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In the last four decades, the assisted reproductive technology (ART) field has witnessed advances, resulting in improving pregnancy rates and diminishing complications, in particular reduced incidence of multiple births. These improvements are secondary to advanced knowledge on embryonic physiology and metabolism, resulting in the ability to design new and improved culture conditions. Indeed, the incubator represents only a surrogate of the oviduct and uterus, and the culture conditions are only imitating the physiological environment of the female reproductive tract. In vivo, the embryo travels through a dynamic and changing environment from the oviduct to the uterus, while in vitro, the embryo is cultured in a static fashion. Importantly, while culture media play a critical role in optimising embryo development, a large host of additional factors are equally important. Additional potential variables, including but not limited to pH, temperature, osmolality, gas concentrations and light exposure need to be carefully controlled to prevent stress and permit optimal implantation potential. This manuscript will provide an overview of how different current culture conditions may affect oocyte and embryo viability with particular focus on human literature.

Abstract Objective To generate an effective embryo prediction model and identify a non-invasive evaluation method by analyzing microRNAs (miRNAs) in embryo culture medium. Design Analysis of microRNA profiles from spent culture medium of blastocysts with good morphology that did or did not result in pregnancy. Setting Clinical and experimental research. Patients Sixty patients who underwent thawed embryo transfer of blastocysts after intracytoplasmic sperm injection. Intervention(s) None. Main outcome measure(s) The association of miRNA abundance levels secreted by blastocysts in culture medium and implantation success. Results Our RNA sequencing analysis found a total of 53 differentially expressed miRNAs in the culture media of pregnancy and non-pregnancy groups. Twenty-one miRNAs were analyzed for their potential to predict implantation success. Eight miRNAs (hsa-miR-191-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-509-3p, hsa-miR-378a-3p, hsa-miR-28-3p, hsa-miR-512-5p, and hsa-miR-181a-5p) were further extracted from the results of a logistic regression analysis of qPCR Ct values. A prediction model for high-quality blastocysts was generated using the eight miRNAs, with an average accuracy of 0.82 by 5-fold cross validation. Conclusion We isolated blastocyst miRNAs that may predict implantation success and created a model to predict viable embryos. Increasing the number of investigated cases and further studying the effect of each miRNA on embryonic development is needed to refine the miRNA-based predictive model.

Oocyte developmental competence declines in women aged 35 and older resulting in many women resorting to IVF. The present study determined whether adding Granulocyte-macrophage colony-stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence in a mouse model of advanced maternal age. Oocytes from 12-14 month C57BL6 J × CBA mice were treated with 10 ng/ml of GM-CSF during IVM, and embryo development, mitochondrial activity, spindle formation and chromosomal alignment were examined. The addition of GM-CSF tended to increase fertilisation rates (76.19 vs. 82.03%; P = 0.07) but did not affect cumulus expansion compared with control. The addition of GM-CSF also increased blastocysts rates (51.10 vs. 61.52%; P < 0.01) and the number of good quality blastocysts (33.31 vs. 44.13%; P < 0.05) present at 96 h of culture as well as inner cell mass (12.64 vs. 15.62 ; P < 0.01) and total cell number (42.98 vs. 48.78 ; P < 0.05). GM-CSF treatment also increased mitochondrial membrane potential two to three fold in the outer (2.86 vs. 0.97; P < 0.001), intermediate (3.25 vs. 0.89; P < 0.001) and peri nuclear areas (3.62 vs. 1.08; P < 0.001). GM-CSF treatment did not influence spindle formation or chromosomal alignment. Together our results indicate that the addition of GM-CSF during IVM may improve oocyte quality in women of advanced maternal age.

Prepubertal animals are often preferred as sources of oocytes for assisted reproductive technologies (ARTs) in laboratory mice, but the normality and developmental competence of these oocytes remain controversial. This study systematically examined in vitro fertilization competence, embryo development, and fetal development after embryo transfer (ET) using oocytes from C57BL/6J mice aged 3 to 10 weeks. Oocytes were collected from superovulated females, fertilized, and cultured in vitro for 96 h or transferred at 2-cell stage to recipient females. Additionally, fetal development was compared between unfrozen and frozen-thawed in vitro-fertilized 2-cell embryos across different age groups. The number of ovulated oocytes per animal decreased with age, while the percentage of morphologically normal oocytes was highest in 3-week-old mice (99%) compared to older ages (70-86%, P < 0.05). Although fertilization percentages were consistently high (≥ 97%), blastocyst development in vitro, the nuclear counts of blastocysts and fetal development after ET were lowest for embryos from 3-week-old mice. Development of frozen-thawed embryos to fetuses was significantly reduced compared to unfrozen embryos in all age groups, except for those from 10-week-old mice. These findings suggest that oocytes from prepubertal mice, particularly from 3-week-old mice, are less developmentally competent than those from older mice. Therefore, the age of animals for oocyte source should be carefully considered based on the specific requirements of the research or ART applications.

This article reports a case of Massilia timonae, an understudied Gram-negative rod, in the culture media of an embryo produced by intracytoplasmic sperm injection into the oocyte cytoplasm. Cultivation was monitored with a time-lapse technology. Microbial contamination can cause embryo developmental arrest.

Research Question: Are there significant differences between cumulative live birth rates (CLBR) after short or extended embryo culture when comparisons are performed per cycle?, Design: This French national study included all IVF cycles performed from January 2016 to December 2019 with at least one cleaved embryo at day 2. The day 2/3 and day 5/6 groups were identified using the National Biomedicine Agency register. Only attempts involving the vitrification method were included. CLBR was assessed with 1 and 2 years of follow-up using a logistic regression model. The evolution of CLBR per cycle was compared over 1 year of follow-up., Results: In total, 133,250 cycles met the inclusion criteria (70,528 and 62,722 in the day 2/3 and day 5/6 groups). In multivariate analysis including several patient and cycle characteristics, the CLBR per cycle was significantly lower in the day 5/6 compared with the day 2/3 group. A significantly higher rate of fresh embryo transfer cancellation was observed in the day 5/6 compared with the day 2/3 group. The evolution of the CLBR was significantly different in favour of the day 2/3 group in cases with three or fewer day 2 embryos, whatever the patient's age, in their two first attempts as well as in their third or further attempts., Conclusions: Overall, the nationwide results per cycle suggest that extended embryo culture until the blastocyst stage, even when used in combination with vitrification, could not improve live birth rates. Moreover, where three or fewer day 2 embryos are obtained, it might be preferable to use the short embryo culture strategy., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Can the best culture medium be determined by mouse embryo testing? Zygotes/embryos of four different mouse strains: inbred Balb/c (n = 79) and C57Bl/6 (n = 95), F1 hybrid DBA/2*C57Bl/6 (n = 133), and outbred ICR (n = 69) were incubated in vitro in four culture media: 1-Step Culture Medium (VitaVitro), CSC medium (Irvine Scientific), Sage 1-Step medium (Origio), G-TL medium (Vitrolife). The embryos were cultured under standard conditions at atmospheric oxygen: 37℃; 6% CO2, 100% humidity. Standard MEA-test parameters (number of expanded blastocysts on E4.5), as well as additional characteristics (embryo development on E2.5-E4.5, number of hatching and hatched blastocysts on E4.5, embryo arrest and degeneration) were assessed for each of the strain-medium combinations. The results were compared across strains as well as across media. Embryo development depended on both the media and the mouse strain. Embryos from inbred and outbred mice were more sensitive to suboptimal culture conditions compared to the hybrid strain; this was reflected in reduced blastocyst and hatching rates, as well as an increased percentage of arrested and degraded embryos. The choice of optimal media depended strongly on the strain: CSC was better for Balb/c, Sage 1-step for C57Bl/6, while both were preferred for hybrids, and G-TL for outbreds. VitaVitro was quite good for hybrids and performed worse for other strains. Overall, the embryos of each strain behaved differently in different media, and it was not possible to make a real preference for any of them over human embryos based on the results of any single mouse strain. Only a pooled sample of different mouse strains can be used for comprehensive media MEA testing. MEA testing with any strain of mice is doomed to error due to their specific culture requirements. Hybrid mice are too reproductively efficient and cannot be used to distinguish media based on their subtle differences. Outbred and inbred mice are too sensitive and may be delayed or degraded in culture media that is well suited to the requirements of human embryos. Combined analysis of multiple mouse strains should be used to test media more fully and may serve as a model for the heterogeneity of human embryos in IVF clinics., Competing Interests: Declarations. All manipulations with animals were carried out in accordance with the European Convention for the Protection of Vertebrate Animals, Strasbourg, March 18.III.1986, and the Rules of Laboratory Practice in the Russian Federation. Approval was obtained from the Bioethics committee of Institute of Gene Biology of Russian Academy of Sciences (IGB RAS). Conflict of Interest: None of the authors has any conflict of interest to declare., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Background: This study aimed to analyse the expression of microRNA-223 (miR-223) in embryo culture medium and its correlation with pregnancy outcomes., Methods: Two hundred and two patients undergoing in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) were divided into clinical pregnancy group (n = 101) and non-pregnant group (n = 101). The baseline data, clinical indicators, and the expression level of miR-223 in the embryo medium were compared between the two groups. Logistic regression analysis was used to analyse the relationship between each index and the pregnancy outcome. Receiver operator characteristic curve was carried out to evaluate the differential ability of miR-223 in pregnancy status. Bioinformatics methods were used to identify the target genes of miR-223 and elucidate their functions., Results: Compared with pregnancy group, the non-pregnancy group exhibited a reduction in miR-223 expression ( p  < 0.001). Multivariate analysis revealed that miR-223 reduction was an independent factor for pregnancy failure ( p  < 0.05). The ROC curve demonstrated the discriminative capability of miR-223 in distinguishing pregnancy and non-pregnancy. In addition, bioinformatics analysis indicated that the target genes of miR-223 were predominantly located in the endocytic vesicle membrane and were primarily enriched in adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathways., Conclusion: In this study, levels of miR-223 in the embryo culture medium predicted pregnancy outcomes in subjects undergoing IVF/ICSI. Low expression of miR-223 was a risk factor for adverse pregnancy outcomes in subjects.

Mechanisms controlling trophoblast (TB) proliferation and differentiation during embryo implantation are poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD173074-treated pluripotent stem cell-derived trophoblast cells (BAP) are two widely employed, contemporary models to study TB development and function, but how faithfully they mimic early TB cells has not been fully examined. We evaluated the transcriptomes of TB cells from BAP and TSC and directly compared them with those from peri-implantation human embryos during extended embryo culture (EEC) between embryonic days 8 to 12. The BAP and TSC grouped closely with TB cells from EEC within each TB sublineage following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences in transcriptional programs existed within each TB sublineage. We also validated the presence of six genes in peri-implantation human embryos by immunolocalization. Our analysis reveals that both BAP and TSC models have features of peri-implantation TB s, while maintaining minor transcriptomic differences, and thus serve as valuable tools for studying implantation in lieu of human embryos., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts?, Summary Answer: Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality., What Is Known Already: Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial., Study Design, Size, Duration: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers., Participants/materials, Setting, Methods: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics., Main Results and the Role of Chance: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10)., Limitations, Reasons for Caution: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes., Wider Implications of the Findings: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment., Study Funding/competing Interest(s): None., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Excessive consumption of fructose-sweetened foods and beverages is a growing concern worldwide. Studies have demonstrated that fructose consumption before and during pregnancy can result in adverse outcomes such as decreased decidualization, increased fetal losses, and low birth weight. The study investigated the teratogenic effects of fructose on rat embryos during organogenesis using whole embryo culture. Within the scope of the study, 4 groups were formed as control, low, medium, and high-dose fructose (HDF) with 10 embryos in each group. The 9.5-day-old rat embryos were cultured with different concentrations of fructose (1, 5 and 10 mM) for 48 h and the possible effects of fructose were examined using morphological scoring, histochemistry, immunofluorescence, and TUNEL methods. According to the analyses, protein synthesis and proliferation were decreased, vascular formation was suppressed, and apoptosis was increased in embryos exposed to fructose, especially at concentrations of 5 mM and above. According to the morphological scoring results, it was determined that heart, hind limb, and somite development were retarded in all experimental groups compared to the control group, developmental retardation increased in direct proportion to fructose concentration, and also significant malformations were observed in all parameters examined in the HDF group. In addition, analysis of yolk sac diameter, head length, crown rump length and somite numbers showed that these parameters were significantly decreased in all experimental groups. End of the study, it was concluded that fructose at concentrations of 1 mM and above may induce embryonic development retardation and other anomalies by decreasing protein synthesis and cell proliferation, suppressing vascular formation, and increasing apoptosis in embryonic tissues., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: In vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8-16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%)., Methods: Bovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7-8. Expanded blastocysts on days 7-8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing., Results: Under 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted., Conclusion: OES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development., (© 2024. The Author(s).)

Study Question: Could an artificial intelligence (AI) algorithm predict fetal heartbeat from images of vitrified-warmed embryos?, Summary Answer: Applying AI to vitrified-warmed blastocysts may help predict which ones will result in implantation failure early enough to thaw another., What Is Known Already: The application of AI in the field of embryology has already proven effective in assessing the quality of fresh embryos. Therefore, it could also be useful to predict the outcome of frozen embryo transfers, some of which do not recover their pre-vitrification volume, collapse, or degenerate after warming without prior evidence., Study Design, Size, Duration: This retrospective cohort study included 1109 embryos from 792 patients. Of these, 568 were vitrified blastocysts cultured in time-lapse systems in the period between warming and transfer, from February 2022 to July 2023. The other 541 were fresh-transferred blastocysts serving as controls., Participants/materials, Setting, Methods: Four types of time-lapse images were collected: last frame of development of 541 fresh-transferred blastocysts (FTi), last frame of 467 blastocysts to be vitrified (PVi), first frame post-warming of 568 vitrified embryos (PW1i), and last frame post-warming of 568 vitrified embryos (PW2i). After providing the images to the AI algorithm, the returned scores were compared with the conventional morphology and fetal heartbeat outcomes of the transferred embryos (n = 1098). The contribution of the AI score to fetal heartbeat was analyzed by multivariate logistic regression in different patient populations, and the predictive ability of the models was measured by calculating the area under the receiver-operating characteristic curve (ROC-AUC)., Main Results and the Role of Chance: Fetal heartbeat rate was related to AI score from FTi (P < 0.001), PW1i (P < 0.05), and PW2i (P < 0.001) images. The contribution of AI score to fetal heartbeat was significant in the oocyte donation program for PW2i (odds ratio (OR)=1.13; 95% CI [1.04-1.23]; P < 0.01), and in cycles with autologous oocytes for PW1i (OR = 1.18; 95% CI [1.01-1.38]; P < 0.05) and PW2i (OR = 1.15; 95% CI [1.02-1.30]; P < 0.05), but was not significantly associated with fetal heartbeat in genetically analyzed embryos. AI scores from the four groups of images varied according to morphological category (P < 0.001). The PW2i score differed in collapsed, non-re-expanded, or non-viable embryos compared to normal/viable embryos (P < 0.001). The predictability of the AI score was optimal at a post-warming incubation time of 3.3-4 h (AUC = 0.673)., Limitations, Reasons for Caution: The algorithm was designed to assess fresh embryos prior to vitrification, but not thawed ones, so this study should be considered an external trial., Wider Implications of the Findings: The application of predictive software in the management of frozen embryo transfers may be a useful tool for embryologists, reducing the cancellation rates of cycles in which the blastocyst does not recover from vitrification. Specifically, the algorithm tested in this research could be used to evaluate thawed embryos both in clinics with time-lapse systems and in those with conventional incubators only, as just a single photo is required., Study Funding/competing Interests: This study was supported by the Regional Ministry of Innovation, Universities, Science and Digital Society of the Valencian Community (CIACIF/2021/019) and by Instituto de Salud Carlos III (PI21/00283), and co-funded by European Union (ERDF, 'A way to make Europe'). M.M. received personal fees in the last 5 years as honoraria for lectures from Merck, Vitrolife, MSD, Ferring, AIVF, Theramex, Gedeon Richter, Genea Biomedx, and Life Whisperer. There are no other competing interests., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To generate an effective embryo prediction model and identify a non-invasive evaluation method by analyzing microRNAs (miRNAs) in embryo culture medium. Design: Analysis of microRNA profiles from spent culture medium of blastocysts with good morphology that did or did not result in pregnancy. Setting: Clinical and experimental research. Patients: Sixty patients who underwent thawed embryo transfer of blastocysts after intracytoplasmic sperm injection. Intervention(s): None. Main outcome measure(s): The association of miRNA abundance levels secreted by blastocysts in culture medium and implantation success. Results: Our RNA sequencing analysis found a total of 53 differentially expressed miRNAs in the culture media of pregnancy and non-pregnancy groups. Twenty-one miRNAs were analyzed for their potential to predict implantation success. Eight miRNAs (hsa-miR-191-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-509-3p, hsa-miR-378a-3p, hsa-miR-28-3p, hsa-miR-512-5p, and hsa-miR-181a-5p) were further extracted from the results of a logistic regression analysis of qPCR Ct values. A prediction model for high-quality blastocysts was generated using the eight miRNAs, with an average accuracy of 0.82 by 5-fold cross validation. Conclusion: We isolated blastocyst miRNAs that may predict implantation success and created a model to predict viable embryos. Increasing the number of investigated cases and further studying the effect of each miRNA on embryonic development is needed to refine the miRNA-based predictive model. [ABSTRACT FROM AUTHOR]

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages., (© The Author(s) 2024. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

Diosmetin (DIOS), a natural flavonoid monomer derived from lemons and present in various plants such as spearmint and spider moss, exhibits antioxidant, anti-inflammatory, and antiaging properties. Nonetheless, its impact on early embryonic development in pigs remains unexplored. This study aimed to determine the influence of DIOS supplementation in an in vitro culture (IVC) medium on porcine embryo development and to elucidate the underlying mechanisms. Findings revealed that embryos cultured in IVC medium with 0.1 μM DIOS demonstrated an increased blastocyst formation rate, higher total cell number, reduced LC3B and CASPASE3 levels, elevated Nrf2 levels, decreased ROS, and enhanced GSH and mitochondrial membrane potential at the 4-cell embryonic stage. Additionally, the expression of proapoptotic genes (CAS3, CAS8, and BAX) and autophagy-related genes (BECLIN1, ATG5, LC3B, and P62) was downregulated, whereas the expression of embryonic development-related genes (CDK1 and CDK2), antioxidant-related genes (SOD1 and SOD2), and mitochondrial biogenesis-related genes (NRF2) was upregulated. These findings suggest that DIOS promotes early embryonic development in pigs by mitigating oxidative stress and enhancing mitochondrial function, thereby reducing autophagy and apoptosis levels., (© 2024 Wiley Periodicals LLC.)

Can a set of metabolites present in embryo culture media correlate with embryo implantation? Case-control study in two phases: discovery phase (101 samples) and validation phase (169 samples), collected between 2018 and 2022, with a total of 218 participants. Culture media samples with known implantation outcomes were collected after blastocyst embryo transfer (including both PGT and non-PGT cycles) and were analyzed using chromatography followed by mass spectrometry. The spectra were processed and analyzed using statistical and machine learning techniques to identify biomarkers associated with embryo implantation, and to develop a predictive model. In the discovery phase, 148 embryo implantation biomarkers were identified using high resolution equipment, and 47 of them were characterized. Our results indicate a significant enrichment of tryptophan metabolism, arginine and proline metabolism, and lysine degradation biochemical pathways. After transferring the method to a lower resolution equipment, a model able to assign a Metabolite Pregnancy Index (MPI) to each embryo culture media was developed, taking the concentration of 36 biomarkers as input. Applying this model to 20% of the validation samples (N=34) used as the test set, an accuracy of 85.29% was achieved, with a PPV (Positive Predictive Value) of 88% and a NPV (Negative Predictive Value) of 77.78%. Additionally, informative results were obtained for all the analyzed samples. Metabolite concentration in the media after in vitro culture shows correlation with embryo implantation potential. Furthermore, the mathematical combination of biomarker concentrations using Artificial Intelligence techniques can be used to predict embryo implantation outcome with an accuracy of around 85%., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Objective: To investigate the ideal time in culture to optimize embryo cell-free deoxyribonucleic acid (cfDNA) analysis in frozen-thawed blastocysts undergoing noninvasive preimplantation genetic testing for aneuploidy (PGT-A). Cell-free DNA is released into the spent blastocyst media (spent media) by the embryo. However, the optimal timing to determine maximal cfDNA in the case of frozen-thawed blastocysts undergoing noninvasive PGT-A remains to be elucidated., Design: In this prospective observational study, 135 spent media and corresponding whole blastocysts were collected from January 2021 through March 2022., Setting: Private fertility clinics., Patients: Day-5 frozen-thawed blastocysts were cultured for 8 hours (Day-5 Short) or 24 hours (Day-5 Long), whereas day-6 frozen-thawed blastocysts were cultured for 8 hours (Day-6 Short). The spent media and whole blastocysts were then collected for further analysis. Spent media and whole blastocysts were amplified using whole genome amplification and sequenced using next-generation sequencing., Main Outcome Measures: Informativity and concordance rates between cfDNA in spent media and whole blastocyst DNA were compared according to the different times in culture., Results: When comparing time in culture, informativity rates for spent media were significantly higher for Day-5 Long and Day-6 Short (>91%) compared with the Day-5 Short group (<60%). A similar trend was observed for cases with and without a previous PGT-A. Regarding blastocyst expansion grade, informativity rates were lower on Day-5 Short compared with Day-5 Long and Day-6 Short, regardless of expansion degree. This decrease was significant for Gardner-grade expansion grades 3, 4, and 5-6. In addition, for a similar time in culture, the grade of expansion did not have an impact on the informativity rates. For concordance rates, no significant differences were observed among the 3 groups. In all cases, concordance rates were 90.5% for Day-5 Short, 93.6% for Day-5 Long, and 92.3% for Day-6 Short. No impact of the expansion grade was observed on concordance rates., Conclusion: Noninvasive PGT-A in frozen-thawed blastocysts yields very high concordance rates with whole blastocysts, possibly limiting the need for invasive PGT-A and making it available for a wider range of patients., Competing Interests: Declaration of Interests G.A. has nothing to disclose. M.B. has nothing to disclose. C.M.G.-P. has nothing to disclose. L.N.-S. is an employee of Igenomix. E.V.Z. has nothing to disclose. J.A.C. is an employee of Igenomix. C.S. has nothing to disclose. D.S. reports support from Igenomix who ran niPGT samples for the submitted work; speakers bureau for Igenomix and EMD Serono; Igenomix Scientific Advisory Board. C.R. is an employee of Igenomix., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Despite the cytotoxicity and embryotoxicity previously reported artesunate is a recommended drug to treat malaria for adults, children, and women in the first trimester of pregnancy. To address the putative effects of artesunate on female fertility and preimplantation embryo development, when the pregnancy is not detectable yet, artesunate was added to the oocyte in vitro maturation and in vitro embryo development of bovine. Briefly, in experiment 1 the cumulus-oocyte complexes (COCs) were in vitro matured for 18 h with 0.5, 1, or 2 µg/mL of artesunate or not (negative control) and then checked for nuclear maturation and subsequent embryo development. In experiment 2, the COCs were in vitro matured and fertilized without artesunate, which was added (0.5, 1, or 2 µg/mL) from the 1st to the 7th day of embryo culture along with a negative and a positive control group with doxorubicin. As a result, the use of artesunate on oocyte in vitro maturation did not differ from the negative control ( p  > 0.05) regarding nuclear maturation, cleavage, and blastocyst formation. Also, artesunate on in vitro embryo culture did not differ from negative control ( p  > 0.05) regarding cleavage and blastocyst formation, except for positive control, with doxorubicin ( p  < 0.05). In conclusion, under the conditions investigated, there was no evidence of artesunate toxicity on oocyte competence and the preimplantation period of in vitro embryo development in the bovine model, however, artesunate use still should be taken carefully as the outcome of implantation after oocytes and blastocysts exposure to artesunate remains unknown.

Research Question: Does artificial shrinkage before fresh blastocyst transfer improve clinical pregnancy rates in IVF?, Design: In this monocentric prospective, randomized, double-blind, controlled pilot study, 150 couples undergoing fresh single-blastocyst transfer were randomized between 20 May 2018 and 22 February 2022. In the artificial shrinkage group (AS group), a single laser pulse was directed to the cellular junction of the trophectoderm on the opposite side of the inner cell mass in each blastocyst. IVF outcomes were clinical pregnancy, multiple pregnancy and live birth rates. Cell-free DNA (cfDNA) concentration was also measured by quantitative real-time PCR in the blastocyst culture medium., Results: In total, 142 couples underwent fresh single-blastocyst transfer: control group, no artificial shrinkage, n = 47; and AS group, artificial shrinkage, n = 95; An intention-to-treat (ITT) analysis was employed. After a reassessment and the exclusion of patients with major protocol deviations, 139 couples underwent fresh single-blastocyst transfer under optimal conditions: control group, n = 47; and AS group, n = 92; a per-protocol analysis was used here. The clinical and laboratory characteristics were not significantly different between the groups. The clinical pregnancy rate was similar in the control and AS groups (ITT: 48.9% versus 49.5%, P = 0.97; per protocol: 48.94% versus 51.1%, P = 0.89). The multiple pregnancy rate and the live birth rate were also similar between the groups. No significant differences in gestational age, birthweight or proportion of male/female newborns were observed. The concentration of cfDNA in the blastocyst culture medium was not associated with IVF outcome., Conclusions: Large-scale randomized controlled trials are required to confirm these preliminary results., (Copyright © 2024. Published by Elsevier Ltd.)

Research Question: Can microbes vertically transmit from semen and follicular fluid to embryo culture media during assisted reproductive technology (ART) treatment?, Design: Spent embryo culture media (SECM), seminal fluid and follicular fluid samples were collected from 61 couples with infertility undergoing ART treatment at the Prince of Wales Hospital, Hong Kong SAR, China. Metagenomic analysis was conducted using 16s rRNA sequencing to identify the source of microbes in SECM, correlation between the semen microbiome and male infertility, and correlation between the follicular fluid microbiome and female infertility., Results: Microbial vertical transmission into SECM was reported in 82.5% of cases, and semen was the main source of contamination in conventional IVF cases. The increased abundances of Staphylococcus spp. and Streptococcus anginosus in semen had negative impacts on total motility and sperm count, respectively (P < 0.001). Significant increases in abundance of the genera Prophyromonas, Neisseria and Facklamia were observed in follicular fluid in women with anovulation, uterine factor infertility and unexplained infertility, respectively (P < 0.01). No significant correlation was found between the bacteria identified in all sample types and ART outcomes, including fertilization rate, embryo development, number of available embryos, and clinical pregnancy rate., Conclusion: Embryo culture media can be contaminated during ART treatment, not only by seminal microbes but also by follicular fluid and other sources of microbes. Strong correlations were found between specific microbial taxa in semen and sperm quality, and between the follicular fluid microbiome and the aetiology of female infertility. However, no significant association was found between the microbiomes of SECM, semen and follicular fluid and ART outcomes., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Study Question: Does the morphological quality on Day 3 influence the pregnancy outcomes of euploid blastocysts?, Summary Answer: The morphological quality on Day 3 affects the clinical pregnancy rate (CPR) and live birth rate (LBR) of low-quality euploid blastocysts., What Is Known Already: The morphological grading of Day 3 embryos affects the pregnancy outcome of cleavage-stage embryos and is an excellent indicator to predict embryo development potential. However, it is still unclear whether morphological quality on Day 3 is associated with pregnancy outcomes of the euploid blastocyst., Study Design, Size, Duration: This retrospective cohort study comprised 1275 patients who received single euploid blastocyst transfer between January 2016 and August 2021 at a tertiary teaching hospital., Participants/materials, Setting, Methods: Patients were grouped into two groups according to the morphological grading on Day 3 of transferred blastocysts: high-quality (HQ, including Grades I and II) Day 3 embryos and low-quality (LQ, Grade III) Day 3 embryos. The primary outcomes were CPR and LBR. Interactions of development days (Day 5 and Day 6) and morphological quality (high- and low-quality) of blastocysts with morphological quality of Day 3 embryos on pregnancy outcomes were tested in the stratified analysis and logistic regression models. The multivariate logistic regression analysis was conducted to investigate the independent effect of the morphological quality of Day 3 embryos on pregnancy outcomes after adjusting for potentially confounding factors., Main Results and the Role of Chance: The CPR and LBR of the HQ Day 3 embryos group were statistically higher than those of the LQ Day 3 embryos group (CPR: 59.73% versus 49.70%, respectively, P = 0.015; LBR: 49.73% versus 41.21%, respectively, P = 0.041). The development days of blastocysts did not exhibit a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.648) and LBR (P for interaction = 0.925). The morphological quality of blastocysts exhibits a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.020) and LBR (P for interaction = 0.012). After adjusting for potential confounders, the HQ Day 3 embryo group was positively associated with the CPR (adjusted odds ratio (aOR): 2.10, 95% CI: 1.31-3.36, P = 0.002) and LBR (aOR: 1.97, 95% CI: 1.20-3.25, P = 0.008) of LQ blastocysts. However, the morphological quality on Day 3 was not significantly associated with the CPR (aOR: 0.95, 95% CI: 0.58-1.55, P = 0.835) and LBR (aOR: 0.86, 95% CI: 0.53-1.40, P = 0.550) of HQ blastocysts., Limitations, Reasons for Caution: Selection and confounding bias introduced by the retrospective design cannot be completely eliminated in this study, although multivariable logistic analysis was conducted to adjust for potential confounders. Also, some subgroups had small sample sizes, which may reduce statistical power. Moreover, participants in our study only received single euploid blastocyst transfer, and whether the results could apply to blastocysts with unknown ploidy status is unclear., Wider Implications of the Findings: This study found that the morphological quality on Day 3 was significantly associated with the CPR and LBR of LQ blastocysts; Therefore, when only LQ euploid blastocysts are available for transfer, blastocysts derived from HQ Day 3 embryos are recommended., Study Funding/competing Interest(s): No external funding was obtained. The authors have no conflicts of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: For in vitro fertilization (IVF), mitochondrial DNA (mtDNA) levels in the trophectodermal (TE) cells of biopsied blastocysts have been suggested to be associated with the cells' developmental potential. However, scholars have reached differing opinions regarding the use of mtDNA levels as a reliable biomarker for predicting IVF outcomes. Therefore, this study aims to assess the association of mitochondrial copy number measured by mitoscore associated with embryonic developmental characteristics and ploidy., Methods: This retrospective study analyzed the developmental characteristics of embryos and mtDNA levels in biopsied trophectodermal cells. The analysis was carried out using time-lapse monitoring and next-generation sequencing from September 2021 to September 2022. Five hundred and fifteen blastocysts were biopsied from 88 patients undergoing IVF who met the inclusion criteria. Embryonic morphokinetics and morphology were evaluated at 118 h after insemination using all recorded images. Blastocysts with appropriate morphology on day 5 or 6 underwent TE biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis involved generalized estimating equations, Pearson's chi-squared test, Fisher's exact test, and Kruskal-Wallis test, with a significance level set at P < 0.05., Results: To examine differences in embryonic characteristics between blastocysts with low versus high mitoscores, the blastocysts were divided into quartiles based on their mitoscore. Regarding morphokinetic characteristics, no significant differences in most developmental kinetics and observed cleavage dysmorphisms were discovered. However, blastocysts in mitoscore group 1 had a longer time for reaching 3-cell stage after tPNf (t3; median: 14.4 h) than did those in mitoscore group 2 (median: 13.8 h) and a longer second cell cycle (CC2; median: 11.7 h) than did blastocysts in mitoscore groups 2 (median: 11.3 h) and 4 (median: 11.4 h; P < 0.05). Moreover, blastocysts in mitoscore group 4 had a lower euploid rate (22.6%) and a higher aneuploid rate (59.1%) than did those in the other mitoscore groups (39.6-49.3% and 30.3-43.2%; P < 0.05). The rate of whole-chromosomal alterations in mitoscore group 4 (63.4%) was higher than that in mitoscore groups 1 (47.3%) and 2 (40.1%; P < 0.05). A multivariate logistic regression model was used to analyze associations between the mitoscore and euploidy of elective blastocysts. After accounting for factors that could potentially affect the outcome, the mitoscore still exhibited a negative association with the likelihood of euploidy (adjusted OR = 0.581, 95% CI: 0.396-0.854; P = 0.006)., Conclusions: Blastocysts with varying levels of mitochondrial DNA, identified through biopsies, displayed similar characteristics in their early preimplantation development as observed through time-lapse imaging. However, the mitochondrial DNA level determined by the mitoscore can be used as a standalone predictor of euploidy., (© 2024. The Author(s).)

An effective tool to assess embryo quality in the assisted reproduction clinical practice will enhance successful implantation rates and mitigate high risks of multiple pregnancies. Potential biomarkers secreted into culture medium (CM) during embryo development enable rapid and noninvasive methods of assessing embryo quality. However, small volumes, low biomolecule concentrations, and impurity interference collectively preclude the identification of quality-related biomarkers in single blastocyst CM. Here, we developed a noninvasive trace multiomics approach to screen for potential markers in individual human blastocyst CM. We collected 84 CM samples and divided them into high-quality (HQ) and low-quality (LQ) groups. We evaluated the differentially expressed proteins (DEPs) and metabolites (DEMs) in HQ and LQ CM. A total of 504 proteins and 189 metabolites were detected in individual blastocyst CM. Moreover, 9 DEPs and 32 DEMs were identified in different quality embryo CM. We also categorized HQ embryos into positive implantation (PI) and negative implantation (NI) groups based on ultrasound findings on day 28. We identified 41 DEPs and 4 DEMs associated with clinical implantation outcomes in morphologically HQ embryos using a multiomics analysis approach. This study provides a noninvasive multiomics analysis technique and identifies potential biomarkers for clinical embryo developmental quality assessment.

Research Question: Is the total duration of spontaneous blastocyst collapse to re-expansion before biopsy related to ploidy and live birth rates after single euploid blastocyst transfer?, Design: This was a retrospective cohort study of 600 preimplantation genetic testing cycles for aneuploidy (PGT-A) cycles, involving 2203 biopsied blastocysts, at a large reproductive medicine centre. Features of spontaneous blastocyst collapse from full to expanded stage, before biopsy, were observed using an embryoscope viewer for embryos cultured in a time-lapse incubator. In total, 568 cycles of frozen blastocyst transfers, either single euploid or mosaic, were performed. Correlations between collapse features and PGT-A outcomes were evaluated, as well as live birth rate, following euploid embryo transfer., Results: Blastocysts with lower morphological quality or delayed development had significantly higher rates of collapse, multiple collapses, and a longer duration of collapse to re-expansion. After controlling for confounders, such as oocyte age, morphological quality of blastocyst, and day of biopsy, multivariate logistic regression revealed that the total duration of collapse to re-expansion was an independent predictor of lower euploidy rate; the multivariate OR was 0.85 (95% CI 0.77-0.95; P = 0.00). Furthermore, even with euploid embryo transfer, the probability of a live birth decreased as the total duration of collapse to re-expansion increased; the multivariate OR was 0.79 (95% CI 0.64-0.98; P = 0.033)., Conclusion: The total duration of blastocyst collapse to re-expansion could be used as a predictor of lower euploidy and live birth rate. When developing blastocyst algorithms for pregnancy prediction, the duration of spontaneous blastocyst collapse should be included as a significant variable., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: To explore if a day 7 blastocyst is predictive of the reproductive potential of sibling day 5 or day 6 blastocysts?, Methods: Retrospective cohort of autologous frozen embryo transfers (FET), February 2019 to April 2022. Cycles divided into groups 1 to 5, according to the day of embryo cryopreservation and the presence of a day 7 blastocyst sibling within the cohort: group 1/group 2-day 5 blastocyst without/with a day 7 sibling, group 3/group 4-day 6 blastocyst without/with a day 7 sibling, group 5-day 7 blastocyst. Clinical, ongoing pregnancy and miscarriage rates, cycle, and patient characteristics are reported. Multivariable generalized estimating equations (GEE) logistic regression analysis accounts for confounders and assesses the effect of a sibling day 7 blastocyst on ongoing pregnancy rates of day 5 or day 6 blastocyst FETs., Results: Ongoing pregnancy rates are 38.4%, 59.5%, 30.8%, 32.7%, and 4.4% in groups 1-5, respectively. When correcting for maternal age, number of oocytes retrieved and discarded per cohort, and ploidy, embryos cryopreserved on either day 6 or day 7 have reduced odds of ongoing pregnancy after FET compared to day 5 blastocysts (OR = 0.76, IQR [0.61-0.95], p-value = 0.01). However, the presence of a day 7 sibling does not significantly affect odds of ongoing pregnancy of day 5 or day 6 blastocysts compared to the same-day blastocyst without a day 7 sibling (p-value = 0.20 and 0.46, respectively). This finding is consistent within both the Preimplantation Genetic Testing for Aneuploidy (PGT-A) unscreened and screened (euploid) embryo subgroups., Conclusions: Day of embryo cryopreservation significantly affects ongoing pregnancy rates. However, day 7 embryos within a cohort do not affect the reproductive potential of sibling day 5 and day 6 blastocysts, suggesting that slow embryo development is an embryo-specific trait., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To evaluate whether laser-mediated assisted hatching (AH) performed on vitrified/warmed blastocysts before embryo transfer can improve live birth rate., Design: The "pArtiaL zonA pelluciDa removal by assisteD hatchINg of blastocysts (ALADDIN)" is a 2-center comparative study with a parallel randomized controlled design., Setting: University hospital., Patients: Participants were recruited between September 2018 and November 2021. They were aged 18-39 years, underwent nondonor in vitro fertilization cycles, and were scheduled for elective single embryo transfer with vitrified/warmed blastocysts. Those with uterine abnormalities, body mass index of >35 kg/m 2 , severe male factor infertility, or performing preimplantation genetic testing were excluded., Intervention: Assisted hatching was performed using a 1,480 nm diode laser, removing approximately one-third of the zona pellucida with continuous 0.2 ms pulses applied from the 1-5 o'clock positions., Main Outcome Measures: The primary outcome was the live birth rate. Secondary end points included clinical pregnancy, miscarriage, multiple pregnancies, preterm births, obstetric and neonatal complications, and congenital anomalies., Results: Overall, 698 participants met the inclusion criteria and were randomized: 352 patients were assigned to the AH arm and 346 to the control arm. Of the participants, 105 (29.8%) and 101 (29.2%), respectively, achieved a live birth after treatment. The relative risk of live birth in patients with vitrified/warmed blastocysts treated with AH was 1.02 (95% confidence interval, 0.86-1.19). Exploratory subgroup analyses for women's age, recruiting centers, indications for in vitro fertilization, method of insemination, blastocyst quality, and days of blastocyst development failed to highlight any clinical situation that could benefit from AH in thawed blastocysts., Conclusion: In patients undergoing frozen embryo transfer with vitrified/warmed blastocysts, laser AH does not improve the live birth rate. Further studies are required to rule out milder but potentially interesting benefits in specific subgroups of patients., Trial Registration: ClinicalTrials.gov: NCT03623659., Competing Interests: Declaration of Interests E.P. reported grants and personal fees from MSD, grants from Ferring, IBSA, TEVA, and Gedeon Richter, grants and personal fees from Merck. P.V. received honoraria for editorial engagements. E.S. received honoraria for presentations at meetings from IBSA and Gedeon Richter, and also is handling 2 research grants from Ferring and IBSA. A.A. has nothing to disclose. M.R. has nothing to disclose. C.G. has nothing to disclose. V.B. has nothing to disclose. G.B. has nothing to disclose. M.P. has nothing to disclose. E.R. has nothing to disclose. S.F. has nothing to disclose. A.P. has nothing to disclose., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the positive predictive value and false positive risk of copy number variations (CNV's) detected in cell free deoxyribonucleic acid (DNA) from spent culture media for nonviable or aneuploid embryos., Design: Diagnostic/prognostic accuracy study., Patient(s): Patients aged 35 and younger with an indication for IVF-ICSI and elective single frozen embryo transfer at a single, private IVF center., Intervention: Embryo selection was performed according to the conventional grading, blinded to noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) results. After clinical outcomes were established, spent culture media samples were analyzed., Main Outcome Measures: Prognostic accuracy of CNVs according to niPGT-A results to predict nonviability or clinical aneuploidy., Results: One hundred twenty patients completed the study. Interpretations of next-generation sequencing (NGS) profiles were as follows: 7.5% (n = 9) failed quality control; 62.5% (n = 75) no CNVs detected; and 30% (n = 36) abnormal copy number detected. Stratification of abnormal NGS profiles was as follows: 15% (n = 18) whole chromosome and 15% (n = 18) uncertain reproductive potential. An intermediate CNV was evident in 27.8% (n = 5) of the whole chromosome abnormalities. The negative predictive value for samples with no detected abnormality was 57.3% (43/75). Whole chromosome abnormality was associated with a positive predictive value of 94.4% (17/18), lower sustained implantation rate (5.6%, 1/18), and higher relative risk (RR) for nonviability compared with no detected abnormalities (RR 2.21, 95% CI: 1.66-2.94). No other CNVs were associated with significant differences in the sustained implantation or RRs for nonviability. Unequal sex chromosome proportions suggested that maternal contamination was not uncommon. A secondary descriptive analysis of 705 supernumerary embryos revealed proportions of NGS profile interpretations similar to the transferred cohort. Significant median absolute pairwise differences between certain subcategories of CNV abnormalities were apparent., Conclusion: Whole chromosome abnormalities were associated with a high positive predictive value and significant RR for nonviability. Embryos associated with other CNVs had sustained implantation rates similar to those with no abnormalities detected. Further studies are required to validate the clinical applicability of niPGT-A., Clinical Trial Registration Number: clinicaltrials.gov (NCT04732013)., Competing Interests: Declaration of Interests G.N. is a shareholder in The Fertility Partners (TFP), the parent company of Olive Fertility Centre. S.R. has minority ownership interests in Sequence46. S.T. has minority ownership interests in Sequence46; C.W. has minority ownership interests in Sequence46. Thermo Fisher Scientific is a vendor to Sequence46 but does not have any other affiliations with the authors. Thermo Fisher provided consumables for the NGS methods required for the study but no direct financial support., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To explore the correlation between mitochondrial quantity and the blastocyst development timeline as well as their respective contributions to early pregnancy., Methods: A retrospective study was conducted using a dataset comprising 2,633 embryos that underwent preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2023. The study was divided into three subsets to address distinct aspects: the representativeness of a single trophectoderm (TE) biopsy for mitochondrial quantity (n=43), the correlation between morphokinetic features and mitochondrial quantity (n=307), and the association analysis among mitochondrial quantity, blastocyst timeline factor, and reproductive outcomes (n=2,283). Distribution assessment of mitochondrial quantity across an individual blastocyst involved the identification within multiple biopsies and spent culture media. Timeline evaluation included correlating mitochondrial quantity with time-lapse datasets. Finally, multivariate logistic regression models, incorporating potential effectors alongside mitochondrial quantity, were employed to analyze their respective contributions to early pregnancy endpoints., Results: Of distribution assessment, mitochondrial quantity exhibited an even distribution across the entire trophectoderm (Spearman's ρ=0.82), while no detectable mtDNAs in the corresponding spent culture media. Then the timeline correlation study revealed significant association between mitochondrial quantity and blastocyst features of both the day of expanded blastocyst formation (95% Confidence intervals, CIs: 0.27~4.89, p=0.03) and the timing of expanded blastocyst formation (tEB) (95% CIs: -0.24~-0.01, p=0.04) in the regression model, indicating a strong dependency between mitochondrial quantity and the blastocyst development timeline. For the contribution to early pregnancy, multivariate logistic regression models showed that the day of expanded blastocyst formation contributed to four endpoints persistently: positive for HCG (odd ratio, OR: 0.71, p=0.006), gestational sac (OR: 0.78, p=0.04), fetal heartbeat (OR: 0.71, p=0.004), and progression to 14 weeks (OR: 0.69, p=0.002). Contrastingly, no notable correlation was observed between the mitochondrial quantity and these endpoints., Conclusions: Strong interaction was observed between mitochondrial quantity and the blastocyst timeline, particularly the timing of expanded blastocyst formation. It suggests that the primary determinant influencing pregnancy outcomes lies in the time-dependent parameter of blastocyst rather than in the specific mitochondrial quantity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chuang, Chou, Kuan, Liu, Kao, Wu, Lai, Hsieh, Liang, Chen and Chen.)

This study examines the expression of three microRNAs (hsa-miR-661, hsa-miR-21-5p, hsa-miR-372-5p) in spent pre-implantation embryos culture media to identify possible new non-invasive biomarkers of embryo competence, predictive of development to the blastocyst stage. A preliminary analysis on 16 patients undergoing IVF cycles was performed by collecting and stored spent culture media on the fifth/sixth day of embryo culture. Expression of miRNAs was evaluated according to the embryos' fate: 1) NE/DG: non-evolved or degenerate embryos; 2) BLOK: embryos developed to the blastocyst stage. Preliminary results revealed a higher miRNAs expression in NE/DG spent media. To elucidate the roles of these miRNAs, we employed a robust bioinformatics pipeline involving: 1) in-silico miRNA Target Prediction using RNAHybrid, which identified the most-likely gene targets; 2) Construction of a Protein-Protein Interaction network via GeneMania, linking genes with significant biological correlations; 3) application of modularity-based clustering with the gLay app in Cytoscape, resulting in three size-adapted subnets for focused analysis; 4) Enrichment Analysis to discern the biological pathways influenced by the miRNAs. Our bioinformatics analysis revealed that hsa-miR-661 was closely associated with pathways regulating cell shape and morphogenesis of the epithelial sheet. These data suggest the potential use of certain miRNAs to identify embryos with a higher likelihood of developing to the blastocyst stage. Further analysis will be necessary to explore the reproducibility of these findings and to understand if miRNAs here investigated can be used as biomarkers for embryo selection before implantation into the uterus or if they may be reliable predictors of IVF outcome., Competing Interests: Declaration of Competing Interest No potential conflict of interest was reported by the authors., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

The quality of the recipient cytoplasm was reported as a crucial factor in maintaining the vitality of SCNT embryos and SCNT efficiency for dairy cows. Compared with oocytes matured in vivo, oocytes matured in vitro showed abnormal accumulation and metabolism of cytoplasmic lipids. L-carnitine treatment was found to control fatty acid transport into the mitochondrial β-oxidation pathway, which improved the process of lipid metabolism. The results of this study show that 0.5 mg/ml L-carnitine significantly reduced the cytoplasmic lipid content relative to control. No significant difference was observed in the rate of oocyte nuclear maturation, but the in vitro developmental competence of SCNT embryos was improved in terms of increased blastocyst production and lower apoptotic index in the L-carnitine treatment group. In addition, the pregnancy rate with SCNT embryos in the treatment group was significantly higher than in the control group. In conclusion, the present study demonstrated that adding L-carnitine to the maturation culture medium could improve the developmental competence of SCNT embryos both in vitro and in vivo by reducing the lipid content of the recipient cytoplasm., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest; financial; personal or other relationship with other people and organization., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Objective: Cryopreservation has some adverse effects on embryos including cell metabolism reduction, mitochondria and plasma membrane damage, excess production of 'Reactive Oxygen Species' and damage to DNA. In the present study. In this study we assessed the effect of coenzyme Q10 as an exogenous antioxidant on mouse embryos following cryopreservation., Methods: We collected mice embryos at the morula stage from uterine horns on the third day of gestation. The morulae were divided into 9 groups (1 control, 2 vehicles and 6 experimental), then vitrified. The culture and/or vitrification media of the experimental groups were supplemented by 10 or 30 μM of CoQ10. After one week, the embryos were warmed and then cultured. After 48 hours of embryo culture, the blastocyst rate, total cell number, viability; and after 72 hours of embryo culture, we assessed the hatching rate., Results: Blastocyst rate and hatching rate were significantly reduced in the groups containing 30 μM CoQ10 supplemented culture media compared to other groups (p<0.05). The hatching rate in the groups containing 10 μM CoQ10 supplemented in both culture and vitrification media was significantly higher than in the other groups (p<0.05). In groups containing 10 μM CoQ10 supplemented culture media, the viability was higher than that in the other groups (p<0.05)., Conclusions: It seems that CoQ10 in a dose-dependent manner is able to improve hatching rate and viability following cryopreservation through its antioxidant and anti-apoptotic properties, and through the production of ATP.

Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 μM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 μM, with no embryo formation occurring at 24 μM. Further analysis showed that embryos treated with 12 μM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells., Competing Interests: Declaration of competing interest Authors have no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Before blastocyst development, embryos undergo morphological and metabolic changes crucial for their subsequent growth. This study aimed to investigate the relationship between morula compaction and blastocyst formation and the subsequent chromosomal status of the embryos., Methods: This retrospective cohort study evaluated embryo development (n = 371) using time-lapse imaging; 94 blastocysts underwent preimplantation genetic testing for aneuploidy (PGT-A)., Results: The embryos were classified as fully (Group 1, n = 194) or partially (Group 2, n = 177) compacted. Group 1 had significantly higher proportions of good- and average-quality blastocysts than Group 2 (21.6% vs. 3.4%, p = 0.001; 47.9% vs. 26.6%, p = 0.001, respectively). The time from the morula stage to the beginning and completion of compaction and blastocyst formation was significantly shorter in Group 1 than in Group 2 (78.6 vs. 82.4 h, p = 0.001; 87.0 vs. 92.2 h, p = 0.001; 100.2 vs. 103.7 h, p = 0.017, respectively). Group 1 embryos had larger surface areas than Group 2 embryos at various time points following blastocyst formation. Group 1 blastocysts had significantly higher average expansion rates than Group 2 blastocysts (653.6 vs. 499.2 μm 2 /h, p = 0.001). PGT-A revealed a higher proportion of euploid embryos in Group 1 than in Group 2 (47.2% vs. 36.6%, p = 0.303)., Conclusion: Time-lapse microscopy uncovered a positive relationship between compaction and blastocyst quality and its association with embryo ploidy. Hence, compaction evaluation should be prioritized before blastocyst selection for transfer or cryopreservation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Study Question: Do embryos displaying abnormal cleavage (ABNCL) up to Day 3 have compromised live birth rates and neonatal outcomes if full blastulation has been achieved prior to transfer?, Summary Answer: ABNCL is associated with reduced full blastulation rates but does not impact live birth rates and neonatal outcomes once full blastulation has been achieved., What Is Known Already?: It is widely accepted that ABNCL is associated with reduced implantation rates of embryos when transferred at the cleavage stage. However, evidence is scarce in the literature reporting birth outcomes from blastocysts arising from ABNCL embryos, likely because they are ranked low priority for transfer., Study Design, Size, Duration: This retrospective cohort study included 1562 consecutive autologous in vitro fertilization cycles (maternal age 35.1 ± 4.7 years) performed at Fertility North, Australia between January 2017 and June 2022. Fresh transfers were performed on Day 3 or 5, with remaining embryos cultured up to Day 6 before vitrification. A total of 6019 embryos were subject to blastocyst culture, and a subset of 664 resulting frozen blastocysts was included for live birth and neonatal outcome analyses following single transfers., Participants/materials, Setting, Methods: ABNCL events were annotated from the first mitotic division up to Day 3, including direct cleavage (DC), reverse cleavage (RC) and <6 intercellular contact points at the 4-cell stage (<6ICCP). For DC and RC in combination, the ratios of affected blastomeres over the total number of all blastomeres up to Day 3 were also recorded. All pregnancies were followed up until birth with gestational age, birthweight, and sex of the baby being recorded., Main Results and the Role of Chance: Full blastulation rates for embryos showing DC (19.5%), RC (41.7%), <6ICCP (58.8%), and mixed (≥2) ABNCL types (26.4%) were lower than the rates for those without ABNCL (67.2%, P < 0.01 respectively). Subgroup analysis showed declining full blastulation rates with increasing ratios of combined DC/RC affected blastomeres over all blastomeres up to the 8-cell stage (66.2% when 0 affected, 47.0% when 0.25 affected, 27.4% when 0.5 affected, 14.5% when 0.75 affected, and 7.7% when all affected, P < 0.01). However, once full blastulation had been achieved, no difference was detected between DC, RC, <6ICCP, and no ABNCL blastocysts following single frozen transfers in subsequent live birth rates (25.9%, 33.0%, 36.0% versus 30.8%, P > 0.05, respectively), gestational age (38.7 ± 1.6, 38.5 ± 1.2, 38.3 ± 3.5 versus 38.5 ± 1.8 weeks, P > 0.05, respectively) and birthweight (3343.0 ± 649.1, 3378.2 ± 538.4, 3352.6 ± 841.3 versus 3313.9 ± 509.6 g, P > 0.05, respectively). Multiple regression (logistic or linear as appropriate) confirmed no differences in all of the above measures after accounting for potential confounders., Limitations, Reasons for Caution: Our study is limited by its retrospective nature, making it impossible to control every known or unknown confounder. Embryos in our dataset, being surplus after selection for fresh transfer, may not represent the general embryo population., Wider Implications of the Findings: Our findings highlight the incremental impact of ABNCL, depending on the ratio of affected blastomeres up to Day 3, on subsequent full blastulation. The reassuring live birth and neonatal outcomes of ABNCL blastocysts imply a potential self-correction mechanism among those embryos reaching the blastocyst stage, which provides valuable guidance for clinical practice and patient counseling., Study Funding/competting Interest(s): This research is supported by an Australian Government Research Training Program (RTP) Scholarship. All authors report no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing in vivo-derived embryos continued their development in vitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Objective: To examine the relationship between the day of embryo cryopreservation and large for gestational age (LGA) infants in women undergoing frozen embryo transfers (FETs) after cryopreservation on days 2-7 after fertilization and to compare the risk of the day of embryo cryopreservation to other possible risk factors of LGA after FET cycles., Design: Retrospective cohort study., Setting: Society of Assisted Reproduction Clinical Outcomes Reporting System., Patients: Women undergoing FET cycles., Intervention: Day of cryopreservation., Main Outcome Measure: Singleton LGA infant., Results: A total of 33,030 (18.2%) FET cycles in the study group (n = 181,592) resulted in LGA infants during the study period of 2014-2019. There was an increase in LGA risk when cryopreservation was performed from day 2 (13.7%) to days 3-7 (14.4%, 15.0%, 18.2%, 18.5%, and 18.9%). In the log-binomial model, the risk increased compared with days 2-3 combined when cryopreservation was performed on days 5-7 (adjusted relative risk [aRR] 1.32, 95% confidence interval [CI] 1.22-1.44 for day 5, aRR 1.34, 95% CI 1.23-1.46 for day 6, and aRR 1.42, 95% CI 1.25-1.61 for day 7). Other factors most associated with LGA risk in the log-binomial model were preterm parity of >3 compared with 0 (aRR 1.82, 95% CI 1.24-2.69) and body mass index (BMI) of >35 kg/m 2 compared with normal weight (aRR 1.94, 95% CI 1.88-2.01). Increasing gravity, parity, BMI, number of oocytes, and embryo grade were also associated with LGA in this model. Asian, Black, Hispanic, and combined Hawaiian and Pacific Islander were protective factors in the model compared with White patients. Low BMI (<18.5 kg/m 2 ) was also considered a protective factor in the model compared with normal BMI., Conclusion: Duration of embryo culture was associated with an increased risk of LGA in this study cohort when controlling for known confounders such as maternal BMI and parity. This study sheds new light on the possible link between FET and LGA infants., Competing Interests: Declaration of interests B.D.P. reports honoraria as an Examiner for the American Board of Obstetrics and Gynecology outside the submitted work. A.R. has nothing to disclose. N.S. reports funding from NICHD grant award HD R25 075737 outside the submitted work. M.S. has nothing to disclose., (Published by Elsevier Inc.)

Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups ( p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts.

IL-6 plays an important role in oogenesis in humans. However, at the preimplantation stage, IL-6 production and the role in embryo development remain unclear. In this study, IL-6 concentrations in single-embryo media were analyzed. In addition, the association between IL-6 production and blastocyst formation was investigated. Single-embryo culture media from 194 embryos were collected on day 6 after fertilization and divided into four groups according to the developmental stage of the corresponding embryo, as follows: cleavage stage group, morula-early blastocyst group, unavailable full blastocyst group, and available full blastocyst group. IL-6 concentrations were significantly lower in the cleavage stage group than in the morula-early blastocyst group (p = 0.009), in the unavailable full blastocyst group (p = 0.003), and in the available full blastocyst group (p < 0.001). Logistic regression analysis showed that IL-6 concentration in single-embryo medium was significantly associated with blastocyst formation (odds ratios β1 = 1.876, 95% CI 1.433 to 2.644, p < 0.0001). Therefore, IL-6 was produced by human preimplantation embryos throughout the preimplantation stage and may play a role in embryo development., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

The Association for the Study of Reproductive Biology (ASEBIR) Interest Group in Embryology (in Spanish 'Grupo de Interés de Embriología') reviewed key morphokinetic parameters to assess the contribution of time-lapse technology (TLT) to the ASEBIR grading system. Embryo grading based on morphological characteristics is the most widely used method in human assisted reproduction laboratories. The introduction and implementation of TLT has provided a large amount of information that can be used as a complementary tool for morphological embryo evaluation and selection. As part of IVF treatments, embryologists grade embryos to decide which embryos to transfer or freeze. At the present, the embryo grading system developed by ASEBIR does not consider dynamic events observed through TLT. Laboratories that are using TLT consider those parameters as complementary data for embryo selection. The aim of this review was to evaluate review time-specific morphological changes during embryo development that are not included in the ASEBIR scoring system, and to consider them as candidates to add to the scoring system. [ABSTRACT FROM AUTHOR]