Blastocoel Fluid Research Articles

Current research status and clinical applications of noninvasive preimplantation genetic testing: A review.

Noninvasive preimplantation genetic testing (ni-PGT) is conducted by obtaining genetic information from embryos through the analysis of free DNA released by embryos in spent embryo culture medium or blastocoel fluid. Compared to conventional preimplantation genetic testing relying on trophectoderm biopsy, ni-PGT is characterized by its noninvasiveness. It has demonstrated early advancements in the detection of embryonic chromosomal aneuploidies and the diagnosis of monogenic diseases, showcasing considerable potential for clinical application. However, there are substantial controversies in the literature concerning the reliability of ni-PGT, the source of cell-free DNA, and maternal contamination. This paper elaborates on the principles, research advancements, effectiveness, and limitations of ni-PGT to provide a basis for clinical applications.

A study on methods for preimplantation genetic testing (PGT) on in vivo- and in vitro-produced equine embryos, with emphasis on embryonic sex determination

Two methods for preimplantation genetic testing (PGT) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocoele fluid aspiration (BFA). While TCB is widely applied for both in vivo- and in vitro-produced embryos, BFA has been mostly utilized for in vivo-produced embryos. Alternative methods for PGT, including analysis of cell-free DNA (CFD) in the medium where in vitro-produced embryos are cultured, have been reported in humans but not for equine embryos. In Experiment 1, in vivo- (n = 10) and in vitro-produced (n = 13) equine embryos were subjected to BFA, cultured for 24 h, then subjected to TCB, and cultured for additional 24 h. No detrimental effect on embryonic diameter or re-expansion rates was observed for either embryo group (P > 0.05). In Experiment 2, the concordance (i.e., agreement on detecting the same embryonic sex using two techniques) among BFA, TCB, and the whole embryo (Whole) was studied by detecting the sex-determining region Y (SRY) or testis-specific y-encoded protein 1 (TSPY) (Y-chromosome), and androgen receptor (AR; X-chromosome) genes using PCR. Overall, a higher concordance for detecting embryonic sex was observed among techniques for in vivo-produced embryos (67–100 %; n = 14 embryos) than for in vitro-produced embryos (31–92 %; n = 13 embryos). The concordance between sample types increased when utilizing TSPY (77–100 %) instead of SRY (31–100 %) as target gene. In Experiment 3, CFD analysis was performed on in vitro-produced embryos to determine embryonic sex via PCR (SRY [Y-chromosome] and amelogenin – AMEL [X- and Y-chromosomes]). Overall, CFD was detected in all medium samples, and the concordance between CFD sample and the whole embryo was 60 % when utilizing SRY and AMEL genes. In conclusion, equine embryos can be subjected to two biopsy procedures (24 h apart) without apparent detrimental effects on embryonic size. For in vivo-, but not for in vitro-produced equine embryos, BFA can be considered a potential alternative to TCB for PGT. Finally, CFD can be further explored as a non-invasive method for PGT in in vitro produced equine embryos.

Temporal Evaluation of a Minimally Invasive Method of Preimplantation Genetic Testing for Aneuploidy (mi-PGT-A) in Human Embryos

Preimplantation genetic testing for aneuploidy (PGT-A) has become a useful approach for embryo selection following IVF and ICSI. However, the biopsy process associated with PGT-A is expensive, prone to errors in embryo ploidy determination, and potentially damaging, impacting competence and implantation potential. Therefore, a less invasive method of PGT-A would be desirable and more cost-effective. Noninvasive methods for PGT-A (ni-PGT-A) have been well-studied but present limitations in terms of cf-DNA origin and diagnostic accuracy. Minimally invasive pre-implantation genetic testing (mi-PGT-A) for frozen-thawed embryo transfer is a promising, less studied approach that utilizes a combination of spent culture media (SCM) and blastocoelic fluid (BF)-derived cell-free (CF)-DNA for genetic testing. This study aimed to optimize the effectiveness of mi-PGT-A for aneuploidy diagnosis by investigating the optimal temporal sequence for this protocol. SCM+BF was collected at either 48 or 72 h of culture after thawing day 3 preimplantation embryos. cf-DNA in the SCM+BF was amplified, analyzed by next-generation sequencing (NGS) and compared with results from the corresponding whole embryos (WEs) obtained from human embryos donated for research. Fifty-three (42 expanded blastocysts, 9 early blastocysts, and 2 morula) WE and SCM+BF samples were analyzed and compared. The overall concordance rate between SCM+BF and WE was 60%. Gender and ploidy concordance improved with extended culture time from 48 h (73% and 45%) to 72 h (100% and 64%), respectively. These results demonstrate that SCM+BF-derived cf-DNA can be successfully used for mi-PGT-A. Our findings indicate that longer embryo culture time prior to SCM+BF-derived cf-DNA analysis improves DNA detection rate and concordance with WEs and decreases the proportion of false positive results.

Comparing the advantages, disadvantages and diagnostic power of different non-invasive pre-implantation genetic testing: A literature review.

To improve embryo transfer success and increase the chances of live birth in assisted reproductive methods, there is a growing demand for the use of pre-implantation genetic testing (PGT). However, the invasive approaches used in PGT have led to in vitrofertilization failure and abortions, increasing anxiety levels for parents. To address this, non-invasive PGT methods have been introduced, such as the detection of DNA in blastocoel fluid of blastocysts and spent culture media (SCM). These methods have proven to be minimally invasive and effective in detecting aneuploidy in the chromosomes of human embryos. This review aims to explore the different approaches to pre-implantation diagnosis, including invasive and non-invasive methods, with a particular focus on non-invasive PGT (niPGT). The search strategy involved gathering data from scientific databases such as PubMed, Google Scholar, and Science Direct using relevant keywords. The search was conducted until January 2023. In total, 22 studies have successfully reported the detection and amplification of cell-free DNA in the embryonic SCM. It is important to note that niPGT has some limitations, which include differences in indicators such as cell-free DNA amplification rate, concordance, level of maternal DNA contamination, sensitivity, and specificity between SCM samples and biopsied cells. Therefore, more extensive and detailed research is needed to fully understand niPGT's potential for clinical applications.

Intrinsic and Extrinsic Apoptotic Pathways May Dictate In Vitro Fertilization Outcomes in Women of Advanced Maternal Age [ID 2683425

INTRODUCTION: Women of advanced maternal age (AMA) (≥35 years) are at an increased risk for a decline in ovarian reserve and have a decreased rate of live birth after one cycle of in vitro fertilization (IVF). Current practice recommends preimplantation genetic testing for aneuploidy (PGT-A) to women of AMA to increase live birth outcomes. However, recent studies have found that conventional IVF was non-inferior to PGT-A. Identifying additional biomarkers may provide another metric for selecting the most viable embryo. METHODS: Blastocoel fluid-conditioned media was collected from day 5 IVF embryos that underwent PGT-A. RNA was purified from pooled blastocoel fluid samples from four groups based on implantation status and maternal age, and then cDNA was synthesized. RT-qPCR was used to assess expression of CASP3, CASP7, CASP8, and BCL2L12 levels in a total of 64 embryos. RESULTS: CASP3 and CASP7 expression were higher in the media from IVF embryos with positive implantation outcomes from patients aged less than 35 years. There was increased CASP8 expression in media from IVF embryos from AMA patients regardless of implantation status. CONCLUSION: Preliminary analysis suggests increased CASP8 expression is associated with successful implantation outcomes in AMA patients. This suggests that CASP8 could be activated in preimplantation embryos as an aging response as found in AMA media samples. One explanation is that CASP8 is associated with aneuploidy in the AMA samples. Further research will determine whether expression and activation of CASP8 are required for positive implantation outcomes and be used as an additional embryo selection tool for other AMA patients utilizing IVF.

Evaluation of blastocyst re-expansion, quality in relation to storage temperature, and sexing using blastocoel fluid after manual perforation with a hand-held needle involving in vivo produced equine embryos

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 μL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at −20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C).

Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy

The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques. While trophectoderm (TE) biopsies are frequently used for genotyping, cell-free DNA (cfDNA) found in blastocoele fluid (BF) arises as a less-invasive method. Moreover, the blastocoel collapse produced by BF aspiration could be beneficial for embryo cryotolerance. This study was conducted to test the BF as a source of cell free-DNA (cfDNA) and to compare the BF to the TE biopsy in terms of sexing efficiency/accuracy, embryo survival and gene expression after vitrification/warming. IVP day 7 expanded blastocysts were artificially collapsed by aspiration of BF (VIT-Collapsed) or biopsied (VIT-Biopsied). After sample collection, embryos were vitrified/warmed by the Cryotop method and individually cultured in vitro. Intact fresh non-vitrified and vitrified/warmed blastocysts served as Fresh Control and VIT-Control, respectively. After sex identification of BF or TE biopsies and the corresponding surviving embryos, amplification efficiency and sexing accuracy were assessed. There were no differences between the BF and TE biopsy samples in terms of sexing accuracy or efficiency. Although all vitrified groups showed lower post-warming re-expansion rates (p < 0.05), the blastocyst re-expansion rates in the VIT-Collapsed group were comparable to those in the Fresh Control group whereas biopsied blastocysts showed the lowest (p < 0.05) re-expansion rates. VIT-Collapsed blastocysts had hatching rates that were comparable to those of Fresh Control blastocysts but significantly higher than those of the other vitrification treatments. Proapoptotic gene BAX was overexpressed in VIT-Biopsied embryos, whereas BCL2 transcripts were more abundant in the VIT-Collapsed group. On the other hand, VIT-Biopsied embryos showed altered ATP1B1- and AQP3-mRNA levels. The analysis of the cfDNA present in the BF is an efficient, minimally invasive approach to sex IVP cattle embryos. Besides, the artificial collapse of blastocoel prior to vitrification resulted in higher re-expansion and hatching ability than when embryos were vitrified after being biopsied.

Healthy Live Births after the Transfer of Mosaic Embryos: Self-Correction or PGT-A Overestimation?

The implementation of next generation sequencing (NGS) in preimplantation genetic testing for aneuploidy (PGT-A) has led to a higher prevalence of mosaic diagnosis within the trophectoderm (TE) sample. Regardless, mosaicism could potentially increase the rate of live-born children with chromosomic syndromes, though available data from the transfer of embryos with putative PGT-A mosaicism are scarce but reassuring. Even with lower implantation and higher miscarriage rates, mosaic embryos can develop into healthy live births. Therefore, this urges an explanation for the disappearance of aneuploid cells throughout development, to provide guidance in the management of mosaicism in clinical practice. Technical overestimation of mosaicism, together with some sort of "self-correction" mechanisms during the early post-implantation stages, emerged as potential explanations. Unlike the animal model, in which the elimination of genetically abnormal cells from the future fetal lineage has been demonstrated, in human embryos this capability remains unverified even though the germ layer displays an aneuploidy-induced cell death lineage preference with higher rates of apoptosis in the inner cell mass (ICM) than in the TE cells. Moreover, the reported differential dynamics of cell proliferation and apoptosis between euploid, mosaic, and aneuploid embryos, together with pro-apoptosis gene products (cfDNA and mRNA) and extracellular vesicles identified in the blastocoel fluid, may support the hypothesis of apoptosis as a mechanism to purge the preimplantation embryo of aneuploid cells. Alternative hypotheses, like correction of aneuploidy by extrusion of a trisomy chromosome or by monosomic chromosome duplication, are even, though they represent an extremely rare phenomenon. On the other hand, the technical limitations of PGT-A analysis may lead to inaccuracy in embryo diagnoses, identifying as "mosaic" those embryos that are uniformly euploid or aneuploid. NGS assumption of "intermediate copy number profiles" as evidence of a mixture of euploid and aneuploid cells in a single biopsy has been reported to be poorly predictive in cases of mosaicism diagnosis. Additionally, the concordance found between the TE and the ICM in cases of TE biopsies displaying mosaicism is lower than expected, and it correlates differently depending on the type (whole chromosome versus segmental) and the level of mosaicism reported. Thus, in cases of low-/medium-level mosaicism (<50%), aneuploid cells would rarely involve the ICM and other regions. However, in high-level mosaics (≥50%), abnormal cells in the ICM should display higher prevalence, revealing more uniform aneuploidy in most embryos, representing a technical variation in the uniform aneuploidy range, and therefore might impair the live birth rate.

Alteration of the embryonic microenvironment and sex-specific responses of the preimplantation embryo related to a maternal high-fat diet in the rabbit model.

The maternal metabolic environment can be detrimental to the health of the offspring. In a previous work, we showed that maternal high-fat (HH) feeding in rabbit induced sex-dependent metabolic adaptation in the fetus and led to metabolic syndrome in adult offspring. As early development representing a critical window of susceptibility, in the present work we aimed to explore the effects of the HH diet on the oocyte, preimplantation embryo and its microenvironment. In oocytes from females on HH diet, transcriptomic analysis revealed a weak modification in the content of transcripts mainly involved in meiosis and translational control. The effect of maternal HH diet on the embryonic microenvironment was investigated by identifying the metabolite composition of uterine and embryonic fluids collected in vivo by biomicroscopy. Metabolomic analysis revealed differences in the HH uterine fluid surrounding the embryo, with increased pyruvate concentration. Within the blastocoelic fluid, metabolomic profiles showed decreased glucose and alanine concentrations. In addition, the blastocyst transcriptome showed under-expression of genes and pathways involved in lipid, glucose and amino acid transport and metabolism, most pronounced in female embryos. This work demonstrates that the maternal HH diet disrupts the in vivo composition of the embryonic microenvironment, where the presence of nutrients is increased. In contrast to this nutrient-rich environment, the embryo presents a decrease in nutrient sensing and metabolism suggesting a potential protective process. In addition, this work identifies a very early sex-specific response to the maternal HH diet, from the blastocyst stage.

Concentration of hCG in Blastocoel Fluid is Independent of Embryo Morphology and PGT-A Results: Could it be a Significant Biomarker for a Competent Embryo?

The aim of this research was to develop a quantitative sandwich-strip ELISA was developed on a nitrocellulose membrane with anti-H alpha and anti-hCG beta antibodies. The detection antibody was conjugated to horseradish peroxidase, and a quantitative sandwich-strip ELISA with luminol substrate was developed following optimization with hCG. Concentration of hCG in the BF was determined for 101 embryo biopsies (Study 1) and 96 embryo biopsies (Study 2) respectively, and the results were compared with the previously recorded embryo morphology and PGT-A results. hCG was present in 73.6% of all BF specimens analysed in these two studies, and a weak correlation between hCG levels and both embryo morphology and PGT-A outcome was observed. Additionally, the receiver operating characteristic (ROC) curves show separately that both embryo morphology and PGT-A do not correlate with hCG concentrations. This indicates that the presence of hCG produced in the BF by the embryo is a separate, independent marker of embryo quality.

SCM is potential resource for non-invasive preimplantation genetic testing based on human embryos single-cell sequencing

The ongoing development of assisted reproductive technologies has provided hope to individuals struggling with infertility, promising the potential for a healthy pregnancy. One significant innovation in field of pre-implantation genetic screening (PGS) requires the biopsy of embryos or oocytes, which has potential implications for the health and development of the resultant offspring. Therefore, a non-invasive approach to preimplantation genetic screening is highly sought after. The clinical application of non-invasive preimplantation genetic testing (ni-PGT) is currently limited, with its sensitivity and specificity requiring further investigation. In this study, we used 218 human embryos for single-cell whole genome amplification (WGA), along with ni-PGT of blastocoele fluid (BF) and spent culture medium (SCM). Whole blastocyst (WB), trophectoderm biopsy (TB), and inner cell mass (ICM) from embryo biopsies were used as controls to track genomic signal alterations. Our results showed that the overall genome similarity between SCM and ICM was higher than that of BF. Apart from the Y chromosome, both SCM and ICM demonstrated numerous variant sites across other chromosomes.Further categorization of gene variants in these two sample types revealed that missense variants were the most prevalent, single nucleotide polymorphisms were more common than insertions or deletions, and C > T was the dominant single nucleotide variants in both ICM and SCM. Lastly, we found that the mutant genes in SCM and ICM had different biological functions and pathways. This study indicates that SCM provides a more effective source of embryonic DNA for preimplantation genetic screening, offering a novel reference point for genetic screening research.

Noninvasive genetic screening: current advances in artificial intelligence for embryo ploidy prediction

This review discusses the use of artificial intelligence (AI) algorithms in noninvasive prediction of embryo ploidy status for preimplantation genetic testing in invitro fertilization procedures. The current gold standard, preimplantation genetic testing for aneuploidy, has limitations, such as an invasive biopsy, financial burden, delays in results reporting, and difficulty in results reporting, Noninvasive ploidy screening methods, including blastocoel fluid sampling, spent media testing, and AI algorithms using embryonic images and clinical parameters, are explored. Various AI models have been developed using different machine learning algorithms, such as random forest classifier and logistic regression, have shown variable performance in predicting euploidy. Static embryo imaging combined with AI algorithms have demonstrated good accuracy in ploidy prediction, with models such as Embryo Ranking Intelligent Classification Algorithm and STORK-A outperforming human grading. Time-lapse embryo imaging analyzed by AI algorithms has also shown promise in predicting ploidy status; however, the inclusion of clinical parameters is crucial to improving the predictive value of these models. Mosaicism, an important aspect of embryo classification, is often overlooked in AI algorithms and should be considered in future studies. The integration of AI algorithms into microscopy equipment and Embryoscope platforms will facilitate noninvasive genetic testing. Further development of algorithms that optimize clinical considerations and incorporate minimal-necessary covariates will also enhance the predictive value of AI in embryo selection. Artificial intelligence-based ploidy prediction has the potential to improve pregnancy rates and reduce costs in invitro fertilization cycles.

P-743 Semen samples quality and blastocysts diversity after PGT-A

Abstract Study question Does semen samples quality contribute to blastocyst diversity by correlating with the presence of DNA in the blastocoelic fluid (BF)? Summary answer Semen samples quality is associated with the detection of DNA in BFs from both euploid and aneuploid blastocysts What is known already DNA was detected by whole genome amplification (WGA) in BFs from expanded blastocysts dependently on the blastocyst chromosome condition (assessed by PGT-A on trophectoderm (TE) cells), being more frequent in aneuploid blastocysts. TE-euploid blastocysts with positive BF-WGA also seem to have less chances to implant compared to those with failed BF-WGA. The extrusion of abnormal cells from the embryo proper was proposed as a mechanism triggered by the mosaic embryo to eliminate abnormal cells. Severe male factor samples are at risk of causing embryo mosaicism. Possible association between semen quality and DNA in BFs has not yet been investigated. Study design, size, duration This retrospective cohort study included 115 patients (maternal age 37.9±4.1), which underwent PGT-A in the last three years. The aim of the study was to evaluate whether the detection of DNA in BFs was correlated with semen samples indices. Both TE-euploid and TE-aneuploid blastocysts were considered for the analysis and stratified according to the semen samples quality as normozoospermic (N), moderate oligoasthenoteratozoospermic (m-OAT) and severe OAT (s-OAT) following the 2021 WHO criteria. Participants/materials, setting, methods BF and trophectoderm (TE) biopsies were collected from high grade expanded blastocysts before vitrification, and submitted to WGA. Amplification after WGA was evaluated by loading an aliquot of the amplified product onto a 1.5% agarose gel. 24-chromosome analysis was performed on TE biopsies. To assess the impact of semen samples quality on the detection of DNA in BFs, a multiple logistic regression analysis was performed correcting for maternal age as a confounding factor. Main results and the role of chance The BF from 590 blastocysts (265 TE-euploid and 325 TE-aneuploid) were submitted to WGA. In TE-euploid blastocysts, 125 derived from N semen samples, 52 from m-OAT, and 88 from s-OAT. The incidence of positive BF-WGA was proportional to the severity of the male factor condition and occurred in 48.0% of blastocysts originated from N samples, in 55.8% of blastocysts derived from m-OAT, and in 60.2% of blastocysts from s-OAT. In TE-aneuploid blastocysts, 178 originated from N semen samples, 80 from m-OAT, and 67 from s-OAT. The incidence of positive BF-WGA showed an opposite trend compared with the TE-euploid group being detected in 64.0% of blastocysts originated from N samples, in 66.3% of blastocysts derived from m-OAT, and in 52.2% of blastocysts obtained from s-OAT. The relationship between semen quality and positive BF-DNA amplification was confirmed by a multiple logistic regression model setting the BF-WGA as the outcome variable. In the TE-euploid blastocyst cohort, s-OAT showed an odd ratio (OR) of 1.78 (95% CI: 1.02-3.14; p=0.045), whereas in the TE-aneuploid blastocyst group, s-OAT displayed an OR of 0.55 (95% CI: 0.31-0.98; p=0.043). Limitations, reasons for caution This is a retrospective cohort study including a limited number of cases where the different categories of sperm samples were not equally represented. Although the results of BF-WGA were evaluated per single blastocyst, they cannot be considered independent variables. Wider implications of the findings In s-OAT samples, abnormal cell elimination is especially active in TE-euploid blastocysts, whereas in TE-aneuploid blastocysts the efficiency of this process is reduced suggesting the difficulty of eliminating abnormal cells in an aneuploid background. BF-DNA amplification is a minimally invasive tool contributing additional knowledge to elucidate blastocyst diversity. Trial registration number None

O-062 MicroRNAs in Blastocoel Fluid: a molecular signature for predicting human embryo implantation potential

Abstract Study question Can microRNA expression in blastocoel fluid (BF) predict embryo implantation potential? Summary answer Up-regulation of miR-106, miR-373, miR-301, miR-320 and miR-525-3p represents the molecular signature characterizing the embryos with high implantation capability. What is known already The discovery of DNA within the BF and in spent embryo culture media, has caused increased interest in the non-invasive preimplantation testing application for monogenic disorders and aneuploidies. Aneuploid embryos have a lower implantation potential, however, chromosomal abnormalities represent only a tiny percentage of the causes of implantation failure. MiRNAs can control all cellular pathways and are involved in pluripotency, self-renewal, and stemness, and their altered regulation affects different human diseases. Study design, size, duration From September 2018 to March 2022, 112 BFsampleswere collected from human embryos on the fifth day of development, before the blastocyst cryopreservation. The samples were classified according to blastocyst grade and the data on implantation outcome and term births were collected for the transferred embryos. We compared the expression profiles of 89 miRNAs, previously identified in BF, between 33 BF from implanted embryos and 30 from non-implanted ones, regardless of blastocyst grade. Participants/materials, setting, methods By custom-designed TaqMan Low-Density Array card (TLDA), we analyzed the expression of 89 miRNAs in 4 different BF samples simultaneously. Differentially expressed (DE) miRNAs were identified by Volcano plot and Significance Analysis of Microarrays (SAM) tests. Bioinformatic analysis was performed to identify the biological role of the DE miRNAs. To evaluate miRNA’s ability for predicting implantation, Pearson’s correlation analyses, classical univariate Receiver Operator Characteristic (ROC) curve analysis, and the optimal cut-off value determination were performed. Main results and the role of chance We found five miRNAs, miR-106, miR-373, miR-301, miR-320 and miR-525-3pup-regulated in BF from implanted blastocysts. The identified miRNAs perform an important role during the first phases of embryo development suggesting that their up-regulation may reflect embryo health. Moreover, four of the five miRNAs showed significant correlation coefficients in both implanted and non-implanted blastocysts, indicating that their expression changes in the same way in the single sample and reflects the potentiality of the embryo to implant. Finally, ROC curve analysis confirmed that our miRNAs could be considered potential biomarkers for implantation. Limitations, reasons for caution Successful implantation requires a close dialogue between the embryo and the endometrium, mediated by different proteins and miRNAs produced by both the embryo and maternal tissues. Determining the quality of the embryo and its implantation potential is not sufficient to predict successful pregnancy outcomes. Wider implications of the findings This study represents the first report correlating miRNA profiles in BFand implantation and suggests that miRNA signature could become an accurate tool to evaluate embryo quality. It could be associated or replaced with the PGT-A to choose the most competent embryo and improve the outcome of assisted reproduction cycles. Trial registration number The study has been approved by theEthical Committee of Azienda Ospedaliero Universitaria Policlinico &amp;quot;G.Rodolico -San Marco&amp;quot; Catania.

P-737 Progress in non-invasive embryo assessment with cell-free nucleic acids in blastocoel fluid

Abstract Study question Can cell-free nucleic acids (cfDNA) from blastocoel fluid (BF) after a trophectoderm (TE) biopsy be used as a non-invasive sample type to assess embryos? Summary answer Both BF and spent media carry comparable, highly fragmented cfDNA and cell-free RNA (cfRNA) which may reveal more about the quality of the embryo. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) is commonly used by IVF clinics to screen embryos for any chromosomal abnormalities during the IVF process. Noninvasive preimplantation genetic testing (ni-PGT) is an emerging category of tools within the field of assisted reproductive technology. Currently, niPGT products have been developed for embryo aneuploidy detection using cfDNA released by the embryo into the spent media. Embryo spent media (ESM) carries cfDNA, which has the potential to be applied to evaluate the quality of the embryo. Previous studies have demonstrated that cfDNA in ESM is highly fragmented to the nucleosome level. Study design, size, duration The quality and quantity of cell free nucleic acid from forty BF-conditioned media (BFCM) samples were analyzed in this study. Embgenix™ Analysis Software was used to analyze and interpret sequencing data. For each sample, the concentration and fragmentation of cfDNA were examined and compared using generalized linear mixed models. To assess RNA quality at a picogram level, reverse-transcribed and amplified cDNA was used to represent the quality of cfRNA in BFCM. Participants/materials, setting, methods Embryos were washed and placed in individual biopsy media droplets for TE biopsy. After biopsy, blastocysts were collapsed, releasing BF into the droplet. The entire droplet was collected as BFCM. The Embgenix ESM Screen Kit was used to prepare sequencing libraries. The SMART-Seq® Single Cell Kit was used to evaluate the RNA quality. The BFCM cfDNA results were also compared to spent media profile that had already been obtained from a different cohort of embryos. Main results and the role of chance Amounts of cfDNA fragmentation detected in BFCM samples was comparable to ESM. Whole-genome amplification (WGA) yields correlated to the amount of cfDNA input but not the degree of cfDNA fragmentation. Informative sequencing reads and computed copy number variations (CNV) plot noise, as evaluated by distribution of log2 ratio spread (DLRS), also showed correlation and stable performance down to ∼2 pg of cfDNA input. We extracted detectable cfRNA from BFCM and created cDNA for RNA quality analysis. Our model demonstrates a significant correlation between cDNA length and RNA quality metrics, with a low DV200 of cfRNA from BFCM samples. The quantity and quality of cfDNA released into spent culture media by a developing embryo are comparable to the cfDNA released into BFCM upon TE biopsy, indicating that BFCM has potential as a noninvasive method for assessing the ploidy status of embryos. WGA yield, informative reads, and DLRS of BFCM samples were likewise comparable to data previously acquired using spent media samples. However, the severely fragmented cfRNA in these samples illustrate the inherent instability of RNA and the difficulty of utilizing RNA analysis to determine embryo health. Assays compatible with small RNA or microRNA (miRNA) are more appropriate for RNA-seq of BFCM. Limitations, reasons for caution Different clinics use varying methods for embryo culture and biopsy; the data reported here is from a single research center. The characteristic of samples may vary between clinics. Our assay estimates RNA quality using amplified cDNA; nonetheless, discrepancies between fragmented RNA and amplified cDNA may exist. Wider implications of the findings In PGT-A, BFCM samples are routinely discarded. Our findings imply BFCM samples contain considerable levels of cell-free nucleic acids which could also be used to screen embryo quality. BFCM samples, therefore, could be analyzed in conjunction with PGT-A to select and/or rank embryos for implantation. Trial registration number Not applicable

P-733 A non-selection study to evaluate non-invasive preimplantation genetic testing for aneuploidy

Abstract Study question How does non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) perform to predict sustained implantation or live birth (SI/LB)? Summary answer Though not comparable to conventional PGT-A, niPGT-A can still be considered an appealing tool for clinical embryo prioritizing given its high negative predictive value (NPV). What is known already To omit the need for the invasive trophectoderm biopsy, niPGT-A which screens embryonic ploidy using embryonic cell free DNA released into the spent embryo culture medium (SEM) or blastocoel fluid (BF) was developed. Although the feasibility of niPGT-A has been shown and healthy live births have been reported from embryos screened by niPGT-A, the predictive value of niPGT-A for embryo transfer outcome has not been established yet and evidence is still lacking as to whether embryo selection by niPGT-A is associated with improved pregnancy outcomes. The answers to these are of vital importance to justify niPGT-A for clinical care. Study design, size, duration This is a single center, blinded, non-selection study consisting of single frozen embryo transfer cycles with a duration of two years, of which 117 cycles (Arm-1) were completed for 83 patients by transferring an embryo that was only morphologically assessed, and 90 cycles (Arm-2) were completed for 67 patients by transferring an euploid embryo that was screened by conventional biopsy-based PGT-A. Participants/materials, setting, methods Embryos were cultured in 25 μL continuous culture medium until blastocyst stage. The “SEM+BF” samples were ongoingly collected and archived at the end of culture. In Arm-1, after pregnancy outcomes were determined, the corresponding “SEM+BF” samples were pulled out for niPGT-A. In Arm-2, niPGT-A was also performed for some cases that did not end up in an SI/LB. NiPGT-A was completed by NICS and the incorporated ChromGo platform (Yikon Genomics). Main results and the role of chance Out of the 117 “SEM+BF” samples in Arm-1, 110 (94%) yielded an informative niPGT-A result. Among these, 46 transferred embryos were determined as “euploid” by niPGT-A and 15 resulted in SI/LB; 32 were determined as “aneuploid” and 27 did not resulted in SI/LB. Therefore, the positive and negative predictive values (PPV and NPV) of niPGT-A were 32.6% (15/46) and 84.4% (27/32), respectively. Another 32 transferred embryos in Arm-1 were determined as “suspected mosaic” and they showed a similar rate of SI/LB per embryo transfer to those “euploid” ones (37.5% vs. 32.6%, P = 0. 8092). Compared to Arm-1 overall, the “transferrable” embryos (“euploid” + “suspected mosaic”) determined by niPGT-A resulted in a much higher rate of SI/LB (34.6% vs. 27.6%, P = 0.3399). In Arm-2, 39 out of the 90 transferred euploid embryos resulted in SI/LB. “Euploid” embryos determined by PGT-A resulted in significantly better rate of SI/LB than “euploid” embryos determined by niPGT-A in Arm-1 (43.3% vs. 32.6%, P = 0.0243). Out of the 51 PGT-A “euploid” embryos that did not end up in SI/LB, 25 were also tested by niPGT-A with 6 being “suspected mosaic” and 3 being “abnormal/aneuploid”, raising the concern of possible false negative of conventional PGT-A. Limitations, reasons for caution The study enrolled a non-selected patient population which resulted in the inclusion of a few patients with a history of repeated implantation failure. Therefore, the PPV of niPGT-A might be underestimated as implantation failure could be attributed to defects in endometrial receptivity rather than in embryo quality. Wider implications of the findings This is the first non-selection study to validate niPGT-A in the setting of continuous culture medium. The high informative rate indicates its feasibility for clinics using a similar culture system. It also forms the foundation of a prospective, randomized controlled trial to further assess the benefit of niPGT-A. Trial registration number Not applicable

P-192 Blastocyst collapse during embryonic development is a marker of low pregnancy potential, independent of other blastocyst evaluation criteria

Abstract Study question Is evaluation of blastocyst collapse during embryonic development an independent marker for ongoing pregnancy in single frozen-thawed blastocyst transfer cycles? Summary answer Collapse of blastocyst more than 20.4% of the area may be a marker of low ongoing pregnancy potential, independent of other evaluation criteria. What is known already Time-lapse monitoring revealed that a section of the extended trophectoderm (TE) is ruptured during development. This TE rupture causes an efflux of blastocoel fluid and a reduction in embryonic volume. This phenomenon is described as collapse. Several studies have reported that the collapse results in a low clinical and ongoing pregnancy potential for the blastocyst. However, the collapsed blastocysts were associated with poor morphological quality. Previous studies have not evaluated whether collapse is an independent predictor of pregnancy based on other blastocyst evaluation criteria. Therefore, it is necessary to determine whether blastocyst collapse is an independent predictor of ongoing pregnancy. Study design, size, duration This retrospective, single-center study was conducted at the TAKAHASHI WOMEN'S CLINIC from January, 2018 to December, 2020. All blastocysts were derived from the ICSI cycles. Time-lapse monitoring using Embryoscope + (Vitrolife) was used to observe the presence and degree of collapse. The degree of collapse was calculated from the embryonic volume at the maximum expansion and the lowest volume after collapse. We analyzed the relationship between 904 cycles of single vitrified-warmed blastocyst transfer and ongoing pregnancy. Participants/materials, setting, methods The ongoing pregnancy rates of collapsed and non collapsed blastocysts were compared using chi-squared test. The cut-off value of the degree of collapse for ongoing pregnancy was calculated using receiver operating characteristic (ROC) curve analysis. The effect of collapse above the cut-off value on ongoing pregnancy was examined using multivariate logistic regression analysis, including confounding factors (patient age, body mass index, inner cell mass (ICM) grade, TE grade, day of blastocyst formation, and expansion stage). Main results and the role of chance During the study period, 329 of the 904 transferred blastocysts exhibited collapse during time-lapse monitoring, and the degree of collapse ranged from 2.10% to 75.90%, with a median of 30.70%. In total, 575 transferred blastocysts were determined to be non collapsed. The mean patient age ( ± standard division) was 36.89 ± 4.77 years in the collapsed blastocyst group and 36.52 ± 4.42 years in the non collapsed blastocyst group (p = 0.151). The proportion of blastocysts with poor morphological grade (&amp;lt;BB ) was higher in the collapsed blastocysts group than in the non collapsed blastocysts group (32.5% vs. 17.91%, p &amp;lt; 0.0001). The ongoing pregnancy rate was significantly lower in the collapsed blastocysts group than in the non collapsed blastocysts group (45.4% vs. 34.0%, p = 0.0009). ROC curve analysis showed that the cut-off value for the degree of collapse for low pregnancy potential was 20.4%. Multivariate logistic analysis with confounding factors revealed that blastocyst collapse above the cut-off value (≥20.4%) had an effect on ongoing pregnancy (adjusted odds ratio: 0.53, 95% confidence interval: 0.34–0.82, p = 0.005) independent of ICM and TE grades, day of blastocyst formation, and expansion stage. Limitations, reasons for caution This retrospective study was conducted at a single fertility center. In addition, the analyzed blastocysts were derived only from the ICSI cycles. Therefore, it is necessary to analyze the effects of collapse in blastocysts derived from C-IVF cycles. Wider implications of the findings Our results demonstrate that ≥20.4% collapse may be an additional marker to consider when selecting blastocysts for transfer. If the blastocysts are of the same grade, it is better to select non collapsed embryos. Trial registration number not affect

Equine blastocyst re-expansion rate, quality, and sex following embryonic collapse using needle perforation of blastocoel and subsequent PCR amplification of blastocoel DNA

Collapse of equine blastocysts following needle perforation of the blastocoel prior to vitrification (Ferris et al., Journal of Equine Veterinary Science. 2016; 100:64-65) and collection and amplification of the free DNA blastocoel fluid may be used for sexing embryos (Herrera et al., Theriogenology. 2015; 83:415-420). The present study aimed to evaluate equine blastocyst re-expansion rate, quality, and sex following manual perforation of the blastocoel and PCR amplification of blastocoel fluid DNA. Embryos (n=19) were collected on day 8 after ovulation, assessed initially and transferred to a 50µL droplet of holding medium in which the blastocoel was manually perforated with a 26g hypodermic needle. Within one minute of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at -20°C until PCR. Some of the collapsed embryos (n=11) were moved and immersed in 2mL of holding medium and cultured in isothermal boxes at 25°C. After 24h, embryo size and quality were reassessed. Post-culture, collapsed blastocysts had partially re-expanded to their initial diameter with a mean expansion rate of 68.8%. Quality of re-expanded embryos was Grade 2 (good, 6/11), Grade 3 (poor, 4/11), and Grade 4 (degenerated, 1/11) (McKinnon,Squires, J. Am. Vet Med. Assoc. 1988; 192:401-406). Non-perforated embryos (n=10) used as controls were initially Grade 1 (excellent, 8/10) and Grade 2 (2/10) and, after 24h of culture, Grade 1 (9/10) and Grade 2 (1/10) with a 25.9% growth rate. Blastocoel fluid from all collapsed embryos (n=19) was subjected to double PCR amplification of the male TSPY gene with blood of an adult male and female horse used as controls. Gene amplification indicated 57.9% (11/19) of embryos were male and non-amplification indicated 31.6% (6/19) of embryos were female. Gene amplification was inconclusive for 10.5% (2/19) of the embryos even though they were completely collapsed. Thus, needle-induced leakage and collapse of the blastocoel resulted in a high rate of blastocysts re-expansion (69%) with many embryos (54%) achieving good quality and potential for transfer as either male or female embryos. In summary, while the number of potentially transferable embryos that recovered from collapse was less than in the control group, the success of these preliminary results warrants further development to decrease damage and increase the culture re-expansion rate and quality of transferable male or female embryos for field application in an equine embryo transfer program.

First report of DNA detection and sex determination in leaked fluid after needle collapse of large equine blastocysts

The current method for sex determination of equine blastocysts involves aspiration of blastocoel fluid followed by PCR, which can be challenging due to the need for specialized equipment and often requires the embryo to be shipped to a separate lab for analysis. In this study, we investigated a new method for sex determination using leakage from manual collapse prior to vitrification, a technique which previously resulted in a pregnancy in our laboratory (1 of 3, unpublished results). We hypothesized that leaked blastocele fluid into the surrounding medium could provide sufficient DNA for amplification and sex determination. From a total of 14 embryos (900 to 1200 μm diameter), 4 were collapsed by micromanipulator-assisted aspiration (Micro group), and 10 were collapsed by manual puncture (Needle group) using a 32-G, penta-point needle (BD Nano, Canada). Additional blastocoel leakage (up to 80% of original volume) was obtained by forced pipetting using a 275 μm flexipet. All manipulations were conducted in TALP without serum. Samples were snap-frozen (30 μl) and stored at -80°C. DNA amplification was performed by multiplex PCR targeted to sex chromosome-linked zinc finger protein genes (ZFx/ZFy) for both sex DNA detection and the Y-encoded testis-specific protein (TSPY) for Y chromosome detection. The primer sequences were ZFx/y-F 5’-ATAATCACATGGAGAGCCACAAGCT-3’, ZFx/y-R 5’-GCACTTCTTTGGTATCTGAGAAAGT–3’, and TSPY-F 5′-GAAGTCAGGCACACCAGTGA-3′, TSPY-R 5′-TAAGGCTGCAGTTGTCATGC-3′, with 445bp and 280bp products, respectively. The reaction was carried out in a 25 μl final volume with 4 μl of PROMEGA Green GOTaq 5X Buffer, 0.2 μM primers, 1 mM MgCl2, 5 μl DNA sample, and 1.25 IU Taq DNA polymerase. The cycle conditions were 4 min 94°C, 40 cycles of 30 sec 94°C, 30 sec 58°C, and 20 sec 72°C followed by final elongation of 1 min 72°C. Equine male and female DNA was purified from whole blood (PROMEGA WizardTM Genomic DNA purification kit) and used as positive controls; female and male band patterns were used as reference. Our results showed that sex was confirmed in 4/4 (100%; 3 male and 1 female) of embryos biopsied by micromanipulation (Micro group) and in 5/10 (50%; 1 male and 4 female) of embryos collapsed by manual needle puncture (Needle group). Although the proportion of sex determination in the manually collapsed group is still low, it could be attributed to sample dilution effects. This report provides proof of concept of DNA detection and sex determination in the medium surrounding manually collapsed embryos without blastocoel aspiration. Our findings suggest that this approach to embryo biopsy for sex determination is promising, but more studies are needed to confirm its effectiveness.

Failure to detect DNA in blastocoel fluid is associated with a higher live birth rate in both PGT-A and conventional IVF/ICSI cycles.

Is the presence of DNA in the blastocoel fluid (BF) of expanded blastocysts, assessed by whole genome amplification (WGA), associated with the clinical outcome at the first transfer? At the first transfer, blastocysts with negative BF-WGA have more chance to implant and to develop to term than those with positive BF-WGA results, both in preimplantation genetic testing for aneuploidies (PGT-A) cycles (where only euploid blastocysts resulting from the chromosomal analysis of trophectoderm (TE) biopsies were transferred) and in IVF/ICSI conventional cycles. Retrospective studies conducted in patients undergoing PGT-A have shown that the incidence of negative BF-WGA was significantly higher in TE-euploid blastocysts than in TE-aneuploid blastocysts. In addition, after the transfer of TE-euploid blastocysts, the ongoing clinical pregnancy rate was significantly higher in the group with negative BF-WGA compared with those with positive BF-WGA. A prospective cohort study including 102 consecutive PGT-A patients (Group 1) and 88 consecutive conventional IVF/ICSI patients (Group 2), was conducted between January 2019 and December 2021. In both groups, BFs were collected from expanded blastocysts of high grade and processed for WGA. DNA amplification was evaluated by agarose gel electrophoresis for the presence (positive BF-WGA) or absence (negative BF-WGA) of a band. Directly after the BF retrieval, blastocysts from Group 1 underwent TE biopsy and vitrification. In Group 2, blastocysts were vitrified immediately after BF collection. In Group 1, only euploid blastocysts were considered for transfer according to the results of TE biopsies. In both groups, the selection of the blastocyst to be transferred was based on BF-WGA results giving priority, if available, to those with negative amplification. The primary outcome investigated was the live birth rate (LBR) at the first transfer. The main variable under investigation was the negative BF-WGA and results were corrected for confounders (maternal and paternal age, number of retrieved oocytes, male factor) by multiple logistic regression analysis. In Group 1, 60 patients transferred negative BF-WGA blastocysts and 42 positive BF-WGA blastocysts, and the LBR at the first transfer was 53.3% and 26.2%, respectively (P = 0.0081). After testing for selected confounders in a multiple logistic analysis, the transfer of blastocysts with negative BF-WGA resulted in an odds ratio of (OR) 3.52 (95% CI: 1.48-8.88, P = 0.0057) compared to transfer of positive BF-WGA blastocysts. In Group 2, at the first transfer 30 deliveries resulted from blastocysts with negative BF-WGA (48.4%) and three from the transfer of positive BF-WGA blastocysts in 26 patients (11.5%; P = 0.0014). Multiple logistic analysis indicated that the transfer of blastocysts with negative BF-WGA resulted in an OR 6.89 (95% CI: 1.98-32.95, P = 0.0056) compared to transfer of positive BF-WGA blastocysts. The LBR per transfer and the cumulative LBR per patient showed the same trend. The study was performed in a single center. The data from this study highlight the heterogeneity of blastocysts of similar morphology, even in those classified as euploid by TE analysis. Failure to detect DNA in BFs after WGA is associated with a significantly higher LBR at the first embryo transfer as well as per transfer and per patient. The processing of the BF by WGA is an easy and cost-effective tool that could become a valuable option to offer patients the highest chances of term pregnancy in the shortest time possible. The study received no funding from external sources. There are no conflicts of interest to declare. N/A.