Endogenous retroviral elements LTR8B and MER65 rewire PSG9 regulation to control trophoblast syncytialization and pre-eclampsia risk.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓The central claim held under reproduction
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Described well enough; reproduced the paper's CENTRAL qualitative claim 1:1 from shipped data + the authors' OWN code, with an honest gene-vs-transcript caveat on the exact figure number. Paper: Singh et al. 2026 Genome Biol, ERVs LTR8B/MER65 -> PSG9 -> trophoblast syncytialization & pre-eclampsia. I ran the authors' own R script ('PSG analysis in Preeclampsia', github.com/Manu-1512/PSG9 @ 0c24ae2) on the authors' SHIPPED gene-level normalized TPM matrix (GSE263305_normalized_Gene_TPM_PE_ctrl.csv: 8 healthy + 10 EO-PE; column layout matches the script's hard-coded 8-ctrl/10-PE groups). Pipeline: ENSG->symbol (GENCODE v44; authors' map not shipped) -> grep '^PSG' -> log2(TPM+1) -> per-gene Wilcoxon -> BH. RESULT: PSG9 is the single most significant PSG of 13 detected and is clearly UPREGULATED in EO-PE (mean TPM 9.60->30.03, log2 delta +1.40, FC 3.13, Wilcoxon p=0.034), reproducing the paper's Fig3 'PSG9 up in EO-PE'. The paper's exact Fig6G value (PSG9-202 secreted isoform: p=0.016, FC=13.61) is a TRANSCRIPT-level statistic; the publicly shipped series matrix is GENE-level only, so it is not the identical statistic: significance reproduces in magnitude (0.034 vs 0.016, both <0.05) but gene-level FC (3.13) is smaller than the isoform FC (13.61) BY CONSTRUCTION, because the gene aggregates the strongly-up secreted -202 with the non-DE membrane-anchored -201 (the paper itself reports -201 as not DE) -- an expected, internally-consistent dilution, not a contradiction. NO fabrication signal: every reproduced value is derivable from shipped data + authors' code. NOT attempted (the hard ~20%): the exact -202 isoform stat (needs raw FASTQ from SRA -> STAR/Salmon transcript-level quant, not shipped at series level); the KO-LTR8B-vs-WT 1,257-DEG analysis (a DIFFERENT accession GSE314215, not the brief's GSE263305); ChIP-seq/ATAC-seq TE-enrichment, chimeric-junction, SCENIC regulons and repeat-expression pipelines (heavier raw-data multi-tool runs); and all wet-lab (qRT-PCR, serum/ELISA PSG9, IHC). Graded partial because the headline figure-level number is only reproducible at gene (not transcript) resolution from shipped data, though the biological claim itself reproduces cleanly.
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Assessment versions
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v1 current initial assessment Score 68assessed: 2026-06-14 ⛓ 0f74314dd6c6
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-14
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-15no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusThe authors hypothesize that endogenous retroviruses bound by the pre-eclampsia-dysregulated transcription factors GATA3 and DLX5 have been co-opted as cis-regulatory elements at the PSG locus, and that their dysregulation rewires PSG9 regulation to control trophoblast syncytialization and contribute to pre-eclampsia risk.
- ★ Retrovirus-derived LTR8B acts as a trophoblast-specific cis-regulatory element within the PSG array, with LTR8B at PSG9 influencing other PSG family members as a master regulator finding
- ★ LTR8B embeds binding sites for trophoblast-specific TFs (GATA3, DLX5, TFAP2A/C) and defines a 3D chromatin domain boundary at the PSG9 locus mechanism
- ★ The retroelement MER65-int provides alternative polyadenylation signals that truncate the ancestral CEACAM transmembrane domain, enabling evolution of secreted PSG variants mechanism
- ★ LTR8B/PSG9 regulates differentiation of multinucleated trophoblasts (syncytialization) and helps determine syncytiotrophoblast identity, like chorionic gonadotropin and syncytin1 finding
- ★ Unique among PSGs, PSG9 produces both secreted and membrane-anchored isoforms finding
- ★ PSG9 is the most upregulated PSG in pre-eclampsia, with levels correlated with GATA3 and DLX5 levels, supporting PSG9 as a predictive PE biomarker finding
- Retroelements contribute to lineage-specific structural and expression evolution of PSG genes finding
- A specific sandwich ELISA was developed to quantify circulating PSG9 in maternal serum resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| ChIP-exonuclease (ChIP-exo) sequencing | SGHPL-4 cells (doxycycline-inducible HA-DLX5) | DLX5 overexpression (TET-inducible) | genome-wide DLX5 transcription factor binding sites | Illumina HiSeq; KAPA Library Quantification Kit |
| CRISPR-Cas9 knockout | trophoblast cells (PSG locus) | LTR8B/PSG9 knockout | PSG expression and TF binding / domain boundary | — |
| 3D chromatin / chromatin conformation analysis | trophoblast cells | none/KO | 3D domain boundary at LTR8B/PSG9 | — |
| single-cell transcriptome and epigenome integrative analysis | human placenta/trophoblast | none | PSG regulation by trophoblast-specific TFs | — |
| RT-qPCR | BeWo, SGHPL-4, hTSC cells and human placenta samples | gene overexpression/knockdown | gene expression normalized to ACTIN (ΔΔCT) | CFX96 Touch system, SsoAdvanced SYBR Green (Bio-Rad) |
| Western blot (antibody specificity and fractionation) | recombinant PSG proteins, BeWo and SGHPL-4 lysates | none / isoform tagging (FLAG/HA) | PSG9 protein detection and isoform subcellular localization | anti-PSG9 ab64425 Abcam; NBP-2 19979 Novus |
| Sandwich ELISA | serum of pregnant women | none | circulating PSG9 concentration (absorbance 450 nm) | Immulon 2 plates; capture NBP1-57656, detection BAP3 mAb |
| shRNA knockdown | BeWo cells | PSG9 knockdown (pLKO.1-shRNA) | PSG9 expression / phenotype | MISSION shRNA |
- ▲ PSG9 is the most upregulated PSG family member in pre-eclampsia
- ▲ PSG9 levels correlate with GATA3 and DLX5 levels in PE
- – GATA3/DLX5/TFAP2A/C binding sites are embedded in LTR8B sequence and define the PSG9 3D domain boundary
- – MER65 alternative polyA signals truncate the ancestral CEACAM transmembrane domain to yield secreted PSG variants
- – PSG9 uniquely possesses both secreted and membrane-anchored isoforms whereas other PSGs are exclusively secreted
- – PSG9 antibody (ab64425) showed no cross-reactivity with other PSGs; bands only in recombinant PSG9 and BeWo/SGHPL-4 lysates
- other 3–8% of pregnancies worldwide (prevalence of pre-eclampsia)
- count PSG1-PSG11 (human); Psg16-33 (mouse) (PSG gene cluster expansion in human vs mouse)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This integrative study combines ChIP-exo chromatin profiling, single-cell transcriptomics and epigenomics, CRISPR-Cas9 functional experiments, RT-qPCR, and sandwich ELISA of clinical serum samples to characterize the LTR8B/PSG9 regulatory axis in trophoblast differentiation and pre-eclampsia risk. Gene expression was quantified by RT-qPCR using the ΔΔCT method normalized to ACTIN; PSG9 serum concentrations were measured by a sandwich ELISA with each value reported as the mean of triplicate assays. The Methods section in the provided full-text excerpt is truncated before any formal inferential statistics, sample sizes, or multiplicity-correction procedures are described, so those fields cannot be fully populated from the available text.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| ΔΔCT relative quantification | RT-qPCR gene expression comparisons across experimental conditions and human placental samples | — | not stated |
| Correlation analysis (method not specified in provided excerpt) | Association between PSG9 protein levels and GATA3/DLX5 expression in pre-eclampsia samples | — | not stated |
| Differential expression analysis (algorithm not named in provided excerpt) | Identification of PSG9 as most upregulated PSG in pre-eclampsia transcriptome data | — | not stated |
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RT-qPCR expression data were normalized using a single housekeeping gene (ACTIN) with the ΔΔCT method↳ Could also: Normalization to the geometric mean of two or more validated reference genes selected by stability algorithms such as geNorm or NormFinder could also be used — Multi-reference normalization reduces sensitivity to expression variation of any single reference gene across the diverse trophoblast states and PE vs. control comparisons examined in the study
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Serum ELISA values are reported as the mean of triplicate assays without a stated measure of dispersion↳ Could also: Reporting mean ± SD (or 95% CI) and the coefficient of variation across replicates could also accompany each value — Explicit dispersion metrics allow readers to assess within-sample assay reproducibility and propagate uncertainty into between-group comparisons
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The correlation between PSG9 levels and GATA3/DLX5 expression is described qualitatively in the available text without a named correlation method or coefficient↳ Could also: Pearson or Spearman correlation with the stated coefficient (r or ρ), 95% CI, and exact p-value could also be reported — A named coefficient with confidence interval makes the direction, magnitude, and precision of the association independently reproducible and comparable across studies
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PSG9 is described as 'the most upregulated PSG in PE' without a named differential expression framework in the provided excerpt↳ Could also: A regularized count-based framework such as DESeq2 or edgeR, reporting log2 fold-changes with Wald test p-values and Benjamini-Hochberg FDR, could also be explicitly cited — Naming the algorithm and FDR-adjustment method ties the 'most upregulated' ranking to a reproducible statistical model with controlled false-discovery rate across all PSG family members tested simultaneously
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Multiple experimental conditions (knockout, overexpression, and controls across several readouts) appear to be compared across the study without a stated family-wise error strategy in the available text↳ Could also: A one-way ANOVA or mixed-effects model with a post-hoc correction (e.g., Tukey HSD for all pairwise, or Dunnett's test vs. a single control) could also be applied — A single omnibus model with a post-hoc correction controls the family-wise type I error rate when several comparisons derive from the same experimental dataset
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PSG9 protein levels in PE patients are compared to controls using ELISA without a stated non-parametric vs. parametric test choice in the provided excerpt↳ Could also: A Mann-Whitney U test (for non-normal distributions or small clinical n) or an independent-samples t-test (with Levene's test for variance equality) could both also be explicitly stated — Declaring the test choice and the rationale for it (e.g., distribution of serum values, sample size) makes the between-group comparison independently evaluable and reproducible
Citation network
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Data lineage
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What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-41796334
Paper: Singh et al. 2026, Genome Biology — "Endogenous retroviral elements LTR8B and MER65 rewire PSG9 regulation to control trophoblast syncytialization and pre-eclampsia risk." DOI 10.1186/s13059-026-03944-z.
Code: https://github.com/Manu-1512/PSG9 @ commit 0c24ae2 (2025-12-16).
Data: GEO GSE263305 (primary human trophoblasts, healthy vs early-onset PE).
Repo character
The repo is the authors' own analysis scripts (R + bash), informal (files named "PSG analysis in Preeclampsia", "featurecount run", "STAR run", etc.; README documents the PSG/PE expression script). Pipelines named across the scripts: STAR (align), Salmon (transcript TPM), featureCounts (gene + TE counts), DESeq2 (DE), and a base-R Wilcoxon/BH comparison for the PSG family.
In scope (pipeline-derived, clearly specified, shipped data)
- C1 — PSG9 gene-level Control-vs-PE expression comparison (Fig 3 theme).
The authors'
PSG analysis in Preeclampsiascript: read shipped gene-level TPM matrix → ENSG→symbol → grep^PSG→ log2(TPM+1) → per-gene Wilcoxon (8 ctrl vs 10 PE) → BH. Input = shipped processed matrixGSE263305_normalized_Gene_TPM_PE_ctrl.csv.gz(colsctrl.rep1..8,PE.rep1..10— exactly the script's group layout). Fully reproducible from shipped data with the authors' own code. This is the 80/20 target. - C1b — full PSG-family stats table (PSG1..PSG11 mean_ctrl, mean_PE, delta, Wilcoxon p, BH padj) as a human-auditable companion output.
Out of scope / not attempted (the hard ~20%)
- PSG9-202 isoform: paper's Fig 6G value (Wilcoxon p=0.016, FC=13.61) is a transcript-level isoform measurement (Salmon transcript TPM). The shipped series matrix is gene-level only; no transcript-level TPM is shipped. Re-deriving it requires aligning the raw FASTQs (SRA) + Salmon transcript quant — deferred. We instead reproduce the gene-level PSG9 comparison and compare direction + significance against the paper, flagging the gene-vs-transcript distinction.
- KO-LTR8B vs WT DEGs (1,257 DEGs, padj<0.01; log2FC=2 / p<1e-6 cutoffs): belongs to a different accession (GSE314215), not the brief's GSE263305. Not attempted.
- ChIP-seq / ATAC-seq TE-enrichment, chimeric-junction, regulon (SCENIC), repeat expression: heavier multi-tool pipelines on raw data — deferred (80/20).
- All wet-lab (qRT-PCR, ELISA/serum PSG9, IHC) — out of scope (non-pipeline).
Compute plan
Light base-R job on «our HPC» (partition std, sg_r env = R 4.3.3 + dplyr + stringr).
ENSG→symbol map built reproducibly from a pinned GENCODE GTF inside the job
(authors' ensembl_human_ID.txt map not shipped). Data on «infra»; only small
result tables + JSON copied back to «host».
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Using the authors' own published R script on their own shipped gene-level TPM matrix (GSE263305), the central claim — PSG9 upregulated in early-onset PE trophoblasts — reproduces cleanly (9.60→30.03 TPM, FC 3.13 UP, Wilcoxon p=0.034, PSG9 = #1 of 13 PSGs), confirming Fig3 and the qualitative direction of Fig6G. The only gap is the exact Fig6G figure number (isoform PSG9-202 p=0.016, FC=13.61), which is transcript-level and not derivable from the shipped gene-level matrix; the gene-level FC is smaller by construction (it dilutes the strongly-up -202 with the non-DE -201, consistent with the paper's own narrative). This is a data-resolution/availability limitation on our side, not an authors' defect or fabrication signal — direction and significance hold throughout. Overall: solid reproduction with one explainable, expected deviation → yellow.
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Reproduction footprint
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