Search Results (17)

A predominant etiological factor in implantation failure and early pregnancy loss is embryonic chromosomal abnormalities. The current clinical standard for determining embryonic ploidy is invasive preimplantation genetic testing. This procedure imposes mechanical stress on embryonic cells during trophectoderm biopsy and fails to significantly improve live birth rates per transfer, likely due to its inability to evaluate the embryo’s implantation potential. Consequently, there is a clear need to develop a non-invasive method, suitable for routine clinical practice, that can simultaneously assess both the ploidy and implantation competence of a blastocyst prior to uterine transfer. Our research group was the first to achieve this by quantifying specific piwiRNAs (piR_016677, piR_017716, piR_020497, piR_015462) in spent culture medium. These data served as the foundation for logistic regression models tailored for day 5 blastocysts, day 6 blastocysts, and blastocysts irrespective of their developmental rate. These models demonstrated high diagnostic accuracy, with specificity ranging from 68% to 100% and sensitivity from 71% to 100%. The rationale for employing these molecules as biomarkers lies in their potential biological roles, which encompass maintaining genomic stability through LINE-1 regulation, as well as direct involvement in critical processes such as cell cycle control, spindle assembly, and cellular adhesion—all of which are imperative for successful implantation. Full article

MicroRNAs, secreted by the embryo in blastocoel fluid (BF) and embryo spent culture medium (SCM), regulate important cellular pathways controlling the stemness of inner cell mass, trophectoderm differentiation, and the dialogue between blastocyst and maternal tissues. In recent years, their role as non-invasive biomarkers of embryo quality has been deeply investigated. We compared the expression profiles of 96 microRNAs between BF and SCM from the same embryos, highlighting the differences between these two compartments. We found 10 and 6 microRNAs specifically expressed in BF and in SCM, respectively; 22 microRNAs significantly up-regulated in BF; and 2 significantly up-regulated in SCM. To investigate the role of SCM microRNAs in implantation, we focused on the microRNAs specifically expressed/up-regulated in SCM and absent in blank medium. We deepened our understanding of SCM microRNA’s biological role by building a network of miRNA–mRNA interaction within the signalling pathways crucial in embryo implantation success. We demonstrated that BF and SCM contain different sets of microRNAs playing different and unique roles in embryo implantation and development. Finally, we suggest that there is not a single “ideal” technique to identify the most competent embryo, but an integrated approach is needed to obtain informative results on the health of the embryo. Full article

MicroRNAs (miRNAs) are short, non-coding RNA molecules that play a crucial role in regulating various biological processes by influencing post-transcriptional gene expression and gene silencing. Background/Objectives: In this study, rabbit embryos were utilised as a model system to investigate potential biomarkers relevant to human embryo development. Seven microRNAs (miRNAs) identified in the embryo culture medium were evaluated as biomarkers by analysing the correlation between their expression levels and the developmental quality of rabbit embryos at days 4 and 6. Methods: We analysed the expression of seven development-specific miRNAs (miR-24-3p, miR-28-3p, miR-103a-3p, miR-181a-5p, miR-191-5p, miR-320a-3p, miR-378a-3p) in 4-day-old and 6-day-old rabbit embryos, along with their culture media. Results: Our findings revealed significant differences in the expression levels of these miRNAs between the 4-day-old and 6-day-old embryos. On the other hand, the expression patterns observed in the culture medium samples showed less variation between the two age groups. Nonetheless, analysis of miRNA expression profiles in the spent culture medium from individually cultured embryos enabled the identification of lower-quality embryos, characterised by smaller size and impaired or delayed development. Conclusions: The detection of these miRNAs in embryo culture medium may serve as a reliable indicator of successful progression to the blastocyst stage. Our experimental results identified specific miRNAs whose expression profiles differ according to embryonic stage and quality, thereby reflecting key developmental milestones. Notably, the detectability of these miRNAs in the medium—without prior RNA isolation—indicates their active secretion into the extracellular environment. By synthesising our findings with the existing literature, we refined a panel of miRNAs essential for the development of implantation-competent embryos in both rabbits and humans. Consequently, we developed a non-invasive assay for predicting implantation and pregnancy outcomes, which may have significant applications in human reproductive medicine. Full article

In this study, we aimed to correlate embryonic ploidy status studied with non-invasive preimplantation genetic testing for aneuploidy with the basic patient characteristics of the infertile couple to gain insight into the effects of parental physical health on embryo ploidy. We recruited 131 couples, who were stratified into 4 groups based on female age. We gathered general patient characteristics of the couple and determined the female’s hormonal status. We included 316 embryos in our study. Embryos were either transferred in the uterus in a fresh cycle or vitrified for later use. We collected spent embryo culture medium on either day 5 or 6 and performed whole genome amplification before using Next Generation Sequencing. Pregnancy outcomes were noted and cross-referenced with patient characteristics and the embryo’s ploidy status in a retrospective manner. While we have indirectly observed a level of maternal contamination, we nevertheless found a significant correlation between embryo ploidy status and cell free deoxyribonucleic acid concentration in spent embryo culture, as well a correlation between female age and embryo ploidy status. We observed a significant correlation between male body mass index and cell free deoxyribonucleic acid concentration in spent embryo culture medium and between male body mass index and pregnancy outcome. We illustrated a connection between male body mass index and cell free deoxyribonucleic acid, independent of female markers. This is the first study to observe not only female but male parameters in correlation to cell free deoxyribonucleic acid. Full article

Recent studies have shown that the metabolome of single embryo culture media is linked to successful pregnancy. In this study, the analysis was expanded to compare the metabolomes of viable and non-viable early-stage embryos and to examine metabolomic markers associated with hatching in viable embryos. The authors hypothesized that the metabolomic profiles of high-quality early blastocysts differ from those of non-viable embryos that reach the blastocyst stage but undergo developmental arrest at later stages. The metabolic profile of 43 spent bovine embryo culture medium samples were analyzed using liquid chromatography–mass spectrometry, covering 189 metabolites, including 40 acylcarnitines, 42 amino acids/biogenic amines, 91 phospholipids, 15 sphingolipids, and the sum of hexoses. Embryos were produced from abattoir-derived oocytes, and the culture medium samples were derived from Grade 1 early blastocysts that progressed to hatching (VBL; n = 10), non-viable early blastocysts that developed to the blastocyst stage but failed to hatch (DBL; n = 12), Grade 1 hatched blastocysts (HBL; n = 16), and plain growth media for control (CM; n = 5). It was observed that methionine sulfoxide (Met-SO) and lysophosphatidylcholine (lysoPC) C24:0 concentrations were significantly lower in the culture media from viable blastocysts compared to those from non-viable blastocysts (p < 0.001). Additionally, blastocysts that resulted in successful hatching had significantly lower levels of phospholipid, arginine (Arg), and methionine-related metabolites that significantly differentiated the control and viable blastocyst culture media from the media containing non-viable embryos. Building on previous studies, there appears to be an overlap in metabolites released during hatching that are also associated with successful pregnancy. The identified biomarkers can aid in assessing an embryo’s developmental potential and enhance embryo selection for transfer or cryopreservation. Full article

Background/Objectives: To assess the ploidy status of embryos via preimplantation genetic testing for aneuploidy (PGT-A), a biopsy of trophectoderm (TE) cells can be performed. However, this approach is considered invasive, and therefore the aim of this study was to identify the optimal sample type and sampling day for non-invasive or minimally invasive PGT-A (ni/miPGT-A) in terms of data quality and concordance rates with TE biopsies derived from the same embryos. Methods: This study was performed using 239 embryo cultures. After optimization using 96 embryos, non-invasive spent culture media (SCM) and a minimally invasive combination of blastocoel fluid and SCM (BF+SCM), along with the corresponding TE samples, were collected from 143 embryos cultured for 5 days (n = 70) or 6 days (n = 73), and all were subjected to ni/miPGT-A with whole-genome amplification followed by next-generation sequencing. Results: The amplification failure rate was lower for SCM samples than for BF+SCM (SCM: 0.7%, 1/143 vs. BF+SCM: 7.7%, 11/143; p = 0.005). The rate of ploidy concordance with TE was significantly higher for SCM samples than for BF+SCM samples (SCM: 83.7%, 118/141 vs. BF+SCM: 58%, 76/131; p < 0.001). Among SCM samples, concordance rates were higher for samples derived from embryos cultured for 6 days (87.5%, 63/72) than for 5 days (79.7%, 55/69). In the embryos cultured for 6 days, discordant cases included five (6.9%) SCM samples with falsely negative (euploid) results that were deemed to be mosaic according to TE and four (5.6%) samples falsely found to be aneuploid. Conclusions: SCM samples derived from embryos cultured for 6 days can be applied in niPGT-A with subsequent verification of aneuploid samples using TE biopsy. Full article

Extracellular vesicles (EVs) are lipid bilayer-bound particles released from cells that cannot replicate on their own, play a crucial role in intercellular communication, and are implicated in various physiological and pathological processes. Within the domain of embryo culture media research, extensive studies have been conducted to evaluate embryo viability by analyzing spent culture medium. Advanced methodologies such as metabolomic profiling, proteomic and genomic analyses, transcriptomic profiling, non-coding RNA assessments, and oxidative status measurements have been employed to further understand the molecular characteristics of embryos and improve selection criteria for successful implantation. In the field of EVs, only a limited number of studies have been conducted on embryo-conditioned medium, indicating a significant gap in knowledge regarding the potential role of EVs in embryo development and implantation. Therefore, this review aims to evaluate current research findings on EVs enriched from animal and human embryo spent medium. By unraveling the potential link between embryo-derived EVs and embryo selection in clinical settings, such research might enhance embryo-selection methods in assisted reproductive technologies, eventually increasing the success rates of fertility treatments and advancing our understanding of mechanisms underlying successful embryo development and implantation in humans. Full article

An embryo culture medium is a specialized set of ambient conditions, technological equipment, and nutrients that embryos require to grow properly. We aimed to investigate the Ki-67, hTERT, and HIF-1α gene expression differences between developing and non-developing embryos in spent embryo culture medium. Ki-67, hTERT, and HIF-1α gene expressions were determined from the spent embryo culture medium containing developing and non-developing embryos of 20 normoresponder patients admitted to the Bahçeci Umut IVF Center. An increase in hTERT gene expression (p < 0.05) and a decrease in HIF-1α gene expression (p < 0.001) were observed in mediums of developing compared to the non-developing embryos. No difference was observed in Ki-67 gene expression (p > 0.05). While there was a correlation between Ki-67 and HIF-1α genes in the non-growing group (r < 0.01); no correlation was observed in the developing group (r > 0.05). Both normoresponder groups will be similar in terms of proliferation rate. The low HIF-1α expression that observed high telomerase activity in embryo development maintains continuity and avoids mechanisms that result in cell death. A molecular study of the embryo development in patients with similar characteristics may help to understand the pathogenesis of the disease and establish a diagnosis and specific treatment. Full article

The evolution of preimplantation genetic testing for aneuploidy (PGT-A) techniques has been crucial in assisted reproductive technologies (ARTs), improving embryo selection and increasing success rates in in vitro fertilization (IVF) treatments. Techniques ranging from fluorescence in situ hybridization (FISH) to next-generation sequencing (NGS) have relied on cellular material extraction through biopsies of blastomeres at the cleavage stage on day three or from trophectoderm (TE) cells of the blastocyst. However, this has raised concerns about its potential impact on embryo development. As a result, there has been growing interest in developing non-invasive techniques for detecting aneuploidies, such as the analysis of blastocoel fluid (BF), spent culture medium (SCM), and artificial intelligence (AI) models. Non-invasive methods represent a promising advancement in PGT-A, offering the ability to detect aneuploidies without compromising embryo viability. This article reviews the evolution and principles of PGT-A, analyzing both traditional techniques and emerging non-invasive approaches, while highlighting the advantages and challenges associated with these methodologies. Furthermore, it explores the transformative potential of these innovations, which could optimize genetic screening and significantly improve clinical outcomes in the field of assisted reproduction. Full article

Despite advances in in vitro fertilization (IVF), there is still a lack of non-invasive and reliable biomarkers for selecting embryos with the highest developmental and implantation potential. Recently, small non-coding RNAs (sncRNAs) have been identified in biological fluids, and extracellular sncRNAs are explored as diagnostic biomarkers in the prediction of IVF outcomes. To determine the predictive role of sncRNAs in embryo quality and IVF outcomes, a systematic review and meta-analysis was performed. Articles were retrieved from PubMed, EMBASE, and Web of Science from 1990 to 31 July 2022. Eighteen studies that met the selection criteria were analyzed. In total, 22 and 47 different sncRNAs were found to be dysregulated in follicular fluid (FF) and embryo spent culture medium (SCM), respectively. MiR-663b, miR-454 and miR-320a in FF and miR-20a in SCM showed consistent dysregulation in two different studies. The meta-analysis indicated the potential predictive performance of sncRNAs as non-invasive biomarkers, with a pooled area under curve (AUC) value of 0.81 (95% CI 0.78, 0.844), a sensitivity of 0.79 (95% CI 0.72, 0.85), a specificity of 0.67 (95% CI 0.52, 0.79) and a diagnostic odds ratio (DOR) of 8 (95% CI 5, 12). Significant heterogeneity was identified among studies in sensitivity (I2  =  46.11%) and specificity (I2  =  89.73%). This study demonstrates that sncRNAs may distinguish embryos with higher developmental and implantation potentials. They can be promising non-invasive biomarkers for embryo selection in ART. However, the significant heterogeneity among studies highlights the demand for prospective multicenter studies with optimized methods and adequate sample sizes in the future. Full article

Embryo selection is needed to optimize the chances of pregnancy in assisted reproduction technology. This study aimed to validate non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using a routine IVF laboratory workflow. Can niPGT-A combined with time-lapse morphokinetics provide a better embryo-selection strategy? A total of 118 spent culture mediums (SCMs) from 32 couples were collected. A total of 40 SCMs and 40 corresponding trophectoderm (TE) biopsy samples (n = 29) or arrested embryos (n = 11) were assessed for concordance. All embryos were cultured to the blastocyst stage (day 5 or 6) in a single-embryo culture time-lapse incubator. The modified multiple annealing and looping-based amplification cycle (MALBAC) single-cell whole genome amplification method was used to amplify cell-free DNA (cfDNA) from the SCM, which was then sequenced on the Illumina MiSeq system. The majority of insemination methods were conventional IVF. Low cfDNA concentrations were noted in this study. The amplification niPGT-A and conventional PGT-A was 67.7%. Based on this study, performing niPGT-A without altering the daily laboratory procedures cannot provide a precise diagnosis. However, niPGT-A can be applied in clinical IVF, enabling the addition of blastocysts with a better prediction of euploidy for transfer. Full article

In this study we evaluated possible differences in metabolomic profiles of spent embryo culture media (SECM) of human embryos with distinct morphology, karyotype, and implantation outcomes. A total of 153 samples from embryos of patients undergoing in vitro fertilization (IVF) programs were collected and analyzed by HPLC-MS. Metabolomic profiling and statistical analysis revealed clear clustering of day five SECM from embryos with different morphological classes and karyotype. Profiling of day five SECM from embryos with different implantation outcomes showed 241 significantly changed molecular ions in SECM of successfully implanted embryos. Separate analysis of paired SECM samples on days three and five revealed 46 and 29 molecular signatures respectively, significantly differing in culture media of embryos with a successful outcome. Pathway enrichment analysis suggests certain amino acids, vitamins, and lipid metabolic pathways to be crucial for embryo implantation. Differences between embryos with distinct implantation potential are detectable on the third and fifth day of cultivation that may allow the application of culture medium analysis in different transfer protocols for both fresh and cryopreserved embryos. A combination of traditional morphological criteria with metabolic profiling of SECM may increase implantation rates in assisted reproductive technology programs as well as improve our knowledge of the human embryo metabolism in the early stages of development. Full article

Despite the improvements in biotechnological approaches and the selection of controlled ovarian hyperstimulation protocols, the resulting pregnancy rate from in vitro fertilization (IVF) protocols still does not exceed 30–40%. In this connection, there is an acute question of the development of a non-invasive, sensitive, and specific method for assessing the implantation potential of an embryo. A total of 110 subfertile couples were included in the study to undergo the IVF/ICSI program. Obtained embryos for transfer into the uterine cavity of patient cohort 1 (n = 60) and cohort 2 (n = 50) were excellent/good-quality blastocysts, and small noncoding RNA (sncRNA) content in the corresponding spent culture medium samples at the morula stage (n = 43) or at the blastocyst stage (n = 31) was analyzed by deep sequencing followed by qRT-PCR in real time. Two logistic regression models were developed to predict the implantation potential of the embryo with 100% sensitivity and 100% specificity: model 1 at the morula stage, using various combinations of hsa_piR_022258, hsa-let-7i-5p, hsa_piR_000765, hsa_piR_015249, hsa_piR_019122, and hsa_piR_008112, and model 2 at the blastocyst stage, using various combinations of hsa_piR_020497, hsa_piR_008113, hsa-miR-381-3p, hsa_piR_022258, and hsa-let-7a-5p. Protein products of sncRNA potential target genes participate in the selective turnover of proteins through the ubiquitination system and in the organization of the various cell cytoskeleton and nucleoskeleton structures, regulating the activity of the Hippo signaling pathway, which determines the fate specification of the blastomers. Full article

Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrimidine metabolism) in the CM group, suggesting a beneficial effect of CM on embryo development. The metabolomic study carried out in a microfluidic environment confirms our hypothesis on the potential of uterine epithelial cells to enhance blastocyst development. Further investigations are required to highlight specific pathways involved in embryo development and implantation. Full article

As part of the optimization of assisted reproductive technology programs, the aim of the study was to identify key small noncoding RNA (sncRNA) molecules that participate in maternal-to-zygotic transition and determine development potential and competence to form a healthy fetus. Small RNA deep sequencing followed by quantitative real-time RT-PCR was used to profile sncRNAs in 50 samples of spent culture medium from morula with different development potentials (no potential (degradation/developmental arrest), low potential (poor-quality blastocyst), and high potential (good/excellent quality blastocyst capable of implanting and leading to live birth)) obtained from 27 subfertile couples who underwent in vitro fertilization. We have shown that the quality of embryos at the morula stage is determined by secretion/uptake rates of certain sets of piRNAs and miRNAs, namely hsa_piR_011291, hsa_piR_019122, hsa_piR_001311, hsa_piR_015026, hsa_piR_015462, hsa_piR_016735, hsa_piR_019675, hsa_piR_020381, hsa_piR_020485, hsa_piR_004880, hsa_piR_000807, hsa-let-7b-5p, and hsa-let-7i-5p. Predicted gene targets of these sncRNAs included those globally decreased at the 8-cell–morula–blastocyst stage and critical to early embryo development. We show new original data on sncRNA profiling in spent culture medium from morula with different development potential. Our findings provide a view of a more complex network that controls human embryogenesis at the pre-implantation stage. Further research is required using reporter analysis to experimentally confirm interactions between identified sncRNA/gene target pairs. Full article