Embryo Implantation Potential Research Articles

The morphokinetic signature of human blastocysts with mosaicism and the clinical outcomes following transfer of embryos with low-level mosaicism

BackgroundGenetic mosaicism is commonly observed in human blastocysts. Embryos’ morphokinetic feature observed from time-lapse monitoring (TLM) is helpful to predict the embryos’ ploidy status in a non-invasive way. However, morphokinetic research on mosaic embryos is extremely limited. Moreover, transfer of mosaic embryos is a new attempt in reproductive medicine, while studies regarding the clinical and neonatal outcomes following transfer of embryos with different levels and types of mosaicism are needed. This study aimed to investigate the morphokinetic characteristics of mosaic blastocysts, uncover clinical outcomes of mosaic embryos, and evaluate the effect of level and type of mosaicism on transfer outcomes.ResultsA total of 923 blastocysts from 229 preimplantation genetic testing cycles were cultured in TLM incubators in a single fertilization center between July 2016 and July 2021. Multivariate logistic regression models showed mosaic embryos had significantly shorter time to reach morula when compared with euploid (P = 0.002), mosaic with aneuploid (P = 0.005), and aneuploid (P = 0.005) embryos after adjusting the potential confounders. KIDScore is an artificial intelligence scoring program from time lapse incubation system to predict embryo implantation potential. Mosaic with aneuploid embryos had significantly lower KIDScore than euploid (P = 6.47e−4), mosaic (P = 0.005), and aneuploid (P = 0.004) embryos after adjustment. Meanwhile, we compared the clinical outcomes following transfer of low-level (< 50%) mosaic embryos (N = 60) with euploid embryos (N = 1301) matched using propensity scoring collected from September 2020 to January 2023. Mosaic embryos had significantly lower clinical pregnancy rate (41.67% vs. 57.65%, P = 0.015) and live birth rate (38.33% vs. 51.35%, P = 0.048) than the euploid embryos. Subgroup analyses showed the whole, segmental, and complex chromosome mosaic embryos had the similar clinical outcomes.ConclusionsThe shortened time to reach morula in mosaic embryos and the low KIDScore in mosaic with aneuploid embryos revealed innovative clues to embryo selection with the non-invasive TLM and provided new insights into biological mechanism of chromosomal abnormality. The analyses of overall and subgroups of mosaic embryo transfer outcomes helped to optimize embryo transfer scheme for in-vitro fertilization procedures. Multi-center prospective studies with large sample sizes are warranted to validate our results in the future.

Morphological assessment of oocyte quality during assisted reproductive technology cycle.

Following the advancement of medically assisted reproduction (MAR) technology, and the rationale to extend the culture to the blastocyst stage, performing elective single embryo transfer (eSET), gamete quality and assessment have acquired large relevance in ART. Embryo quality is strictly correlated with gametes quality and culture conditions. Oocyte maturity assessment is therefore imperative for fertilization and embryo evolution. Mature oocytes at the metaphase II stage result in a higher fertilization rate compared to immature oocytes. Indeed, oocyte morphology evaluation represents an important and challenging task that may serve as a valuable prognostic tool for future embryo development and implantation potential. Different grading systems have been reported to assess human embryos, however, in many cases, it is still a major challenge to select the single embryo to transfer with the highest implantation potential. Further, eSET has conferred a challenge to embryologists, who must try to enhance embryo culture and selection to provide an adequate success rate, whilst reducing the overall number of embryos transferred. Above the standard morphological assessment, there are several invasive or non-invasive approaches for embryo selection such as preimplantation genetic testing, time-lapse technology, proteomics and metabolomics, as well as oxygen utilization and analysis of oxidative stress in culture medium. This short review is not designed to be a comprehensive review of all possible features that may influence oocyte quality. It does give, however, a brief overview and describes the prognostic value of the morphological characteristics of human oocytes on their developmental capacity following ART treatments.

Blastocysts from partial compaction morulae are not defined by their early mistakes

Research questionIs partial compaction during morula formation associated with an embryo's developmental ability and implantation potential? DesignRetrospective analysis of data from 196 preimplantation genetic testing for aneuploidy (PGT-A) cycles. Embryos starting compaction were grouped according to the inclusion or not of all the blastomeres in the forming morula (full compaction or partial compaction). The possible effect of maternal age and ovarian response on compaction was analysed. Morphokinetic characteristics, blastocyst formation rate, morphology and cytogenetic constitution of the obtained blastocysts were compared. Comparisons of reproductive outcomes after the transfer of euploid blastocysts from both groups were established. Finally, in a subset of embryos, the chromosomal constitution concordance of the abandoned cells and the corresponding blastocyst through trophectoderm biopsies was assessed. ResultsA total of 430 embryos failed to include at least one cell during compaction (partial compaction group [49.3%]), whereas the 442 remaining embryos formed a fully compacted morula (full compaction group [50.7%]). Neither female age nor the number of oocytes collected affected the prevalence of partial compaction morulae. Morphokinetic parameters were altered in embryos from partial compaction morulae compared with full compaction. Although an impairment in blastocyst formation rate was observed in partial compaction morulae (57.2% versus 70.8%, P < 0.001), both chromosomal constitution (euploidy rate: partial compaction [38.4%] versus full compaction [34.2%]) and reproductive outcomes (live birth rate: partial compaction [51.9%] versus full compaction [46.2%]) of the obtained blastocysts were equivalent between groups. A high ploidy correlation of excluded cells-trophectoderm duos was observed. ConclusionsPartial compaction morulae show a reduced developmental ability compared with full compaction morulae. Resulting blastocysts from both groups, however, have similar euploidy rates and reproductive outcomes. Cell exclusion might be a consequence of a compromised embryo development regardless of the chromosomal constitution of the excluded cells.

Effects of different oocyte cytoplasmic granulation patterns on embryo development and euploidy: a sibling oocyte control study.

This study evaluated the relationship between cytoplasmic granulation patterns and the developmental potential of mature sibling oocytes. Data from 54 cycles of preimplantation genetic tests for structural rearrangement from July 2019 to June 2022 were analyzed. In total, 564 embryos were cultured using a time-lapse system. Sibling oocytes were divided into four groups based on cytoplasmic granulation patterns: fine granulation (FG) group (n = 177), central granulation (CG) group (n = 183), dispersed granulation (DG) group (n = 161), and uneven granulation (UG) group (n = 43). The CG group was further divided into three groups (grades I, II, and III) based on the tertile of the ratio of central granular distribution area to oocyte area. Fertilization rate, embryo morphokinetics, chromosomal ploidy, and clinical outcomes of the groups were compared. No significant differences were observed in morphokinetic parameters, fertilization rate, embryo production, blastocyst formation, and aneuploidy rates among the different cytoplasmic-granulation pattern groups. However, embryos derived from CG oocytes showed significantly higher aneuploidy rates in grade III compared to grade I (86.21% vs 61.54%, P = 0.036) or grade II (86.21% vs 56.00%, P = 0.013). Thirty embryos were transferred to the uteri of female patients and the clinical pregnancy and live birth rates did not significantly differ among groups. Cytoplasmic granulation patterns may not affect embryo fertilization, development speed, and aneuploidy rates. However, a higher grade of CG may be associated with increased aneuploidy rates. Larger sample sizes are required to explore the impact of oocyte cytoplasmic granulation patterns on embryo implantation potential.

Elevated high-mannose N-glycans hamper endometrial decidualization

Decidualization of endometrial stromal cells is a hallmark of endometrial receptivity for embryo implantation, and dysfunctional decidualization is associated with pregnancy failure. Protein glycosylation is an important posttranslational modification that affects the structure and function of glycoproteins. Our results showed that high-mannose epitopes were elevated in the decidual tissues of miscarriage patients compared with early pregnant women by Lectin microarray. Furthermore, the level of mannosyl-oligosaccharide α-1,2 mannosidase IA (MAN1A1), a key enzyme for high-mannose glycan biosynthesis, was decreased in the decidual tissues of miscarriage patients. Screening of lncRNAs showed that lncNEAT1 level was increased in the serum and decidua of miscarriage patients, and negatively correlated with MAN1A1 expression. The results also revealed that specific binding of lncNEAT1 with nucleophosmin (NPM1)-SP1 transcription complex inhibited MAN1A1 expression and hampered endometrial decidualization and embryo implantation potential. The study suggests the new insights into the function of high-mannose glycans/MAN1A1 modification during endometrial decidualization.

The effect of amorphous calcium carbonate as a culture media supplement on embryonic development of murine sibling embryos.

The aim of this study was to compare the addition in culture media of stabilized amorphous calcium carbonate (ACC) versus calcium chloride (CaCl2) or calcium carbonate in crystalline form (CCC) on growth rates among sibling mouse embryos. We evaluated the effect of different ACC concentrations on the rates of embryo compaction at 60 h, blastocyst rate at 84 h and percentage of fully hatched at 108 h following hCG injection. As ACC is stabilized by tripolyphosphate (TPP), we also evaluated the addition of TPP alone to the culture media. Finally, we compared supplemented ACC culture media to one-step SAGE and Irvine cleavage media. The results revealed that ACC accelerates the compaction and blastocyst rates, as well as the percentage of fully hatched embryos in a dose-dependent manner, with an increased positive effect at 2.5 mM. The magnitude of the effect for ACC-supplemented media on the embryo developmental rate was between 30 to 40% (p < 0.01) faster for each stage, compared to both SAGE and Irvine one-step standard media. Embryos cultured with SAGE or Irvine media with or without supplementation of CaCl2 or CCC, did not produce the same improvements as observed with ACC. In conclusion, the ACC demonstrates a rapid modulation effect for restoring media optimal pH. ACC can inhibit cathepsin B activity during in vitro culture of fibroblast cells. The beneficial impact of ACC on cleavage mouse embryos is likely due to an improved buffering effect causing slower pH media variations, which may enhance quality and implantation potential of embryos following in vitro culture.

P-590 Similar mean follitropin delta daily/total doses yield satisfactory ICSI outcomes in different subgroups of maternal age and body mass index: a “real-world” experience with Rekovelle®

Abstract Study question Can similar mean follitropin delta daily/total doses yield satisfactory ICSI outcomes in different subgroups of maternal age and body mass index (BMI)? Summary answer Similar mean follitropin delta daily/total doses yield satisfactory number of oocytes and maturity rates, and pregnancy rates in different subgroups of maternal age and BMI. What is known already Rekovelle® (follitropin delta) is a novel recombinant human FSH (rFSH) expressed from a host cell line of human fetal retinal origin (PER.C6®) and is the first commercially available rFSH product derived from human cell lines. Rekovelle® allows the individualization of the initial dose of gonadotropin using predictive response factors to COS, such as anti-müllerian hormone (AMH) levels and body weight. In clinical practice different approaches can be adopted to obtain the most satisfactory response to controlled ovarian stimulation (COS). The objective of this study was to describe data on “real-world” Rekovelle® administration, regarding the response to COS and ICSI outcomes. Study design, size, duration This non-interventional study based on secondary use of data included patients undergoing ICSI in a private university-affiliated IVF center from Jan/2018 to Dec/2021. Each patient or cycle had to meet the following inclusion criteria: pre-menopausal women, undergoing COS with Rekovelle® (16 ug) daily for ICSI; diagnosed with infertility, or with partners diagnosed with male infertility factors, eligible for ICSI using fresh sperm from partner ejaculation; presenting with both ovaries. Participants/materials, setting, methods Ovarian response to stimulation and ICSI outcomes were described for 362 ICSI cycles. The primary outcome measures were the numbers of retrieved oocytes and maturity rates, and the secondary outcome were the ongoing pregnancy rates per fresh and per fresh and/or frozen-thawed embryo transfer. The results obtained with the population enrolled in the ESTHER-1 trial (extern), and a population stimulated with follitropin alpha 300 IU (on site) were recorded (data not shown, to be presented). Main results and the role of chance The mean total dose of follitropin delta administered in subgroups of age and BMI were as follows: age ≤35 y-old 156µg, 36-39 y-old 158 µg, and ≥40 y-old 157µg, and BMI: &amp;lt;18.5 165µg, 18.5-24.9 156µg, 25.0-24.9 159µg, and ≥30 171µg. Patients in the Follitropin delta group showed acceptable outcomes in different age and BMI groups (Table1). Adequate embryo morphokinetic development and euploidy rates were also observed, suggesting that the modified protocol does not interfere with oocyte/embryo competences and embryo implantation potential. This was further corroborated by satisfactory cumulative pregnancy rates. Limitations, reasons for caution Given its descriptive nature, we have focused on the number and maturity stage of retrieved oocytes instead of clinical ICSI outcomes. The use of historical cohort groups is another drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings Patients in different subgroups of age and BMI stimulated with similar mean follitropin delta daily and total doses showed satisfactory number of retrieved oocytes and maturity rates, and cumulative pregnancy rates without increasing OHSS, providing a rationale for conducting clinical trials to confirm the benefits of using this modified protocol. Trial registration number N/A

O-063 Accurate mitochondrial DNA quantification clarifies the clinical value of measuring mtDNA in trophectoderm biopsy specimens

Abstract Study question Can accurate mitochondrial DNA (mtDNA) quantification of trophectoderm (TE) biopsy specimens provide insights into the biology and viability of blastocyst-stage human embryos? Summary answer mtDNA quantity in TE cells is correlated with embryo morphology and shows alterations associated with aneuploidy. However, measurement does not significantly improve embryo viability assessment. What is known already Mitochondria are essential organelles, responsible for producing ATP. Changes in the amount of mtDNA in blastocysts biopsy specimens have been reported in association with embryo implantation potential, leading to proposals that mtDNA quantification might serve as a useful biomarker of embryo viability. However, results from clinical studies to explore this possibility have yielded contradictory data, due in part to deficiencies of the molecular methods used for mtDNA measurement. We sought to clarify what quantification of mtDNA can tell us about embryo biology and viability by developing and applying a method that we believe to be the most accurate ever devised. Study design, size, duration This study involved the analysis of samples collected during the course of routine preimplantation genetic testing for aneuploidy (PGT-A). The IVF treatments and embryo biopsies were undertaken at two different clinics, while chromosomal analyses were carried out at a single reference laboratory. Mitochondrial data was subsequently analysed in a university setting. The embryos analysed were derived from a broad population of patients referred for PGT-A (average age 38.7 years; range 25-47). Participants/materials, setting, methods 651 blastocysts from 133 couples underwent trophectoderm biopsy on day-5 or day-6. The specimens were analysed using a highly validated real-time PCR method, which was used to quantify three distinct sites in the mtDNA and 198 loci in the nuclear genome. The measurement of multiple independent loci provided outstanding sensitivity and accuracy. The nuclear loci were used to normalise the mtDNA data, adjusting for differences in the number of cells in the biopsy specimens. Main results and the role of chance The method developed displayed extraordinary sensitivity and accuracy when quantifying mtDNA. Lower mtDNA quantities were associated with day-6 biopsy (p &amp;lt; 0.0001), extent of blastocyst expansion (p &amp;lt; 0.0001), and superior TE morphology, although the latter was not statistically significant (p = 0.09). mtDNA levels were higher in aneuploid embryos (p &amp;lt; 0.0001), independent on patient age. However, the difference was not sufficient to be considered diagnostic. There was no correlation between mtDNA level and the chances of blastocyst implantation in this dataset. Lower mtDNA levels, previously reported to be associated with higher probabilities of embryo implantation, were most often observed in embryos of excellent morphological grade and likely reflect the increased TE cell numbers of such embryos. Little if any mtDNA replication occurs during preimplantation development and consequently the mtDNA content is divided amongst an ever-growing number of cells, meaning less mtDNA per cell. In this context, mtDNA quantification of blastocyst biopsy specimens provides a highly sensitive measure of TE cellularity, but probably provides little additional benefit for embryo selection beyond conventional morphological grading. However, the fact that higher mtDNA quantities were observed in aneuploid embryos, may indicate that subtle differences in TE cellularity exist in abnormal embryos, which are not fully captured by traditional morphological assessment. Limitations, reasons for caution Previous studies suggested that some blastocysts have greatly elevated mtDNA levels and that such embryos are not viable. In this study, only 5% of embryos were considered outliers in terms of mtDNA quantity. Unfortunately, none of these embryos were transferred, so the potential of these embryos could not be assessed. Wider implications of the findings The quantification of mtDNA in trophectoderm biopsies has sometimes been used for the prioritisation of embryos for transfer. While our results confirm existence of biologically interesting associations between mtDNA and aneuploidy, and a relationship with certain aspects of embryo morphology, measurement of mtDNA seems unlikely to significantly improve embryo selection. Trial registration number not applicable

P-399 Oocyte vitrification does not impair clinical outcomes in women of advanced maternal age. An analysis of 1268 cycles using fresh and vitrified oocytes

Abstract Study question Does oocyte vitrification impair clinical outcomes in women of advanced maternal age (AMA, &amp;gt;35 years old)? Summary answer Up to the age of 39 years, clinical outcomes were comparable amongst cycles using fresh and vitrified oocytes. What is known already Primarily recommended for young women, oocyte vitrification is the most efficient method for elective fertility preservation. Nevertheless, various social and economic circumstances often compel women to undergo fertility preservation when already at an AMA. Recent studies in mouse suggest that AMA increases the vulnerability of oocytes to molecular and subcellular damage during vitrification, ultimately compromising embryo viability and implantation potential. However, the effects of oocyte vitrification on human preimplantation development and clinical outcomes in women of AMA currently remain unknown. Study design, size, duration This is a retrospective cohort single-center study. We compared outcomes of 1268 patients undergoing their first fresh ICSI cycle using autologous fresh (n = 1087) or vitrified oocytes (n = 181), performed between January 2019 and October 2022. All vitrified oocytes were obtained following elective fertility preservation for age-related fertility decline. Outcomes were stratified according to maternal age at oocyte pick-up: control group (≤35) and AMA groups (36-37, 38-39 and ≥40 years). Participants/materials, setting, methods We compared outcomes between fresh and vitrified oocytes across matching maternal age groups. These included fertilization rates, proportion of viable embryos on day 3, clinical pregnancy and live birth rates. Univariate and multivariate analyses (2-contrast linear and logistic regression) were used to establish correlations. Analyses were adjusting for sperm origin (partner or donor), day of transfer (3 or 5), number of embryos transferred and endometrial age. All p values &amp;lt;0.05 were considered significant. Main results and the role of chance Sperm donation and homologous cycles were equally distributed among the study groups (45% sperm donor and 55% male partner across the entire cohort). In our cohort, blastocyst transfer was performed less frequently in older women, 23.8% for women ≥40 years compared to 45.6% for women ≤35 years (p &amp;lt; 0.0001). When comparing oocyte status across matching maternal age groups, fertilization rates were ∼10% lower when vitrified oocytes were used (p &amp;lt; 0.05). Our adjusted analysis confirmed this negative impact of oocyte vitrification on fertilization rates. Oocyte vitrification also affected ongoing cleavage, as the proportion of viable embryos on day 3 was significantly lower across all groups when vitrified oocytes were used (p &amp;lt; 0.05). Nevertheless, this effect did not translate to clinical pregnancy rates, which were comparable across all maternal age groups regardless of oocyte status. As expected, live birth rate decreased steadily with advancing maternal age (from 38.5% in women ≤35 to 15.0% in women ≥40). While our adjusted analysis showed a negative effect of oocyte vitrification on live birth rates for patients ≥40 (OR: 0.60, 95%CI: 0.18-2.06), we observed no such effect in AMA patients ≤39 years. Limitations, reasons for caution The main limitation of this study is its retrospective design. The low number of women ≥40 undergoing fertility presentation (n = 39) warrants careful interpretation. Our results cannot be generalized to all patient populations and indications for fertility preservation. Wider implications of the findings Clinical outcomes were comparable between fresh and vitrified oocytes up to the age of 39 years. While oocyte vitrification before 35 years old remains the most established and effective method for preventing age-related fertility decline, our findings offer a valuable clinical resource for counselling AMA patients considering elective fertility preservation. Trial registration number Not applicable

P-644 The mitochondrial DNA copy number in cumulus cells associate with age and AMH level but do not predict implantation potential of embryos

Abstract Study question is there correlation between the mtDNA copy number in cumulus cells and anti-Mullerian hormone level,female age, oocyte quality, embryo morphology, ploidy and blastocyst implantation rate? Summary answer A positive correlation of mtDNA quantity in CCs with the AMH,female age was revealed.There was no correlation between mtDNA quantity and embryo morphology,ploidy,implantation rate What is known already Recent studies have suggested that age-related decreased competence of oocytes may be due to low quantity of mtDNA copy number. Moreover, quantification of mtDNA in CCs may serve as an predictor of blastocysts viability. The purpose of this study is to investigate relative levels of mtDNA in the OCCCs in association with female age, ovarian reserve, embryo morphology, ploidy and blastocyst implantation rate. Study design, size, duration Prospective clinical study performed on 470 CCs retrieved from 72 advanced reproductive age patients undergoing ART treatment with intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A). Out of the 130 obtained blastocysts 56 embryos were diagnosed as aneuploid, and 74 as euploid. Presently, 51 frozen euploid embryos were transferred (FET). All transferred euploid blastocysts (n = 51) divided into 2 groups: 1 group (n = 21) - implanted embryos, 2 group (n = 30) - non-implanted. Participants/materials, setting, methods Inclusion criteria: age 35-45years;BMI: 18 – 24,9kg/m2; FSH ≤15IU/ml; normal female/male karyotype; non-smokers. Exclusion criteria: genital endometriosis III-IV;severe extragenital pathology; polycystic ovary syndrome; chronic endometritis; &amp;gt; 96% of sperm with abnormal morphology (WHO criteria). MtDNA was assessed by using a quantitative real-time polymerase chain reaction technique. DNA from the trophectoderm samples were amplified and subjected to aneuploidy analysis using array comparative genomic hybridization. In statistic analysis was used Pearson’s correlation coefficients and Fisher’s exact test; p &amp;lt; 0.05 was considered significant. Main results and the role of chance The median age was 37.8 years old (range, 35–45), the mean level of anti-Mullerian hormone (AMH) was 2.66±1.09, and the mean body mass index was 22.3±1.5. A positive correlation of the relative level of mtDNA in the CCs with the patients' age (p = 0.008) and AMH levels (p = 0.003) was revealed. There was no statistically significant correlation between mtDNA copy number and embryo morphology on day 5 (p = 0.7). There was a tendency to increase mtDNA copy number in group 1 vs. group 2, 390 and 299, respectively (p &amp;gt; 0.05). In this study we didn’t find relationship between median mtDNA content of CCs and embryos ploidy (356 vs. 325, in euploid (n = 74) and aneuploidy (n = 56) blastocyst, respectively, p &amp;gt; 0.05). Limitations, reasons for caution Our study was carried out in a relatively small subset of participants and obtained embryos. Correspondingly, the results obtained cannot be readily extrapolated on other groups of patients and need to be confirmed in larger trials. Wider implications of the findings This study showed that mtDNA quantification in CCs isn’t a useful biomarker for prediction embryos implantation potential or ploidy. MtDNA content in CCs correlated only with female age and AMH level. Trial registration number *

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.

O-193 Opening the black box: why do euploid blastocysts fail to implant? A systematic review and meta-analysis

Abstract Study question What are the embryonic, maternal, paternal, clinical and laboratory features associated with the live-birth-rate (LBR) after euploid blastocyst transfer? Summary answer Delayed blastulation, poor blastocyst quality, maternal age ≥38 years, obesity, previous repeated-implantation-failure (RIF), poor or multiple manipulations may reduce the LBR per euploid blastocyst transfer. What is known already Euploidy assessed at the blastocyst stage through comprehensive-chromosome-testing (CCT) technologies during IVF cycles with preimplantation genetic testing for aneuploidies (PGT-A) represents the strongest predictor of embryo implantation potential. Nevertheless, its positive predictive value hardly exceeds 50-60%. The current gap of knowledge on the causes of euploid blastocysts’ reproductive failure is known as “the black box of implantation”. Study design, size, duration Systematic search conducted up to August-2021 with the keywords “(blastocyst OR day 5 embryo OR day 6 embryo OR day 7 embryo) AND (euploid OR chromosomally normal OR preimplantation genetic testing) AND (implantation OR implantation failure OR miscarriage OR abortion OR live birth OR biochemical pregnancy OR recurrent implantation failure)”. Among 1608 items, prospective/retrospective studies and randomized controlled trials (RCTs) investigating associations with LBR and/or miscarriage rates (MR) after non-mosaic euploid blastocyst transfers were scrutinized. Participants/materials, setting, methods 335 retrospective, 30 prospective papers and 7 RCTs were included. PRISMA guideline, PICO model, and ROBINS-I and ROB 2.0 scoring for bias, were adopted. Funnel plots and trim and fill methods were used to visually inspect bias regarding the LBR. Categorical data were combined with a pooled-OR. The random-effect model was used for meta-analyses. Between-study heterogeneity was addressed using I2. Whenever not suitable for the meta-analysis, the included studies were simply described for their results. Main results and the role of chance Inner-cell-mass C-grade (7 studies, OR:0.37, 95%CI:0.27-0.52, p &amp;lt; 0.01), trophectoderm C-grade (9 studies, OR:0.53, 95%CI:0.43-0.67, p &amp;lt; 0.01), overall blastocyst quality &amp;lt;BB (8 studies, OR:0.4, 95%CI:0.24-0.67, p &amp;lt; 0.01) developmental delay (18 studies, OR:0.56, 95%CI 0.49-0.63, p &amp;lt; 0.01) and, by qualitative analysis, some morphodynamic abnormalities pinpointed through time-lapse microscopy (abnormal cleavages, spontaneous collapse, longer tM, tB, and blastulation) were all associated with poorer reproductive outcomes. Slightly lower LBR was reported for women ≥38 years (7 studies, OR:0.87, 95%CI:0.85-1.00, p = 0.05), while maternal BMI&amp;gt;30 involved both lower LBR (2 studies, OR:0.66, 95%CI:0.55-0.79, p &amp;lt; 0.01) and higher MR (2 studies, OR:1.8, 95%CI:1.08-2.99, p = 0.02). The experience of previous RIF was also associated with lower LBR (3 studies, OR:0.72, 95%CI:0.55-0.93, p = 0.01). By qualitative analysis, abnormal progesterone level prior to transfer impaired LBR and MR. Among clinical policies, vitrified-warmed transfer was more effective than fresh (2 studies, OR:1.56, 95%CI:1.05-2.33, p = 0.03). Lastly, multiple vitrification-warming cycles (2 studies, OR:0.41, 95%CI 0.22-0.77, p &amp;lt; 0.01) and, by qualitative analysis, high biopsy cellularity may slightly impact the LBR, while simultaneous zona opening and biopsy protocol showed higher LBR than the day3 hatching-based protocol (3 studies, OR:1.41, 95%CI:1.18-1.69). Limitations, reasons for caution The associations outlined mostly issue from retrospective and/or few studies or studies with a limited sample size, therefore the level of evidence is low or very low. Moreover, some findings represent “prognosis without promise” because the poorer outcome of euploid blastocysts is not clinically actionable (e.g., women ≥38 years). Wider implications of the findings Future research should target: (i) the mechanisms involved in reproductive aging beyond de novo chromosomal abnormalities, and how environment and nutrition may accelerate or exacerbate their consequences; (ii) uterine evaluation and blastocyst-endometrial dialogue; (iii) standardization of embryo assessment and IVF protocols; (iv) additional (non-)invasive tools for embryo selection. Trial registration number http://www.crd.york.ac.uk/PROSPERO/ (registration number CRD42021275329)

P-213 The effects of vitrified embryos transported on IVF clinical outcomes: one center experience

Abstract Study question Does the transportation of vitrified embryos affect the survival rate and in vitro fertilization (IVF) outcomes compared to those produced on-site? Summary answer The regulated transport of cryopreserved embryos has shown no detrimental effects on embryo survival rate or pregnancy outcomes. What is known already Reproductive medicine has substantially evolved in the last decades. A growing number of patients have stored gametes or embryos following IVF treatments; contemporary innovations in cryopreserving techniques revolutionized the IVF laboratory. However, the IVF industry is changing; clinics worldwide are merging with larger international companies, and patients choose to travel abroad for fertility treatments, which requires the movement of cryopreserved embryos over long distances. So far, no exhaustive evidence has been provided regarding the possible detrimental effects on cryopreserved embryos due to their transportation. Study design, size, duration This retrospective observational study assessed the outcomes of 608 single frozen embryo transfers at Instituto Valenciano de Infertilidad (IVI) Rome (Italy) between February 2021 and March 2022, assessing survival and IVF outcomes. Participants/materials, setting, methods Data from N = 608 patients undergoing frozen embryo transfer (FET) from autologous or donated oocytes were analysed. Single blastocysts transferred at IVI Rome (Group A, n = 440), were compared to those generated and vitrified at IVI Spain clinics, and subsequently transported to IVI Rome (Group B, n = 168). The transport system was managed by the same truck company. Frozen embryos from abroad were rigorously checked and later thawed by a team of expert embryologists. Main results and the role of chance Analysis of baseline population characteristics showed a significant difference in female patients’ age between Group A and Group B (39.78 ± 5.16 vs 42.47 ± 4.70; p &amp;lt; 0.01). The highest incidence of heterologous cycles was in Group B (45.4% vs 82.14%; p &amp;lt; 0.01). No significant differences were found between the age of the oocyte donors, male partners, or the patients’ BMI. Considering IVF outcomes, we found no statistically significant difference comparing Group A and B respectively for embryo survival rates after thawing (N = 430/440, 97.7% vs. N = 165/168, 98.21%, p = 0.71), pregnancy rate (N = 221/440, 50.23% vs. N = 77/168, 45.83%, p = 0.33), clinical pregnancy rate (N = 200/440, 45.45% vs. N = 62/168, 36.90%, p = 0.06), and cumulative miscarriage rate (N = 42/221, 19,00% vs. 22/77, 28.57%, p = 0.07). The sub-analysis considering embryos screened for aneuploidies using preimplantation genetic testing for aneuploidies (PGT-A) showed no statistical differences across the different groups. The logistic regression analysis showed that PGT, semen parameters, endometrial thickness, type of endometrial preparation and age, do not affect embryo survival and IVF outcomes. Limitations, reasons for caution The retrospective nature was the principal limitation. One single fertility Company (IVI-RMA) and its network have been considered, which could bias the results, although we employed different clinics in two different countries. Wider implications of the findings Regulated transport of vitrified human embryos between clinics in different countries are safe and reliable, with no adverse effects on embryo survival after thawing and IVF outcomes following FET. Embryo transportation did not affect embryo implantation potential, offering opportunities for patients to transfer biological samples within different laboratories. Trial registration number not applicable

Prediction model for day 3 embryo implantation potential based on metabolites in spent embryo culture medium

BackgroundMetabolites in spent embryo culture medium correlate with the embryo’s viability. However, there is no widely accepted method using metabolite dada to predict successful implantation. We sought to combine metabolomic profiling of spent embryo culture medium and clinical variables to create an implantation prediction model as an adjunct to morphological screening of day 3 embryos.MethodsThis investigation was a prospective, nested case-control study. Forty-two day 3 embryos from 34 patients were transferred, and the spent embryo culture medium was collected. Twenty-two embryos implanted successfully, and the others failed. Metabolites in the medium relevant to implantation were detected and measured by Liquid Chromatography-Mass Spectrometry. Clinical signatures relevant to embryo implantation were subjected to univariate analysis to select candidates for a prediction model. Multivariate logistical regression of the clinical and metabolomic candidates was used to construct a prediction model for embryo implantation potential.ResultsThe levels of 13 metabolites were significantly different between the successful and failed groups, among which five were most relevant and interpretable selected by Least Absolute Shrinkage and Selection Operator regression analysis. None of the clinical variables significantly affected day 3 embryo implantation. The most relevant and interpretable set of metabolites was used to construct a prediction model for day 3 embryo implantation potential with an accuracy of 0.88.ConclusionsDay 3 embryos’implantation potential could be noninvasively predicted by the spent embryo culture medium’s metabolites measured by LC-MS. This approach may become a useful adjunct to morphological evaluation of day 3 embryos.

Real-time image and time-lapse technology to select the single blastocyst to transfer in assisted reproductive cycles

The success of an assisted reproduction cycle should be the achievement of a healthy singleton live birth following the replacement of one embryo. Therefore, one of the most critical points for embryologists has been the selection criteria and how to choose the best embryo to transfer with high implantation potential. In this vein, morphological evaluation has been historically the method applied. However, this practice relies on a limited number of single observations and is associated with high operator variability. Recently, a major innovation in embryo culture has been the introduction of a new type of incubator with integrated time-lapse monitoring, which enables the embryologist to analyze the dynamic events of embryo development, from fertilization to blastocyst formation. This novel practice is quickly growing and has been implemented in many IVF clinics worldwide. Therefore, the main aim of this review is to illustrate the benefits of time-lapse technology in a modern embryology laboratory. In particular, we discuss the blastocyst collapse(s) event and morphometric blastocyst assessment and analyse their association with embryo viability and implantation potential.

Evaluation of fragmented embryos implantation potential using time-lapse technology.

To examine the implantation potential of fragmented embryos that underwent morphokinetic evaluation in a time-lapse incubator. A retrospective study analyzing 4210 Day 5 embryos which were incubated in a time-lapse incubator, between 2013 and 2019. Embryos with more than 5% fragmentation (379 embryos) were included in the study. Embryos selected using the general model and re-examined by our in-house model. Embryo fragmentation percentage was documented from the first cell-division (start fragmentation) to its maximal percentage (final fragmentation), and the ratio between them (fragmentation worsening). Data were analyzed with relation to embryo development, embryos transfer or freezing, clinical pregnancy, and live birth rates. Embryo fragmentation and morphokinetics were found to be independent variables for clinical pregnancy achievements. A higher fragmentation worsening was noted among discarded embryos compared to transferred or frozen embryos (p < 0.0001). Advanced maternal age had a significant negative effect on fragmentation (p < 0.001). Missed abortion rates were similar in fragmented embryos that implanted compared with the overall population. Live birth rates were comparable among embryos which were selected for transfer or freezing by their morphokinetics and had different severity of fragmentation. Our study shows that fragmented embryos have a potential to implant and therefore should be selected for transfer. Laboratories which do not use time-lapse incubators for embryo selection, should consider transferring fragmented embryos, since they have an acceptable chance for live birth. Calculation of fragmentation worsening may enhance our ability to predict embryo development. Further research with analysis of more fragmented embryo maybe beneficial. This study was approved by the local ethics committee No. 0010-19 CMC on April 18th, 2019.

The effects of cycloastragenol on bovine embryo development, implantation potential and telomerase activity.

Telomerase reverse transcriptase is a key factor responsible for structural and cellular alterations in aged oocytes and changes in the structure of the zona pellucida and mitochondria. Telomerase expression is reduced in aged cumulus oocyte complexes, and its activation or enhanced expression would be beneficial for in vitro oocyte maturation and in vitro embryo development. This study aimed to investigate telomerase activation by cycloastragenol and its effect on bovine oocyte in vitro maturation, fertilisation, and early embryo development. We used qPCR, Western blot, immunofluorescence, reactive oxygen species (ROS) assay,TUNEL assay, JC-1 assay, and invasion assay to analyse the affect of cycloastragenol (CAG) on bovine oocyte maturation, embryo development, embryo quality and implantation potential. Cycloastragenol treatment of oocytes in in vitro maturation (IVM) media significantly (P <0.05) improved oocyte IVM (90.87%), embryo cleavage (90.78%), blastocyst hatching (27.04%), and embryo implantation potential. Telomerase also interacts with mitochondria, and JC-1 staining results showed significantly (P <0.05) higher mitochondrial membrane potential (ΔΨ m) in the CAG-treated group. Furthermore, the inner cell mass (OCT4 and SOX2) and trophoblasts (CDX2) of the control and CAG groups were examined. Moreover, CAG treatment to primary cultured bovine cumulus cells substantially enhanced telomerase activity. Telomerase activation via cycloastragenol is beneficial for bovine oocyte IVM and for the production of high-quality bovine embryos. Cycloastragenol is a natural telomerase activator, and could be useful as a permanent component of oocyte maturation media.

Quality assurance (QA) for monitoring the performance of assisted reproductive technology (ART) staff using artificial intelligence (AI).

Deep learning neural networks have been used to predict the developmental fate and implantation potential of embryos with high accuracy. Such networks have been used as an assistive quality assurance (QA) tool to identify perturbations in the embryo culture environment which may impact clinical outcomes. The present study aimed to evaluate the utility of an AI-QA tool to consistently monitor ART staff performance (MD and embryologist) in embryo transfer (ET), embryo vitrification (EV), embryo warming (EW), and trophectoderm biopsy (TBx). Pregnancy outcomes from groups of 20 consecutive elective single day 5 blastocyst transfers were evaluated for the following procedures: MD performed ET (N = 160 transfers), embryologist performed ET (N = 160 transfers), embryologist performed EV (N = 160 vitrification procedures), embryologist performed EW (N = 160 warming procedures), and embryologist performed TBx (N = 120 biopsies). AI-generated implantation probabilities for the same embryo cohorts were estimated, as were mean AI-predicted and actual implantation rates for each provider and compared using Wilcoxon singed-rank test. Actual implantation rates following ET performed by one MD provider: "H" was significantly lower than AI-predicted (20% vs. 61%, p = 0.001). Similar results were observed for one embryologist, "H" (30% vs. 60%, p = 0.011). Embryos thawed by embryologist "H" had lower implantation rates compared to AI prediction (25% vs. 60%, p = 0.004). There were no significant differences between actual and AI-predicted implantation rates for EV, TBx, or for the rest of the clinical staff performing ET or EW. AI-based QA tools could provide accurate, reproducible, and efficient staff performance monitoring in an ART practice.

Combining Machine Learning with Metabolomic and Embryologic Data Improves Embryo Implantation Prediction

This study investigated whether combining metabolomic and embryologic data with machine learning (ML) models improve the prediction of embryo implantation potential. In this prospective cohort study, infertile couples (n=56) undergoing day-5 single blastocyst transfer between February 2019 and August 2021 were included. After day-5 single blastocyst transfer, spent culture medium (SCM) was subjected to metabolite analysis using nuclear magnetic resonance (NMR) spectroscopy. Derived metabolite levels and embryologic parameters between successfully implanted and failed groups were incorporated into ML models to explore their predictive potential regarding embryo implantation. The SCM of blastocysts that resulted in successful embryo implantation had significantly lower pyruvate (p<0.05) and threonine (p<0.05) levels compared to medium control but not compared to SCM related to embryos that failed to implant. Notably, the prediction accuracy increased when classical ML algorithms were combined with metabolomic and embryologic data. Specifically, the custom artificial neural network (ANN) model with regularized parameters for metabolomic data provided 100% accuracy, indicating the efficiency in predicting implantation potential. Hence, combining ML models (specifically, custom ANN) with metabolomic and embryologic data improves the prediction of embryo implantation potential. The approach could potentially be used to derive clinical benefits for patients in real-time.

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.