potent spermatogonial stem cells (mSSC), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESC). Similar to ESC, mSSC are capable of differentiating into 3-germ layers in vitro and teratoma formation in vivo. Additionally, mSSC proliferate rapidly and can be transfected more easily than SSC. In contrast to previous reports, we have found that mSSC also have germ-cell-specific micro (mi)RNA and gene expression profiles. Therefore, the aims of this study were to compare the efficiency of mSSC v. ESC to differentiate into germ lineage and produce male gametes, as well as to develop a novel system for the production of genetically modified mice. Mouse mSSC were transfected with a lentiviral vector expressing green fluorescent protein (GFP) and testis-specific gene and maintained in the ESC-culture medium containing leukemia inhibitory factor (LIF). Embryonic bodies (EB) were formed after the cells were detached from the feeder cells. Bone morphogenetic protein (BMP)-4 (10 ng mL˜1) and retinoic acid (RA, 0.1 μM) were added to the ESC-culture medium for 3 days in order to induce differentiation into germ lineage cells. Then, these cells were changed to germ cell-culture medium (Stem-Pro™ containing GDNF; Invitrogen, Carlsbad, CA, USA) and cultured for 3 days. After 6 days, cultured cells were sorted by magnetic activating cell sorting system using specific marker for germ cells, CD-9. Isolated germ lineage cells were transplanted into a busulfan-treated mouse testis for the production of male germ cells. Three to 6 weeks later, the testis and epididymis were collected, and half of the sample was used to perform histological analysis and the other half for the production of intracytoplasmic sperm injection (ICSI)-derived embryos. The statistical significance of differences between the 2 groups was evaluated by Student’s t-test Immunocytochemical and flow cytometrical analysis performed 6 days after differentiation showed that the ratio of germ cell-specific markers in EB derived from mSSC was higher than those from ESC. Moreover, after 3 to 6 weeks of transplantation the testis produced sperms and germ cells expressing GFP. We have successfully produced embryos by ICSI and offspring by embryo transfer into uteri of poster mothers. These results demonstrate that mSSC can be easily differentiated into germ lineage cells compared with ESC and have the potential to generate functional gametes. Therefore, the differentiation and transgenesis of mSSC may be a useful model for production of genetically modified mice. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084923).

Although the precise mechanism of granulosa cell (GC) apoptosis has not yet been clarified, GC died during follicular atresia and luteolysis results from apoptosis have suggested a potential role in human reproduction. Besides, that apoptotic episode may be involved in the outcome of IVF. The objective of this study was to investigate the effects of apoptosis-related genes in the GC of IVF patients regarding fertilization, cleavage, and pregnancy rate. A prospective clinical study Human GCs were obtained from pooled collections of follicular fluid from 56 women with normal FSH levels (aged <40 years) that were undergoing oocyte retrieval for IVF under the indications of tubal factor, male factor, and unexplained cases. The GCs were lysed in a sample buffer. Western blot were performed with the ECL detection system. We designated the active form of apoptosis-related genes as group1(g1), while group2 (g2) showing no active form. The active form of cysteine protease caspase-8 was detected in 39 women (g1) but not in 17 (g2). The percentage of fertilization, cleavage, and pregnancy rate in g1/g2 was 97.6/98.5, 72.0/72.0 and 25.6/52.9 (p=0.0473), respectively. There was a statistically significant difference in the pregnancy rate between the two groups (p<0.05). On the other hand, the active form of caspase-3 was observed in 34 women (g1) but not in 22 (g2). Fertilization, cleavage, and pregnancy rates between g1 and g2 were 97.5/97.9, 75.1/69.7, and 29.4/40.9, respectively. The cleavage fragment of PARP was observed in 33 women (g1), but not in 23 (g2). The fertilization, cleavage, and pregnancy rates in g1/g2 were 96.7/95.6, 73.0/70.0 and 33.3/34.8. Data were analyzed using X2-test and Fisher's exact test (p<0.05). Our study demonstrated higher pregnancy rate revealed group2 than group1. On the other hand, there were no differences in the fertilization, cleavage, and pregnancy rates between group1 and group2 of caspase-3, PARP. Although some authors have not confirmed the existence of a relationship between apoptosis and the outcome of IVF patients, several reports have been published showing relationship. However, it was noted that women with lower percentage of GC apoptosis had higher pregnancy rates. Caspases' activation did not occur until after oocyte activation. The ligation of Fas ligand to Fas in the cellular membrane triggers the activation of caspase 8. Once activated, caspase 8 transduces signal to effector caspases including caspase 3 known to be a major executioner protease. This leads to the degradation of those caspases, cellular substrates, including cytoplasmic structural proteins such as actin and nuclear proteins such as PARP. Interestingly, in Xenopus, the activation of embryonic gene transcription leads to a decrease in embryo susceptibility to apoptosis after the previous increase due to oocyte activation. Although caution must be shown when comparing results obtained with caspase 8, which was associated with pregnancy rate, it has been well demonstrated that caspase 8 and 3 are directly associated with the Fas-mediated apoptosis pathway, as already reported. In all circumstances, further studies are progressed to demonstrate a prognostic role for apoptosis of GC, such as confirming the results in vivo according to the women stimulation protocol.