A pilot study of transcriptomic preimplantation genetic testing (PGT-T): towards a new step in embryo selection?

Is it possible to predict an euploid chromosomal constitution and identify a transcriptomic profile compatible with extended embryonic development from RNA sequencing (RNA-Seq) data? It has been possible to obtain a karyotype comparable to preimplantation genetic testing for aneuploidy (PGT-A), in addition to a transcriptomic signature of embryos which might be suggestive of improved implantation capacity. Conventional assessment of embryo competence, based on morphology and morphokinetic, lacks knowledge of molecular aspects and faces controversy in predicting ploidy status. Understanding the embryonic transcriptome is crucial, as gene expression influences development and implantation. PGT has improved pregnancy rates, but problems persist when high-quality euploid embryos do not reach term. In fact, only around 50-60% implant, of which 10% result in miscarriage. Comprehensive approaches, including RNA-Seq, offer the potential to discover molecular markers of reproductive competence, and could theoretically be combined with extended-embryo culture platforms up to Day 14 that can be utilized as a proxy to study embryo development at post-implantation stages. This prospective pilot cohort study was conducted from March 2023 to August 2023. A total of 30 vitrified human blastocysts with previous PGT-A diagnosis on Day 5 (D5) or Day 6 (D6) of development were analysed: n = 15 euploid and n = 15 aneuploid. Finally, 21 embryo samples were included in the study; the rest (n = 9) were excluded due to poor quality pre-sequencing data (n = 7) or highly discordant data (n = 2). Following warming and re-expansion, embryos underwent a second trophectoderm (TE) biopsy. The embryos were then cultured until day 11 to assess their development. Biopsy analysis by RNA-Seq, studied the differential expressed genes (DEG) to compare embryos which did not or did attach to the plate: unattached embryos (n = 12) versus attached embryos (n = 9). Thus, we also obtained a specific transcriptomic signature of embryos with a "theoretical" capacity for sustained implantation, based on plate attachment on day 11. The digital karyotype obtained by RNA-Seq showed good concordance with the earlier PGT-A data, with a sensitivity of 0.81, a specificity of 0.83, a Cohen's Kappa of 0.66, and an area under the ROC of 0.9. At the gene level, 76 statistically significant DEGs were found in the comparison unattached versus attached embryos (Padj < 0.05; FC > 1). To address the functional implications of these differences, significantly deregulated pathways according to GO and KEGG categories were identified. The mural trophectoderm (TE) of the unattached blastocysts showed 63 significantly deregulated terms, displaying upregulation in autophagy, apoptosis, protein kinase and ubiquitin-like protein ligase activity, and downregulation of ribosome, spliceosome, kinetochore, segregation, and chromosome condensation processes. The overall transcriptomic signature specific to embryos still attached to the plate on day 11 (with a theoretically higher implantation capacity) consists of 501 genes, including: EMP2, AURKB, FOLR1, NOTCH3, LRP2, FZD5, MDH1, APOD, GPX8, COLEC12, HSPA1A, CMTM7, BEX3, which are related to implantation and embryonic development (raw P-value < 0.05; shrunk LFC > 1.1). These findings indicate that it might be possible to identify euploid embryos with a greater capacity for implantation and development, after excluding those embryos that present chromosomal alterations. This study included a small sample size, remarkable variability between samples, and low success rate of RNA amplification. Also, structural chromosomal abnormalities were not included, and it was not possible to diagnose mosaic embryos. TE biopsy does not assure the chromosomal status of the whole embryo. The maximum day for in vitro development was Day 11, and attachment to the plate on this day does not provide a clear indication of implantation capacity and viability, which was not tested in this study. The short-term goals following on from this pilot study is to expand the sample size with embryos of more complex abnormalities, and to perform a prospective in vitro preclinical validation. In a more distant future and with optimal results, this technique could have clinical application, thus increasing clinical outcomes by assessing both chromosomal content and transcriptomic profiling. The Institut Valencià de Competitivitat Empresarial (IVACE) (IMIDCA/2022/39) and Generalitat Valenciana (CIACIF/2021/11) supported the present study. A.C. is an employee of JUNO Genetics. He has received honoraria for an IBSA lecture and a Merck lecture. He is also a minor shareholder of IVIRMA Global. The other authors have no conflicts of interest to declare. N/A.

Ortega-Jaén D ，Mora-Martinez C ，Capalbo A ，Mifsud A ，Boluda-Navarro M ，Mercader A ，Martín Á ，Pardiñas ML ，Gil J ，de Los Santos MJ ... -  《-》

Multi-omics PGT: re-evaluation of euploid blastocysts for implantation potential based on RNA sequencing.

In addition to chromosomal euploidy, can the transcriptome of blastocysts be used as a novel predictor of embryo implantation potential? This retrospective analysis showed that based on differentially expressed genes (DEGs) between euploid blastocysts which resulted and did not result in a clinical pregnancy, machine learning models could help improve implantation rates by blastocyst optimization. Embryo implantation is a multifaceted process, with implantation loss and pregnancy failure related not only to blastocyst euploidy but also to the intricate dialog between blastocyst and endometrium. Although in vitro studies have revealed the characteristics of trophectoderm (TE) differentiation in implanted blastocysts and the function of TE placentation at the implantation site, the precise molecular mechanisms of embryo implantation and their clinical application remain to be fully elucidated. This study involved 102 patients who underwent 111 cycles for preimplantation genetic testing for aneuploidies (PGT-A) between March 2022 and July 2023. The study included 412 blastocysts biopsied at Day 5 [D5] or Day 6 [D6] for patients who underwent PGT-A. The biopsy lysates were split and subjected to DNA and RNA sequencing (DNA- and RNA-seq). One part was used for PGT-A to detect DNA copy number variations, whereas the other part was assessed simultaneously by RNA-seq to determine the transcriptome characteristics. To validate the reliability and accuracy of RNA-seq obtained from this strategy, we initially analyzed the transcriptome of blastocysts with chromosomal aneuploidies. Subsequently, we compared the transcriptomic features of blastocysts with different rates of formation (D5 vs D6) and investigated the network of interactions between key blastulation genes and the receptive endometrium. Then to evaluate the implantation potential of euploid blastocysts, we identified DEGs between euploid blastocysts that resulted in clinical pregnancy (defined as the presence of a gestational sac detected by ultrasound after 5 weeks) and those that did not. These DEGs were then employed to construct a predictive model for optimizing blastocyst selection. The successful detection rate of PGT-A was remarkably high at 99.8%. The RNA data may infer aneuploidy for both trisomy and monosomy. Between the euploid blastocysts that formed on D5 and D6, 187 DEGs were predominantly involved in cell differentiation for embryonic placenta development, the PPAR signaling pathway, and the Notch signaling pathway. These D5/D6 DEGs also exhibited a functional dialog with the receptive phase endometrium-specific genes through protein-protein interaction networks, indicating that the embryo undergoes further differentiation for post-implantation development. Furthermore, a modeling strategy using 280 DEGs between blastocysts leading to successful clinical pregnancies or failing to produce clinical pregnancies was implemented to refine the euploid embryo optimization, achieving areas under the curves of 0.88, 0.71, and 0.84 for the random forest (RF), support vector machine, and linear discriminant analysis models, respectively. Finally, a retrospective analysis of 83 transferred euploid blastocysts using the RF model identified three types of euploid embryos with a decreasing trend in implantation potential. Notably, the implantation rate of the good group was significantly higher than that of the moderate group (88.6% vs 50.0% P = 0.001) and that of the moderate group was higher than that of the poor group (50.0% vs 20.8%, P = 0.035). The sample size was insufficient; thus, a prospective study is needed to verify the clinical effectiveness of the above model. Because we did not analyze blastocysts that led only to biochemical pregnancies but failed clinical pregnancies separately, our classification system still must be modified to screen these embryos. Transcriptomic analysis of blastocysts offers a novel approach for predicting embryo implantation potential, which can be utilized to optimize clinical embryo selection. The ranking system may be effective in reducing the times and costs involved in achieving a clinical pregnancy. This study was funded by the "Pioneer" and "Leading Goose" R&D Program of Zhejiang (No. 2023C03034), the National Natural Science Foundation of China (82101709), and the National Key Research and Development Program for Young Scientists of China (No. 2022YFC2702300). The authors state no competing interests. N/A.

Jin J ，Ma J ，Wang X ，Hong F ，Zhang Y ，Zhou F ，Wan C ，Zou Y ，Yang J ，Lu S ，Tong X ... -  《-》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》

Time will tell: time-lapse technology and artificial intelligence to set time cut-offs indicating embryo incompetence.

Can more reliable time cut-offs of embryo developmental incompetence be generated by combining time-lapse technology (TLT), artificial intelligence, and preimplantation genetics screening for aneuploidy (PGT-A)? Embryo developmental incompetence can be better predicted by time cut-offs at multiple developmental stages and for different ranges of maternal age. TLT is instrumental for the continual and undisturbed observation of embryo development. It has produced morphokinetic algorithms aimed at selecting embryos able to generate a viable pregnancy, however, such efforts have had limited success. Regardless, the potential of this technology for improving multiple aspects of the IVF process remains considerable. Specifically, TLT could be harnessed to discriminate developmentally incompetent embryos: i.e. those unable to develop to the blastocyst stage or affected by full-chromosome meiotic aneuploidies. If proven valuable, this application would prevent the non-productive use of such embryos, thereby improving laboratory and clinical efficiency and reducing patient stress and costs due to unnecessary embryo transfer and cryopreservation. The training dataset involved embryos of PGT-A cycles cultured in Embryoscope with a single media (836 euploid and 1179 aneuploid blastocysts and 1874 arrested embryos; 2013-2020). Selection criteria were ejaculated sperm, own (not donated) fresh oocytes, trophectoderm biopsy and comprehensive-chromosome-testing to diagnose uniform aneuploidies. Out-of-sample (30% of training), internal (299 euploid and 490 aneuploid blastocysts and 680 arrested embryos; 2021-2022) and external (97 euploid, 110 aneuploid and 603 untested blastocysts and 514 arrested embryos, 2018 to early 2022) validations were conducted. A training dataset (70%) was used to define thresholds. Several models were generated by fitting outcomes to each timing (tPNa-t8) and maternal age. ROC curves pinpointed in-sample classification values associated with 95%, 99% and 99.99% true-positive rate for predicting incompetence. These values were integrated with upper limits of maternal age ranges (<35, 35-37, 38-40, 41-42, and >42 years) in logit functions to identify time cut-offs, whose accuracy was tested on the validation datasets through confusion matrices. For developmental (in)competence, the best performing (i) tPNa cut-offs were 27.8 hpi (error-rate: 0/743), 32.6 hpi (error rate: 0/934), 26.8 hpi (error rate: 0/1178), 22.9 hpi (error-rate: 1/654, 0.1%) and 17.2 hpi (error rate: 4/423, 0.9%) in the <35, 35-37, 38-40, 41-42, and >42 years groups, respectively; (ii) tPNf cut-offs were 36.7 hpi (error rate: 0/738), 47.9 hpi (error rate: 0/921), 45.6 hpi (error rate: 1/1156, 0.1%), 44.1 hpi (error rate: 0/647) and 41.8 hpi (error rate: 0/417); (iii) t2 cut-offs were 50.9 hpi (error rate: 0/724), 49 hpi (error rate: 0/915), 47.1 hpi (error rate: 0/1146), 45.8 hpi (error rate: 0/636) and 43.9 hpi (error rate: 0/416); (iv) t4 cut-offs were 66.9 hpi (error rate: 0/683), 80.7 hpi (error rate: 0/836), 77.1 hpi (error rate: 0/1063), 74.7 hpi (error rate: 0/590) and 71.2 hpi (error rate: 0/389); and (v) t8 cut-offs were 118.1 hpi (error rate: 0/619), 110.6 hpi (error rate: 0/772), 140 hpi (error rate: 0/969), 135 hpi (error rate: 0/533) and 127.5 hpi (error rate: 0/355). tPNf and t2 showed a significant association with chromosomal (in)competence, also when adjusted for maternal age. Nevertheless, the relevant cut-offs were found to perform less well and were redundant compared with the blastocyst development cut-offs. Study limits are its retrospective design and the datasets being unbalanced towards advanced maternal age cases. The potential effects of abnormal cleavage patterns were not assessed. Larger sample sizes and external validations in other clinical settings are warranted. If confirmed by independent studies, this approach could significantly improve the efficiency of ART, by reducing the workload and patient impacts (extended culture and cleavage stage cryopreservation or transfer) associated with embryos that ultimately are developmentally incompetent and should not be considered for treatment. Pending validation, these data might be applied also in static embryo observation settings. This study was supported by the participating institutions. The authors have no conflicts of interest to declare. N/A.

Coticchio G ，Bartolacci A ，Cimadomo V ，Trio S ，Innocenti F ，Borini A ，Vaiarelli A ，Rienzi L ，Ahlström A ，Cimadomo D ... -  《-》

Endometrial extracellular vesicles regulate processes related to embryo development and implantation in human blastocysts.

What is the transcriptomic response of human blastocysts following internalization of extracellular vesicles (EVs) secreted by the human endometrium? EVs secreted by the maternal endometrium induce a transcriptomic response in human embryos that modulates molecular mechanisms related to embryo development and implantation. EVs mediate intercellular communication by transporting various molecules, and endometrial EVs have been postulated to be involved in the molecular regulation of embryo implantation. Our previous studies showed that endometrial EVs carry miRNAs and proteins associated with implantation events that can be taken up by human blastocysts; however, no studies have yet investigated the transcriptomic response of human embryos to this EV uptake, which is crucial to demonstrate the functional significance of this communication system. A prospective descriptive study was performed. Primary human endometrial epithelial cells (pHEECs), derived from endometrial biopsies collected from fertile oocyte donors (n = 20), were cultured in vitro to isolate secreted EVs. Following EV characterization, Day 5 human blastocysts (n = 24) were cultured in the presence or absence of the EVs for 24 h and evaluated by RNA-sequencing. EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blot, nanoparticle tracking analysis, and transmission electron microscopy. Human blastocysts were devitrified, divided into two groups (n = 12/group), and cultured in vitro for 24 h with or without previously isolated EVs. RNA-sequencing analysis was performed, and DESeq2 was used to identify differentially expressed genes (DEGs) (FDR < 0.05). QIAGEN Ingenuity Pathway Analysis was used to perform the functional enrichment analysis and integration with our recently published data from the pHEECs' EV-miRNA cargo. Characterization confirmed the isolation of EVs from pHEECs' conditioned culture media. Among the DEGs in blastocysts co-cultured with EVs, we found 519 were significantly upregulated and 395 were significantly downregulated. These DEGs were significantly enriched in upregulated functions related to embryonic development, cellular invasion and migration, cell cycle, cellular organization and assembly, gene expression, and cell viability; and downregulated functions related to cell death and DNA fragmentation. Further, the intracellular signaling pathways regulated by the internalization of endometrial EVs were previously related to early embryo development and implantation potential, for their role in pluripotency, cellular homeostasis, early embryogenesis, and implantation-related processes. Finally, integrating data from miRNA cargo of EVs, we found that the miRNAs carried by endometrial EVs targeted nearly 80% of the DEGs in human blastocysts. This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. This study provides novel insights into the functional relevance of EVs secreted by the human endometrium, and particularly the role of EV-miRNA regulation on global transcriptome behavior of human blastocysts during early embryogenesis and embryo implantation. It provides potential biomarkers that could become useful diagnostic targets for predicting implantation success. This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139), and Instituto de Salud Carlos III and cofounded by the European Social Fund (ESF) "Investing in your future" through the Miguel Servet Program (CP20/00120 [H.F.]; CP19/00149 [I.C.]). The authors have no conflicts of interest to disclose. N/A.

Segura-Benítez M ，Carbajo-García MC ，Quiñonero A ，De Los Santos MJ ，Pellicer A ，Cervelló I ，Ferrero H ... -  《-》