Comprehensive profiling of Small RNAs in human embryo-conditioned culture media by improved sequencing and quantitative PCR methods
Bibliographic record
Abstract
Embryo implantation depends on two primary factors: the quality of the embryo and endometrial receptivity. Small RNAs have been shown to be potent epigenetic regulators influencing cell proliferation, differentiation, and communication even in the context of early embryonic development. However, previous reports are limited to miRNAs and lack sensitivity. Here, we describe a platform for non-invasive small RNA biomarker discovery and validation from embryo-conditioned culture media (ECCM). We hypothesize that small non-coding RNAs (sncRNAs) are secreted by the embryo into the ECCM and test the limit of detection for profiling sncRNA by deep sequencing and quantitative PCR. In the first set of experiments, we evaluated sequencing sensitivity by comparing sncRNA profiles from pools of 10, 5, 3, and single ECCM drops. Next, we performed a similar test for TaqMan qPCR sensitivity by measuring select sncRNAs in 5, 3 and single drop ECCM pools. Finally, we compared the expression of an sncRNA panel by qPCR in single ECCM vs no-embryo control media . We report the first comprehensive sequencing of sncRNAs in ECCM with a sequencing sensitivity of 3 single embryo drops, capturing ~150 miRNAs and an abundance of tRNA-derived small RNAs (tsRNAs). We then profiled 15 sncRNAs by qPCR and determined that the assay maintains sensitivity in single ECCM drops. Finally, we found significant differences in these sncRNA expression between control and ECCM drops. Improving embryo selection is crucial for reducing time to pregnancy. Here we describe a sensitive technique for biomarker discovery by sequencing and qPCR validation in ECCM, demonstrating that the majority of sncRNAs are embryo derived. We also report an abundance of tsRNAs which suggests these sncRNAs may have functions in endometrial-maternal communication beyond the microRNAs which have been described previously.Abbreviations: PGT-A: Preimplantation genetic testing for aneuploidies; ECCM: Embryo-conditioned culture media; sncRNAs: Small non-coding RNAs; miRNAs: microRNAs; EVs: Extracellular vesicles; PCA: Principal component analysis.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.004 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".