Assessment Of Embryo Quality Research Articles

Application of imaging and spectroscopy techniques for grading of bovine embryos - a review.

Although embryo transfers have grown considerably in the cattle industry, the selection of embryos required for successful pregnancies remains a challenging task. Visual inspection of 7th-day embryos using a stereomicroscope, followed by classification based on morphological features is the most commonly practiced procedure. However, there are inaccuracies and inconsistencies in the manual grading of bovine embryos. The objective of this review was to evaluate the potential of imaging and spectroscopic techniques in the selection of bovine embryos. Digital analysis of microscopic images through extracting visual features in the embryo region, and classification using machine learning methods have yielded about 88-96% success in pregnancies. The Raman spectral pattern provides valuable information regarding developmental stages and quality of the embryo. The Raman spectroscopy approach has also been successfully used to determine various parameters of bovine oocytes. Besides, Fourier Transform Infrared (FTIR) spectroscopy has the ability to assess embryo quality through analyzing embryo composition, including nucleic acid and amides present. Hyperspectral Imaging has also been used to characterize metabolite production during embryo growth. Although the time-lapse imaging approach is beneficial for morphokinetics evaluation of embryo development, optimized protocols are required for successful implementation in bovine embryo transfers. Most imaging and spectroscopic findings are still only at an experimental stage. Further research is warranted to improve the repeatability and practicality to implement in commercial facilities.

New Technologies to Improve Embryo Assessment

Embryo quality is a key factor in determining whether an IVF cycle will result in a live birth. Current approaches for assessing embryo quality are subjective (morphology) or invasive (biopsy) and are not always predictive of live birth. Development of an accurate and non-invasive method to assess embryo quality would likely improve IVF success. We have taken a different approach towards developing a non-invasive diagnostic for embryo quality: optical imaging. We use low levels of light to illuminate the embryo and obtain spatial information on cell metabolism as well as biophysical properties of the embryo. Here I will present our work showing that hyperspectral imaging – captures natural autofluorescence emitted by the embryo – can be used to accurately discriminate between euploid and aneuploid mouse blastocysts (inner cell mass). Importantly, we show that this form of imaging is safe: compared to embryos that were not imaged, hyperspectral imaged embryos had similar, and not significantly different, pregnancy and implantation rates, with offspring weight at weaning also comparable. Another potential optical approach to assess embryo quality is digital holographic imaging. To date, little attention has been paid to the physical parameters of an embryo and whether these could be markers of embryo quality. One such physical parameter is refractive index. We have shown that digital holographic microscopy is able to measure very small changes in refractive index between good and poor-quality mouse embryos. Importantly, these optical approaches occur in the absence of stains or exogenous tags. Used alone, or in combination, such optical approaches may prove beneficial in developing to an accurate, non-invasive diagnostic for embryo quality.

#225 : miR-135b Expression Correlated with Aneuploidy in Trophectoderm from IVF Patients: An Approach to NIR-Invasive Screening for Embryo Quality Assessment

Background and Aims: MicroRNAs (miRNAs) are abundantly expressed in rapidly growing and undifferentiated cells, for example, embryonic stem cells. This led to the discovery that miRNAs are significantly expressed in human embryos and that intracellular miRNAs expression patterns differ between euploidy and aneuploid embryos. Our study aims to determine the correlation between miR-35b and embryo chromosomal status. Methods: Thirty-six blastocyst culture media were collected from patients who underwent the In Vito Fertilization (IVF) program followed by Preimplantation Genetic Testing for Aneuploidy (PGT-A) at Dr. Ciptomangunkusumo General Hospital, Jakarta. The trophectoderm cells were biopsied, and half of the spent media culture was evaluated for aneuploidy detection using Next Generation Sequencing (NGS). While the rest of the spent media on day five were analyzed for miR-135b expression using quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). All the data were analyzed using SPSS, and the sensitivity and specificity of the miR-135b were analyzed using the ROC curve. Results: miR-135b relative expression was detected in blastocyst spent blastocyst media culture and not in culture media without embryo. Based on the average of miR-135b, expression was higher in aneuploidy embryos (7.9(0.01-1-45.57)) than embryos with no aneuploidy detected (0.7(0.003-9.65)) and statistically different with p¡0.05. miR-135 relative expression in spent culture media was 10.72-fold higher in aneuploidy compared with an embryo with a normal chromosome. Based on the validation, the sensitivity value was 71.4%, and the specificity was 68.2%, with a cut-off of the relative expression of miR-135b was 1.185-fold. Conclusion: miR-135b detected in IVF spent culture media from blastocyst media culture. The significant expression of miR-135b in an aneuploidy embryo makes miR-135b a potential biomarker for predicting the chromosomal quality of blastocyst embryos.

Debate 4: Morphological Assessment of Embryos is Outdated

Motion: For The Outdated Significance of Morphological Assessment in Embryo Selection and the Rise of Advanced Technologies in Reproductive Medicine This symposium lecture presents a compelling debate, shedding light on the diminishing relevance of traditional static morphological assessment as a primary criterion for embryo. The speaker aims to challenge the long-standing reliance on morphological evaluation and advocate for the integration of cutting-edge technologies as a more reliable and objective means of embryo quality assessment. During the debate, the speaker will examine the limitations of morphological assessment, acknowledging its subjective nature, potential bias, and lack of comprehensive insight into embryonic development. The traditional morphological assessment based solely on visual appearance may not adequately predict an embryo’s true potential for successful implantation and healthy pregnancy. A variety of available new technologies will be highlighted during the lecture, showcasing alternatives to morphological embryo assessment. Attendees will gain insight into how these innovative methods can offer more objective and precise measures of embryo quality, enhancing the success rates of assisted reproductive procedures. The symposium will foster an engaging environment, encouraging active participation and thoughtful discussions among attendees from the reproductive medicine community. Participants will have the opportunity to challenge prevailing notions and explore the implications of adopting new technologies in clinical practice. In summary, this debate aims to catalyze a paradigm shift in embryo evaluation, emphasizing the need to move beyond traditional morphological assessment towards embracing advanced technologies, to optimize embryo selection and lead to improved outcomes in IVF. Motion: Against Morphological Assessment of Embryos is Not Outdated In the era of new technologies and artificial intelligence, looking good has never been more important! On-time progression through specific events is requisite for human preimplantation development and provides important visual insights into the health and physiology of the embryo. The formation of 2 distinct pronuclei, the times and patterns of cleavage, and how the cells compact are all key markers of development. Following compaction, the formation of the blastocyst is not only a temporally regulated event, but also an energetically demanding process, one that reflects the metabolic activity (a biomarker of viability) of the embryo. Further, at the blastocyst stage it is possible to visualise and quantitate the formation of both the inner cell mass (ICM) and trophectoderm, with all parameters of blastocyst development linked to transfer outcomes. The development of time-lapse incubation, which can be described as embryo morphology over time, has led to the incorporation of several key morphological features to support embryo selection, further reflecting the significance of embryo morphology. Interestingly, AI algorithms used for embryo selection reflect predominantly those morphological parameters that are known to affect transfer outcomes, such as blastocyst quality. While only specific molecular tests can accurately determine the precise chromosomal composition of an embryo, key morphological features and events can also be used to tag those embryos at greatest risk of being genetically abnormal. In conclusion, key morphological developmental events indirectly inform us of the physiology of the embryo (degree of blastocyst expansion reflects metabolic activity for example), and hence this is why morphological grading systems (such as the Gardner Grade for blastocysts) have been shown to be effective in embryo selection for over the past 25 years, and further have proven to be as useful as AI or biomarker analysis for the majority of patients.

Discard or not discard, that is the question: an international survey across 117 embryologists on the clinical management of borderline quality blastocysts.

Do embryologists from different European countries agree on embryo disposition decisions ('use' or 'discard') about Day 7 (>144 h post-insemination) and/or low-quality blastocysts (LQB; <Gardner's BB) (i.e. borderline quality blastocysts)? The prevalence of 'discard' answers was 38.7%; nevertheless, embryologists' agreement was overall just fair (Fleiss-k = 0.26). The utilization of LQBs and adoption of culture beyond 144 h post-insemination is increasing worldwide. Although morphology and morphokinetics are associated with embryo developmental competence, previous studies demonstrated significant interobserver variability among embryologists regarding embryo quality assessment and disposition decisions for borderline quality blastocysts. An anonymous survey was run in a large network of IVF centers. A total of 117 embryologists from 6 European countries and 29 IVF centers filled in the survey. Randomly selected anonymous time-lapse videos of 50 Day 7 and/or LQB whole embryo preimplantation development were assessed by the embryologists. The key information on patients/cycles was provided along with each video. All cycles entailed preimplantation genetic testing for aneuploidies. Each embryologist specified whether he/she would have discarded or used ('transfer-fresh'/'cryopreserve'/'biopsy') any embryo. Inter-rater agreement was measured with Fleiss-k. Examiners were asked about their years of experience, center location, average number of cycles and average maternal age, number of colleagues, and use of time-lapse incubators at their centers. All participants were blinded to artificial intelligence (AI) scores generated by two commercially available software packages, chromosomal diagnosis (all blastocysts were tested for aneuploidies), and clinical outcomes after vitrified-warmed euploid single blastocyst transfer. These data were known only by one embryologist not involved in the survey. Participants were Italian (40%, N = 47), Spanish (24%, N = 28), Portuguese (5%, N = 6), Czech (5%, N = 6), Swedish (23%, N = 27), and Icelandic (3%, N = 3). In total, 2263 (38.7%) 'discard' and 3587 (61.3%) 'use' decisions were recorded. Czech, Portuguese, and Italian embryologists expressed lower 'discard' decision rates (mean ± SD 17 ± 7%, range 8-24%; 23 ± 14% range 4-46%; and 27 ± 18% range 2-72%, respectively), while Spanish gave intermediate (37 ± 16% range 4-66%) and Nordic gave higher (67 ± 11% range 40-90%) rates. The prevalence of 'discard' answers was 38.7% out of 5850 choices (mean per embryologist: 39 ± 23% range 2-90%). Only embryologists' country and IVF group were associated with this rate. Overall agreement among embryologists was fair (Fleiss-k = 0.26). The prevalence of 'discard' responses per embryo was 37 ± 24% (range 2-87%). Only the number of sibling blastocysts influenced this rate (i.e. the larger the cohort, the higher the inclination to 'discard'). No difference was shown for the two scores between euploid and aneuploid borderline quality blastocysts, while the embryologists were, by chance, more prone to 'discard' the latter (28.3 ± 21% range 9-71% versus 41.6 ± 24.8% range 2-87%, respectively). The survey included only private IVF clinics located in Europe. Moreover, a key variable is missing, namely patients' access to care. Indeed, all embryologists involved in the survey were part of the same network of private IVF clinics, while the embryo disposition decisions might be different in a public setting. Decision-making by European embryologists regarding Day 7 embryos or LQBs is inconsistent with putative clinical consequences, especially in patients with low prognosis. Although the embryologists could make decisions independent from their local regulations, their mindset and clinical background influenced their choices. In the future, AI tools should be trained to assess borderline quality embryos and empowered with cost-effectiveness information to support embryologists' decisions with more objective assessments. No external funding was obtained for this study. The authors have no conflict of interest to declare. N/A.

The role of serum vitamin D in patients with normal ovarian reserve undergoing the first IVF/ICSI cycle.

The debate over the impact of vitamin D in assisted reproduction continues. The purpose of our study was to assess embryo quality and pregnancy outcomes among groups with different levels of vitamin D after the first in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycle in patients with normal ovarian reserve (NOR). Patients in this retrospective cohort study were divided into three groups: severe vitamin D deficiency group (25OH-D < 10 ng/ml), vitamin D deficiency group (10 ng/ml ≤ 25OH-D < 20 ng/ml), and non-vitamin D deficiency group (25OH-D ≥ 20 ng/ml). The primary outcome was clinical pregnancy, while the secondary outcomes were mature oocytes, oocyte fertilization, available cleavage embryos, available blastocysts, biochemical pregnancy, early abortion, and embryo implantation. A modified Poisson regression model and multiple linear regression analysis were conducted for the multivariate analysis. 264 NOR patients undergoing the first IVF/ICSI cycles were included. For the primary outcome, there was no significant difference in clinical pregnancy between the severe vitamin D deficiency group and the other two groups (vitamin D deficiency group: adjusted RR = 1.026; 0.780 - 1.350; P = 0.854; non-vitamin D deficiency group: adjusted RR = 1.092; 0.743 - 1.605; P = 0.652). For all secondary outcomes, no significant differences were observed among the severe vitamin D deficiency, vitamin D deficiency, and non-vitamin D deficiency groups (P > 0.05). Exploratory subgroup analyses concerning the season of embryo transfer, phase of embryo transferred, and endometrial thickness, as well as the sensitivity analysis using logistic regression models for the primary outcome, revealed comparable clinical pregnancy rates among the groups (P > 0.05). Subgroup analysis concerning ovarian stimulation protocol indicated that in the subgroup of gonadotrophin-releasing hormone (GnRH) antagonist protocol, the clinical pregnancy rate of the non-vitamin D deficiency group was significantly higher than that of the other two groups (P < 0.05). Serum vitamin D level was not associated with embryo quality and pregnancy outcomes for patients with NOR. Further studies with greater sample sizes and a longer follow-up period are needed to elucidate the relationships between vitamin D levels and IVF outcomes.

P-152 Velocity of cytoplasmic movement in trophectodermal cells depends on the keratin cytoskeleton

Abstract Study question Can dysfunction of the keratin cytoskeleton, known to severely hinder postimplantation development of mammalian embryos, be detected by analysis of cytoplasmic movement in trophectodermal cells? Summary answer Depletion of keratins increases cytoplasmic movement in trophectodermal cells in extent depending on the developmental stage and on the location of the trophectodermal cells (mural/polar). What is known already Recent literature data indicate that keratins, intermediate filament proteins, are key regulators of trophectoderm function in mouse and human embryos. Keratin knockouts display trophoblast fragility, placental bleeding, and lethality after implantation. However, depletion of keratin 8 (Krt8) and keratin 18 (Krt18), which are most abundant in preimplantation embryos, does not lead to severe phenotype up to the blastocyst stage at least in mice and cattle, so can easily remain unnoticed. However, it affects the biomechanical properties of the trophectoderm. Analysis of cytoplasmic movement allows for biomechanical assessment of the trophectoderm in a non-invasive way. Study design, size, duration We analyzed 188 mouse embryos (66 control, 60 with Krt8 depletion, and 62 with Krt18 depletion) to quantitatively assess changes in the velocity of cytoplasmic movement caused by a knockdown of Krt8 and Krt18. Additionally, we correlated the velocity of cytoplasmic movement in trophectodermal cells (n = 70 embryos) with the ability to form outgrowths. Appropriate statistical tests were used to determine the statistical significance of the obtained results. A value of p &amp;lt; 0.05 was considered statistically significant. Participants/materials, setting, methods Zygotes were injected with Krt8 and Krt18 siRNAs or with scrambled siRNAs used as a negative control. The efficiency of the knockdown was tested on mRNA and protein levels. The embryos were cultured for 3 or 4 days and the velocity of cytoplasmic movement in polar and mural trophectoderm cells was measured. Additionally, we correlated the cytoplasmic movement velocity in trophectodermal cells in E3.5 embryos with their ability to implant in vitro (i.e., form outgrowths). Main results and the role of chance Our research showed that the velocity of cytoplasmic movement of trophectodermal cells varies depending on the embryo's developmental stage. Namely, the velocity of cytoplasmic movement was higher in E3.5 blastocysts than in E4.5 blastocysts. In addition, the velocity of cytoplasmic movement was higher in polar (8.45 ± 2.64 nm/s in E3.5 and 6.79 ± 1.96 nm/s in E4.5 control embryos) than in mural (6.30 ± 2.30 nm/s in E3.5 and 4.98 ± 1.89 nm/s in E4.5 control embryos) trophectodermal cells. Noteworthy, the depletion of Krt8 and Krt18 increased the velocity of cytoplasmic movement both in the mural and polar trophectodermal cells. However, it appears that changes caused by the depletion of these keratins depend on the embryo's developmental stage. For example, the depletion of Krt18 increased the velocity of cytoplasmic movement in mural trophectodermal cells in E3.5 embryos (9.61 ± 3.80 nm/s), while the depletion of Krt8 - in E4.5 embryos (6.65 ± 2.35 nm/s). In addition, data from our preliminary experiments with in vitro implantation suggested that embryos characterized by the higher cytoplasmic movement velocity were unable to form an outgrowth. The above data suggest that the velocity of cytoplasmic movement depends on the keratin cytoskeleton and probably can be used to predict an embryo’s ability to implant. Limitations, reasons for caution We used mouse embryos, a well-established model of early mammalian embryo development. However, analogous experiments should be repeated on other mammalian species, including humans, if we wish to pursue the possibility that cytoplasmic velocity in trophectodermal cells is an embryo quality marker. Wider implications of the findings Our data indicate that cytoplasmic velocity in trophectodermal cells may be a valuable tool in embryo quality assessment, as it reflects the functionality of the keratin cytoskeleton in embryos as well as correlates with the embryos’ ability to implant. Trial registration number not applicable

O-120 Predicting Embryo Ploidy Status Using Time-lapse Images

Abstract Study question Can deep learning models using time-lapse images of embryo development be used to predict embryo ploidy and provide supplemental information to embryologists for clinical decision-making? Summary answer We developed a general MDBS-Ploidy model that uses time-lapse images and maternal age to predict embryo quality scores and ploidy status. What is known already Ploidy status, or the presence or lack of chromosomal abnormalities, is an important factor in a successful pregnancy. Embryos with abnormalities are classified as aneuploid, whereas those without are euploid. With the advent of artificial intelligence in computer vision and the collection of large IVF-related datasets combining images, videos, and clinical outcomes, a variety of methods have been developed to automatically assess embryo quality and other characteristics using images from time-lapse sequences. To our knowledge, a video classification model validated by heterogeneous data to predict both embryo quality score and ploidy is lacking. Study design, size, duration The training dataset for the model consisted of 1,998 time-lapse sequences captured by the Embryoscope®. Time-lapse image sequences consisted of around 360–420 frames captured during 5 days of development. PGT-A results were used as ground truth labels for all ploidy prediction tasks, with embryos classified as euploid or aneuploid. The dataset also included clinical information such as blastocyst score (BS) and maternal age at the time of oocyte retrieval. Participants/materials, setting, methods The MDBS-Ploidy consists of two steps. The first step is quality score prediction from day-5 time-lapse video input using a Bidirectional Long Short-Term Memory architecture with added output layers for multitasking. In the second step, the predicted score, in addition to maternal age, is used to predict the ploidy status of the embryo using a logistic regression model. We evaluated the performance of the MDBS-Ploidy using area under the receiver-operating-characteristic (AUROC) on a validation dataset. Main results and the role of chance For the MDBS-Ploidy quality score prediction module, the Pearson correlation between scores predicted by the MDBS-Ploidy and ground truth quality scores is 0.70 on the validation dataset, suggesting moderate correlation strength. As for the aneuploidy prediction module, the MDBS-Ploidy can discriminate between euploidy and aneuploidy with an AUROC of 0.76 ± 0.002. The MDBS-Ploidy performs comparably with a model trained on the embryologist-annotated blastocyst score. We replicate these comparative results in external validation datasets as well, namely the Embryoscope+® dataset with 1,000 embryos and the IVI Valencia Spain dataset with 543 embryos. Moreover, the MDBS-Ploidy is completely automated, requiring only time-lapse images from 96 to 112 hpi and maternal age to predict embryo ploidy status. This allows the MDBS-Ploidy to be adapted clinically without interrupting ongoing workflows. The MDBS-Ploidy also provides a certain level of explainability; embryologists can use the intermediate quality score (from the first module) to determine why an embryo is classified as a certain ploidy status. With a recall of 0.84 ± 0.004 for the validation dataset, the MDBS-Ploidy shows promise for successfully selecting euploid embryos. We believe that the MDBS-Ploidy can provide supplemental information for selecting the most viable embryo. Limitations, reasons for caution Our model primarily uses data from time-lapse microscopy. Clinics without access to this technology will be unable to use our model. Wider implications of the findings This model is clinically relevant for making decisions as to whether an embryo may be chromosomally normal. Automated quality score prediction is instrumental to embryologists who are currently annotating embryos manually. Future iterations of the model can theoretically be adopted into clinical practice because it is end-to-end and fully automated. Trial registration number not applicable

O-238 Artificial intelligence algorithms in assisted reproduction: differences in the evaluation of embryos from fresh or vitrified donor oocytes

Abstract Study question Do the Embryoscope and EmbryoscopePlus systems' built-in software evaluate differently embryos from vitrified or fresh oocytes? Summary answer The embryo scores provided by artificial intelligence (AI) algorithms are lower for embryos originated from vitrified/warmed oocytes than for those that come from fresh oocytes. What is known already Time-lapse technology has enabled embryologists to develop several AI-based tools that allow the automation and standardization of embryo quality assessment. EmbryoScope and EmbryoScopePlus systems include deep learning-based models using only time-lapse image sequences (iDAScore v1.0 and v.2.0) and the machine learning-based model (KIDScore D5 v3) which classifies embryos in categories based on cleavage time points and blastocyst appearance. It has been reported that fertilization, pregnancy and implantation rates in vitrified oocytes are comparable to those of fresh oocytes. To our knowledge, this is the first study to test whether artificial intelligence tools assess embryos derived from these oocytes differently. Study design, size, duration This retrospective study includes the analysis of 34950 videos of embryos from fresh and vitrified donor oocytes. Embryos were cultured on EmbryoScope and EmbryoScopePlus Time Lapse systems and morphological and morphokinetic parameters were automatically detected by EmbryoViewer software. Senior embryologists routinely evaluated the embryos according to the ASEBIR criteria. In addition, embryos were graded using IDAScore v1, IDAScore v2 and KIDScoreD5 algorithms in different scores from low to high quality (1-9.9). Participants/materials, setting, methods The scores of embryos from fresh or vitrified donor oocytes obtained with the different algorithms were compared. Specifically, 21533 embryos from fresh and 12710 from vitrified oocytes with the IDAScore v1; 21335 from fresh and 12705 from vitrified with the IDAScore v2; and 13544 from fresh and 6559 from vitrified with the KIDScore v3. In addition, the ASEBIR evaluation performed by embryologists was also compared between embryos from fresh (21968) and vitrified oocytes (12980). Main results and the role of chance Mean scores of embryos coming from fresh oocytes from IDAScore v1, IDAScore v2 and KIDScore v3 were 5.34 ± 2.91, 3.48 ± 2.93, 5.13 ± 2.1 respectively. And mean scores of those from vitrified oocytes were 5.09 ± 2.83, 3.1 ± 2.71, 4.99 ± 2.01. Thus, mean embryo scores obtained by the 3 artificial intelligence algorithms were significantly higher* for embryos from fresh oocytes compared to those from vitrified oocytes. However, this lower embryo assessment of those from vitrified oocytes was not detected when comparing embryo quality according to ASEBIR classification. We hypothesized that it might be because several embryos that had not reached blastocyst stage on D6 were included in this comparison. Comparisons were repeated excluding these embryos, mean scores of embryos from fresh oocytes from IDAScore v1, IDAScore v2 and KIDScore v3 were 7.31 ± 2.12, 5.15 ± 2.79, 5.18 ± 2.07 respectively. Mean scores of those from vitrified oocytes were 7.14 ± 2.12, 4.75 ± 2.70, 5.01 ± 2.01. Again, mean scores of embryos from fresh oocytes were significantly higher* than those from vitrified oocytes. In this analysis, this significant difference* in embryo score was also detected according to the ASEBIR classification. *p&amp;lt;.05 Limitations, reasons for caution This project is limited by its retrospective and single-center nature. A multicenter study could be performed to determine whether these minimal but significant differences in the evaluation of embryos from fresh or warmed oocytes have any impact on clinical outcome. Wider implications of the findings This study shows that embryos from vitrified oocytes have significantly lower scores than those from fresh oocytes. The embryo assessment performed in this study is based on AI models that allow their instantaneous, non-invasive, accurate and objective evaluation. Thus, the impact of these differences on clinical outcome, should be analyzed. Trial registration number not applicable

RNA sequencing-based transcriptome analysis of granulosa cells from follicular fluid: Genes involved in embryo quality during in vitro fertilization and embryo transfer.

Granulosa cells play an important role in folliculogenesis, however, the role of RNA transcripts of granulosa cells in assessing embryo quality remains unclear. Therefore, we aims to investigate that RNA transcripts of granulosa cells be used to assess the probability of the embryonic developmental capacity. This prospective cohort study was attempted to figure out the probability of the embryonic developmental capacity using RNA sequencing of granulosa cells. Granulosa cells were collected from 48 samples in good-quality embryo group and 79 in only poor- quality embryo group from women undergoing in vitro fertilization and embryo transfer treatment. Three samples from each group were used for RNA sequencing. 226 differentially expressed genes (DEGs) were related to high developmental competence of embryos. Gene Ontology enrichment analysis indicated that these DEGs were primarily involved in biological processes, molecular functions, and cellular components. Additionally, pathway analysis revealed that these DEGs were enriched in 13 Kyoto Encyclopedia of Genes and Genomes pathways. Reverse transcription quantitative polymerase chain reaction verified the differential expression of the 13 selected DEGs. Among them,10 genes were differently expressed in the poor-quality embryo group compared to good-quality embryo group, including CSF1R, CTSH, SERPINA1, CYP27A1, ITGB2, IL1β, TNF, TAB1, BCL2A1, and CCL4. RNA sequencing data provide the support or confute granulosa expressed genes as non-invasive biomarkers for identifying the embryonic developmental capacity.

Potential roles of extracellular vesicles as a noninvasive tool for prenatal genetic diagnosis

The rate of infertility is increasing owing to genetic and environmental factors. Consequently, assisted reproductive technology has been introduced as an alternative. Bearing in mind the global trend toward the transfer of only one embryo, there is an increasing trend for assessing embryo quality before transfer through prenatal genetic diagnosis (PGD) tests. This ensures that the best-quality embryos are implanted into the uterus. In the invitro fertilization cycle, PGD is not only used for diseases or quality checks before embryo freezing but also for evaluating unfortunate risks, such as aneuploidy, signs of early abortions, and preterm birth. However, traditional preimplantation genetic testing and screening approaches are invasive and harm the health of both the mother and embryo, raising the risk of miscarriage. In the last decade, embryonic extracellular vesicles (EVs) have been investigated and have emerged as a promising diagnostic tool. In this mini-review, we address the use of EVs as a noninvasive biomarker in PGD to test for biological hazards within the embryo without invading its cells. We summarize the state-of-the-art in the use of the embryo's EV content, genomic DNA, messenger RNA, and microRNA in the spent culture medium and their relationship with embryo quality, successful implantation, and pregnancy.

MicroRNAs as A Biomarker to Predict Embryo Quality Assessment in In Vitro Fertilization.

Embryo selection for in vitro fertilization (IVF) is an effort to increase the success rate of embryo implantation. Factors influencing the success of embryo implantation include embryo quality, endometrial receptivity, embryo characteristics, and maternal interactions. Some molecules have been found to influence these factors, but their regulatory mechanisms are unclear. MicroRNAs (miRNAs) are reported to play an essential role in the embryo implantation process. miRNAs are small non-coding RNAs consisting of only 20 nucleotides that play an essential role in the stability of gene expression regulation. Previous studies have reported that miRNAs have many roles and are released by cells into the extracellular environment for intracellular communication. In addition, miRNAs can provide information related to physiological and pathological conditions. These findings encourage research development in determining the quality of embryos in IVF to increase the implantation success rate. Moreover, miRNAs can provide an overview of embryo-maternal communication and potentially be noninvasive biological markers of embryo quality, which could increase assessment accuracy while reducing mechanical damage to the embryo itself. This review article summarizes the involvement of extracellular miRNAs and the potential applications of miRNAs in IVF.

Metabolic Signature of Energy Metabolism Alterations and Excess Nitric Oxide Production in Culture Media Correlate with Low Human Embryo Quality and Unsuccessful Pregnancy.

Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those in the highest class give rise to successful pregnancy. Previous studies highlighted the potentiality of the analysis of metabolites in human embryo culture media, useful for the selection of embryos for implantation. In the present study, we analyzed in blind 66 human embryo culture media at 5 days after in vitro fertilization with the aim of quantifying compounds released by cell metabolism that were not present as normal constituents of the human embryo growth media, including purines, pyrimidines, nitrite, and nitrate. Only some purines were detectable (hypoxanthine and uric acid) in the majority of samples, while nitrite and nitrate were always detectable. When matching biochemical results with morphological evaluation, it was found that low grade embryos (n = 12) had significantly higher levels of all the compounds of interest. Moreover, when matching biochemical results according to successful (n = 17) or unsuccessful (n = 25) pregnancy, it was found that human embryos from the latter group released higher concentrations of hypoxanthine, uric acid, nitrite, and nitrate in the culture media. Additionally, those embryos that developed into successful pregnancies were all associated with the birth of healthy newborns. These results, although carried out on a relatively low number of samples, indicate that the analysis of the aforementioned compounds in the culture media of human embryos is a potentially useful tool for the selection of embryos for implantation, possibly leading to an increase in the overall rate of ART.

Assessing embryo quality with digital holographic microscopy

A low-powered, non-invasive digital holographic microscopic imaging technique for embryo quality assessment is presented. Two groups of 2 cell-stage embryos cultured in media of different lipid concentrations have been characterized with digital holographic microscopy for lipid aggregation. The study suggests refractive index measurements are reflective of the lipid and dry mass content in embryos thus making DHM a prospective label-free diagnostic tool for quality assessments in assisted reproduction.

Alpha-lipoic acid improves bovine preimplantation blastocyst quality and cryotolerance

In vitro embryo production has grown in recent decades due to its great potential for cattle production. However, the quality of in vitro-produced embryos is lower compared with those produced in vivo. The postfertilization culture environment has a major influence on bovine embryo quality. We hypothesize that the inclusion of the inclusion of alpha-lipoic acid (ALA) in the in vitro culture (IVC) medium during the first 24 h would have positive effects on embryo development in vitro and cryotolerance. The aims of this study were to evaluate the antioxidant effect of ALA in IVC medium for 24 h on bovine zygotes (21 h post in vitro fertilization, IVF), day 2 cleaved embryos (46 h post-IVF), and to assess embryo quality, developmental competence, and cryotolerance after vitrification. In all experiments, IVC medium was the Control, and 2.5 μM ALA was the treatment implemented. Viability and reactive oxygen species (ROS) levels in zygotes and day 2 embryos did not differ from the Control (P > 0.05). Supplementation with ALA increased total blastocyst and hatching rates (P < 0.05). It also improved embryo quality, evidenced by the increased blastocyst total cell number and the percentage of excellent-quality embryos observed (P < 0.05). In embryos cultured with ALA and then vitrified, ALA reduced intracellular ROS levels in warmed blastocysts (P < 0.05). In conclusion, ALA supplementation to IVC medium during 24 h is a new advantage in improving embryo quality for assisted bovine reproduction.

Modeling three sources of uncertainty in assisted reproductive technologies with probabilistic graphical models

Embryo selection is a critical step in assisted reproduction: good selection criteria are expected to increase the probability of inducing a pregnancy. Machine learning techniques have been applied for implantation prediction or embryo quality assessment, which embryologists can use to make a decision about embryo selection. However, this is a highly uncertain real-world problem, and current proposals do not model always all the sources of uncertainty.We present a novel probabilistic graphical model that accounts for three different sources of uncertainty, the standard embryo and cycle viability, and a third one that represents any unknown factor that can drive a treatment to a failure in otherwise perfect conditions. We derive a parametric learning method based on the Expectation–Maximization strategy, which accounts for uncertainty issues.We empirically analyze the model within a real database consisting of 604 cycles (3125 embryos) carried out at Hospital Donostia (Spain). Embryologists followed the protocol of the Spanish Association for Reproduction Biology Studies (ASEBIR), based on morphological features, for embryo selection. Our model predictions are correlated with the ASEBIR protocol, which validates our model. The benefits of accounting for the different sources of uncertainty and the importance of the cycle characteristics are shown. Considering only transferred embryos, our model does not further discriminate them as implanted or failed, suggesting that the ASEBIR protocol could be understood as a thorough summary of the available morphological features.

Self-reparation signs in embryo development

Background and Aim: Time-lapse systems allow IVF laboratories to perform in-depth analysis of the embryo development thanks to the continuous monitorization. Some events impossible to detect with conventional morphologic evaluation, such as reverse cleavage or multinucleation, can be detected using time-lapse. Even though the low scientific evidence, the presence of these events is considered as negative factor when the embryo quality assessment is performed.

Non-Invasive Metabolic Live Cell Imaging of Early Embryo Development Using Novel Adapted Confocal Microscopy in Ageing

Background: Developing non-invasive methods that are reliable to assess embryo quality has been a significant aim for assisted reproductive technologies. Changes in metabolic activity could lead to abnormal embryo development and implantation potential which could be potentially predicted by incorporating non-invasive measurements of metabolism. Metabolic imaging of nicotinamide adenine dinucleotide (NADH) has been well studied in cell lines, however, evidence for its use in embryo development is limited. Measurements of NADH during advanced-reproductive-age could be a useful tool as the auto-fluorescence of NADH is a straightforward representation of mitochondrial function and cellular redox status. Aim: To determine potential differences in NADH levels associated in early embryo development from young and old mice using novel adapted confocal microscopy. Methods: Oocytes from two groups of 20 super-ovulated female mice (Young: 5-8 weeks old, n=10; Old: 45-52 weeks old, n=10) were inseminated through in-vitro fertilisation (IVF). Inseminated oocytes (Young:93; Old:64) were assessed for fertilisation and embryo development to the blastocyst stage. Early embryos (Young:44; Old:34) from both young and old groups were assessed for NADH levels during embryo development every 2 and 3 hours respectively in separated experiments, using arbitrary units of auto-fluorescence (AU). Results: IVF experiments showed no significant differences in fertilisation rates between age groups but with significant differences in blastocyst formation (Young: 90.9%; Old: 66.7%; p[Formula: see text]0.05). Embryos from young and old females showed significantly different NADH levels during embryo development (Young: 29,889 ±864AU; Old: 25,972 ±260AU; p[Formula: see text]0.0001). Zygotes that reached the blastocyst stage in old females had significantly decreased NADH levels compared to young females (Young: 31,508 ±853AU, Old: 26,417 ±186AU; p=0.0002). Conclusion: Our study indicates different levels of NADH activity during early embryo development associated to ageing and blastocyst formation. Non-invasive measurements of NADH could be applied to determine embryo metabolic activity associated to ageing using simple technology.

Embryologist agreement when assessing blastocyst implantation probability: is data-driven prediction the solution to embryo assessment subjectivity?

Embryologist agreement when assessing blastocyst implantation probability: is data-driven prediction the solution to embryo assessment subjectivity?

P-209 Overweight and obese women have slower developing embryos and a lower rate of morphokinetic top-quality blastocysts compared to underweight and normal weight women

Abstract Study question Does maternal body mass index affect embryo morphokinetics and the rate of top-quality blastocysts Summary answer Overweight and obese women had slower developing embryos and a lower rate of top-quality blastocysts compared to underweight and normal weight women What is known already Time-lapse monitoring (TLM) allows for a detailed collection of embryo morphologic and kinetic changes compared to traditional periodic daily assessments. TLM allows for continuous observation of the developing embryo in time intervals for dynamic assessment of embryo morphokinetics. This may overcome any potential and unnecessary environmental fluctuations in comparison to the removal of embryos from the incubator for traditional static morphologic evaluations. There is a continuing strive to assess what factors are involved in embryo morphokinetics. Previous studies found maternal weight (either being underweight or overweight) to possibly be a contributing factor to embryo kinetic development, with conflicting results. Study design, size, duration Embryos derived from GnRH antagonist ICSI cycles between July 2013-December 2020 were included. Embryo division timings and morphokinetic top-quality embryo rates were compared between underweight (&amp;lt;18 kg/m2) normal weight (8.5–25 kg/m2) overweight (25–30 kg/m2 ) and obese (≥30 kg/m2) women. Top-quality embryos with the highest chance to implant were defined: tPNf &amp;lt;24.08, t2&amp;lt;26.6, S2&amp;lt;0.9, t8&amp;lt;56 hours post insemination (hPi) for cleavage embryos and t2&amp;lt;26.6, S2&amp;lt;0.9, t8&amp;lt;56 and tSB&amp;lt;96.6 hPi for blastocysts. Participants/materials, setting, methods Optimal time durations: time to polar body extrusion (tPB2), time to pronucleus fading (tPNf), cleavage timings (t2-t8), synchrony of the second and third cycles (S2 and S3), duration of the second cycle (CC2) time to start blastulation (tSB) were based on an in-house model including embryos with known implantation data (KID). Multivariate logistic regression analysis: age, type of gonadotrophins, number of oocytes aspirated and peak estradiol levels was performed. Pregnancy rates were calculated from transfers. Main results and the role of chance We analyzed 1,953 embryos derived from 705 GnRH-antagonist ICSI cycles; 123 embryos from 43 cycles of underweight women, 888 embryos from 319 cycles of normal weight women, 517 embryos from 186 cycles of overweight women and 425 embryos from 157 cycles of obese women. Mean BMI was 17.3 kg/m2for underweight women, 21.8 kg/m2 for normal weight, 26.6 kg/m2 for overweight and 33.8 kg/m2 for obese women. Maternal age, gravidity and parity were similar between the groups. Pregnancy rates were similar between the groups. Overweight and obese women had a statistically significant longer mean time across cell stages t5-8 and tSB compared to underweight and normal weight women. Overweight and obese patients had the lowest rate of top-quality blastocysts (10.9% and 13.4% respectively) compared with underweight and normal weight women versus (33.9% and 20.1%, respectively), p = 0.0003. Top-quality blastocyst rates remained lower in overweight and obese women after adjusting for maternal age, type of gonadotrophins, number of oocytes aspirated and peak estradiol levels. Limitations, reasons for caution The strengths are a single center study with embryos cultured under the same standardized laboratory conditions and a relatively large number of embryos in each group. Only GnRH-antagonist ICSI cycles were included to standardize the protocol and control for fertilization time. The retrospective nature of the study is a limitation. Wider implications of the findings Developing an in-house model for morphokinetic parameters is an important additive tool for improving embryo selection and assessment of embryo quality. Finding patient characteristics that influence morphokinetic embryo quality are important in further improving IVF success rates. Trial registration number None