Genome-Wide Analysis of DNA Methylation in Buccal Cells of Children Conceived through IVF and ICSI

Early life periconceptional exposures during assisted reproductive technology (ART) procedures could alter the DNA methylation profiles of ART children, notably in imprinted genes and repetitive elements. At the genome scale, DNA methylation differences have been reported in ART conceptions at birth, but it is still unclear if those differences remain at childhood. Here, we performed an epigenome-wide DNA methylation association study using Illumina InfiniumEPIC BeadChip to assess the effects of the mode of conception on the methylome of buccal cells from 7- to 8-year-old children (48 children conceived after ART or naturally (control, CTL)) and according to the embryo culture medium in which they were conceived. We identified 127 differentially methylated positions (DMPs) and 16 differentially methylated regions (DMRs) (FDR < 0.05) with low delta beta differences between the two groups (ART vs. CTL). DMPs were preferentially located inside promoter proximal regions and CpG islands and were mostly hypermethylated with ART. We highlighted that the use of distinct embryo culture medium was not associated with DNA methylation differences in childhood. Overall, we bring additional evidence that children conceived via ART display limited genome-wide DNA methylation variation compared with those conceived naturally.

Non-invasive Metabolomic Profiling of Embryo Culture Medium Using Raman Spectroscopy With Deep Learning Model Predicts the Blastocyst Development Potential of Embryos

Purpose: This study aimed to establish a non-invasive predicting model via Raman spectroscopy for evaluating the blastocyst development potential of day 3 high-quality cleavage stage embryos.Methods: Raman spectroscopy was used to detect the metabolic spectrum of spent day 3 (D3) embryo culture medium, and a classification model based on deep learning was established to differentiate between embryos that could develop into blastocysts (blastula) and that could not (non-blastula). The full-spectrum data for 80 blastula and 48 non-blastula samples with known blastocyst development potential from 34 patients were collected for this study.Results: The accuracy of the predicting method was 73.53% and the main different Raman shifts between blastula and non-blastula groups were 863.5, 959.5, 1,008, 1,104, 1,200, 1,360, 1,408, and 1,632 cm–1 from 80 blastula and 48 non-blastula samples by the linear discriminant method.Conclusion: This study demonstrated that the developing potential of D3 cleavage stage embryos to the blastocyst stage could be predicted with spent D3 embryo culture medium using Raman spectroscopy with deep learning classification models, and the overall accuracy reached at 73.53%. In the Raman spectroscopy, ribose vibration specific to RNA were found, indicating that the difference between the blastula and non-blastula samples could be due to materials that have similar structure with RNA. This result could be used as a guide for biomarker development of embryo quality assessment in the future.

The effects of supplemented sericin on in vitro maturation and preimplantation development of mouse embryos: An experimental study

Background: Mouse embryo culture condition is an essential part of transgenic, reproductive and developmental biology laboratories. Mouse embryonic culture media may have a high risk of serum contamination with pathogens. Objective: To investigate the effect of sericin as an embryo culture medium supplement on in vitro maturation (IVM), in vitro fertilization (IVF), and development of the preimplantation embryo in mice. Materials and Methods: The effects of sericin at three concentrations (subgroups) of 0.1%, 0.5%, and 1% as a medium supplement on IVM, IVF, and in vitro development of mouse embryos were separately investigated and compared with a sericin-free (control) group. The cumulative effect of the three concentrations was evaluated for IVM + in vitro development and IVF + in vitro development as follow-up groups. Results: In the IVM group, compared to the control group, the number of oocysts reaching the MII stage was significantly higher when 1% sericin was used (161/208 = 77.4%). No significant results were observed in the IVF and in vitro development groups with different concentrations of sericin compared to the control group. Among the follow-up groups, in the IVM + in vitro development group, the number of oocytes was higher after passing the IVM and IVF and reaching the blastocysts stage when 1% sericin was used, compared with other sericin subgroups. A significant difference was also noted when compared with the control group (p = 0.048). The IVF + in vitro development study group, on the other hand, did not show any significant relationship. Conclusion: It can be concluded that 1% sericin can be used as a supplement in mouse embryo cultures to improve the IVM rate. Also, based on the findings, sericin appears to be an effective supplement which can have a positive effect on the development of embryos derived from IVM. Key words: Sericin, In vitro maturation, In vitro fertilization, Preimplantation embryo, Culture medium, Mice.

P–146 Differential impact of three embryo culture media for IVF on in vitro development and perinatal outcome: a single-center RCT

Study question This report provides updated data from an RCT determining which embryo culture medium yields optimal IVF outcomes. Summary answer Embryo culture systems used for IVF differentially affected preimplantation development and resultant obstetric and perinatal outcomes, including birthweights of live-born singletons. What is known already Currently, multiple embryo culture medium systems are in use for IVF, raising questions regarding which is optimal. However, the ability of a medium to yield preimplantation embryos is not necessarily indicative of embryo viability. For example, supplementation of medium with serum was commonly used to increase animal blastocyst yields, but this impaired embryonic, fetal, and offspring health. In humans, medium composition and culture duration can influence IVF efficacy and offspring phenotype. Given the importance of culture systems in determining clinical outcomes, existing data regarding differential culture system impacts are insufficient and additional well-designed studies are required. Study design, size, duration Between February 2016 and August 2017, 795 couples undergoing their first autologous clinical IVF cycle and freeze-all strategy were recruited. Participants were randomized via computer-generated tables into three groups. Following standard oocyte retrieval and IVF/ICSI procedures, embryos were cultured using three different culture media, G1 Plus/G2 Plus (G1/G2; Vitrolife), Global Total (GT; LifeGlobal), or Sequential Cleav/Sequential Blast (SC/SB; Origio). Thirty-eight patients exhibiting no 2PN oocytes following insemination or those undergoing fresh embryo transfers were excluded. Participants/materials, setting, methods For patients yielding a single good-quality cleavage-stage (day–2 or day–3) embryo, that cleavage-stage embryo was vitrified. For patients yielding two or more good-quality cleavage-stage embryos, two or less good-quality cleavage-stage embryos were vitrified. The culture period of the remaining embryos was extended, and all good-quality blastocyst-stage (day–5 or day–6) embryos were vitrified. This report presents data for vitrified embryo transfer performed until the end of December 2020. Main results and the role of chance The mean per-cycle vitrified embryo yield (± SD) was comparable between groups for cleavage-stage embryos, but significantly different for blastocyst-stage embryos (G1/G2: 1.69 ± 2.2, GT: 2.53 ± 3.01, SC/SB: 2.04 ± 2.42; P = 0.001). Following vitrified cleavage- or blastocyst-stage embryo transfers, biochemical pregnancy rates were significantly different between groups (G1/G2: 55.6%, GT: 59.1%, SC/SB: 46.2%; P = 0.011). Furthermore, a between-group trend towards different live birth rates was observed (G1/G2: 41.7%, GT: 42.1%, SC/SB: 33.1%; P = 0.063). Of 382 live births, data for first-borns (n = 323; 295 singletons and 14 twin-pairs) are reported here. Perinatal data did not differ significantly between groups for both cleavage- and blastocyst-stage embryo transfers, including gestational age- and gender-adjusted singleton birthweight (z-score). Following multiple linear regression (including selected covariates), adjusted mean singleton birthweights were significantly lower in the G1/G2 and GT groups than in the SC/SB group (by 131 g; P = 0.011 and 110 g; P = 0.032, respectively) and tended to be lower for cleavage-stage embryo transfers than for blastocyst-stage embryo transfers (by 102 g; P = 0.053). Limitations, reasons for caution A larger cohort size and longer-term follow-up are required to verify and further elucidate the impact of embryo culture methods on child health. Such studies will raise awareness regarding the sensitivity of in vitro-cultured human embryos to their environment, ultimately resulting in practices that decrease IVF risks to offspring. Wider implications of the findings: Pregnancy outcome of the medium yielding fewer blastocysts was comparable or superior to that of other media, highlighting the importance of differentiating between the ability to support preimplantation development versus the ability to yield viable embryos. Embryo culture medium had a greater impact than embryo transfer stage on live birthweight. Trial registration number UMIN000020910

O-121 Exploring non-invasive methods to predict Ploidy Status: Combination of blastocyst morphology image analysis and proteomic profiles by using Artificial Neural Networks

Study question Is the blastocyst morphology image analysis combined with the protein content of spent embryo culture medium a suitable way to predict embryo ploidy? Summary answer Morphological variables from blastocyst image analysis combined with IL-6 or MMP-1 concentration in spent culture medium showed more than 80% of accuracy for euploidy prediction. What is known already An artificial intelligence model based on the proteomic profile of euploid embryos and morphological data from blastocyst time-lapse images has been recently published (Bori et al., 2020). The most promising artificial neural network (ANN) algorithm considered 20 morphological variables extracted from image analysis and two proteins detected in embryo culture medium (MMP-1 and IL-6). The overall success rate on blind test data was 72.7% for live birth prediction. The main aim of the present study was to check if the same morphological variables combined with MMP-1 or IL-6 with a cost-effective technique could discriminate between euploid and aneuploid embryos. Study design, size, duration This prospective study included 120 embryos from the preimplantation genetic testing for aneuploidies (PGT-A) program. A single blastocyst image was obtained for each embryo and their spent culture medium was collected on the day 5/6 of embryo development (day of trophectoderm biopsy). Morphological variables were extracted for all the blastocyst. On the other hand, we quantified IL-6 levels of 67 embryos and MMP-1 levels of 53 embryos. Resulting parameters were used to predict PGT-A results. Participants/materials, setting, methods Blastocyst images were imported into Matlab software and segmented into regions of interest. We obtained 20 mathematical variables related to measurements of areas, number of pixels and texture analysis. Chromosome analysis was performed using next-generation sequence technology. In parallel, 20 µL of spent culture medium from each blastocyst was analyzed with ELISA kits (IL-6 or MMP-1). Protein concentrations and morphological variables were used as input data for an ANN associated with genetic algorithms. Main results and the role of chance The euploid rate for the set of embryos included in the IL-6 group was 51.4%. The ANN was trained with 49 embryos and blind tested with 18 embryos. Following results correspond to euploidy prediction on the blind test. The sensitivity, specificity, accuracy and area under the ROC curve (AUC) were: 0.56, 0.78, 0.67 and 0.72 considering only IL-6 values; 0.88, 0.78, 0.83 and 0.61 considering IL-6 values and blastocyst morphological data extracted from the image analysis. The euploid rate for the set of embryos included in the MMP-1 group was 51.9%. The ANN was trained with 39 embryos and blind tested with 14 embryos. Following results correspond to euploidy prediction on the blind test. The sensitivity, specificity, accuracy and AUC were: 0.71, 0.57, 0.64 and 0.67 considering only MMP-1 values; 0.86, 0.86, 0.86 and 0.61 considering MMP-1 values and morphological data extracted from the image analysis. Limitations, reasons for caution The detection limit in protein quantification is the main limitation of our study. The small number of embryos and the specific culture medium used should be considered for the model application. Wider implications of the findings Our preliminary results showed that blastocyst morphology and embryo secretomics could be useful for euploidy prediction by using artificial intelligence techniques. These findings may contribute to the emerging era of non-invasive preimplantation genetic testing (ni-PGT-A). Trial registration number not applicable

P–275 Development of a prediction model using machine learning on small noncoding RNA biomarkers for non-invasive selection of high-quality embryos for the in vitro fertilization process

Study question The aim of the study was to identify molecules in the embryo culture medium as important predictive biomarkers of high-quality embryos Summary answer The study identified 14 canonical iso-miRNA molecules that prognostically determine the quality of the embryo with a prediction accuracy with 95% sensitivity and 80% specificity. What is known already The quality of the embryo for the success of the IVF process is not specifically diagnosed, only morphological features (monitoring in the embryoscope) are considered. Embryo quality selection systems have likely reached their peak. The success rate of the IVF process is only 29%.; it is therefore necessary to look for other biomarkers. The oocyte itself can significantly predict the development of the early embryo,as it is a supplier of RNA and cellular mechanisms. However, collection follicular fluid is technically demanding. The probability of oocyte fertilization does not reach the required percentage therefore other embryological techniques multiply the economic costs. Study design, size, duration Women (n = 734) who visited the IVF centre were recruited for the study. Oocytes were collected from 54 of them and used for IVF. After 4/5-day embryo cultivation, the best quality embryo was selected and used for implantation into the uterus. The culture medium has been collected from 60 embryos during 3 years (2018–2020). Written informed consent was obtained from all patients. The study has been approved by the Ethical committee of the Košice governing region Participants/materials, setting, methods We used fresh/frozen culture media of embryos selected using an embryoscope. Further, information regarding the success of IVF, pregnancy and IVF failure was collected. Culture media libraries of noncoding small RNAs (miRNAs) were examined using massively parallel sequencing on the Illumina platform. Obtained data was processed with freely available bioinformatic tools and machine learning. For methods with different models, the number of predictive biomarkers and specific prognostic-predictive molecules were selected. Main results and the role of chance The main results of the study specifically identify ncRNA molecules that prognostically and predictively select a high-quality embryo suitable for IVF transmission from a low-quality embryo with 95% sensitivity and 80% specificity with an average accuracy of 85% in 4 different models. We also determined the minimum of 14 miRNA as prediction biomarkers. The developed model can predict embryo quality from the culture medium based on ncRNA results from sequence data and set the cut-off value for the expression and significance of individual miRNA molecules with respect to embryo quality. Furthermore, positive and negative correlations of miRNA molecules with different distributions in a high-quality embryo compared to a low-quality embryo were determined. The molecules identified in the embryo culture medium were organized according to their importance, resp. significance based on their significance coefficient. So far, there is no evidence of pending patents regarding the distribution of specific canonical miRNAs and iso-miRNA molecules analysed by massively parallel sequencing in terms of biological competence and embryo quality determination with multifactorial consideration of its variation. This is the first study focused on the success of the IVF process based on embryo quality prediction. Limitations, reasons for caution Exploratory data need to be validated in a larger scale study. Wider implications of the findings: The given miRNA molecules and the software model can be used as a safe,non-invasive diagnostic test for the selection of a highly competent embryo. Canonical and iso-miRNA molecules from the study can be used in other forms of diagnostic assays, such as specific embryo selection probes and, plate hybridization assay. Trial registration number non clinical trials