A stress-responsive morphogenetic program of the uterine epithelium safeguards the establishment of early pregnancy

Reviewer #1 (Public review):

Summary:

This manuscript asks how the uterine lumen is remodeled across the peri-implantation window and whether this remodeling is functionally linked to embryo attachment and subsequent pregnancy establishment. The authors combine whole-organ three-dimensional imaging of optically cleared mouse uteri with single-cell and spatial transcriptomic profiling, conditional deletion of p38α at the uterine-wide versus epithelial-restricted level, and rescue experiments using progesterone and leukemia inhibitory factor. Based on these datasets, the authors propose that the luminal epithelium undergoes a previously underappreciated phase of organ-scale architectural reorganization before attachment, and that a p38α-dependent stress-responsive program coordinates epithelial remodeling together with epithelial-stromal communication required for implantation competence.

Strengths:

By moving beyond local attachment-site morphology to a horn-level representation of luminal topology, the work provides anatomical context that is difficult to reconstruct from conventional section-based approaches and should be broadly useful to the implantation community. The integration of organ-scale morphology with single-cell and spatial transcriptomic datasets, together with genetic perturbation and rescue experiments, adds breadth and increases the potential long-term utility of the dataset for investigators interested in uterine receptivity and embryo-uterine interactions.

Weaknesses:

(1) The whole-uterus analysis of luminal folds and creases requires stronger methodological support. Given the mechanical compliance of the uterine lumen, it is difficult to evaluate from the current description whether dissection, fixation, clearing, and/or mounting could influence the observed luminal topography. This issue is particularly important because several key conclusions depend on the spatial distribution of folds across the uterine horn. A fuller account of tissue handling and reconstruction, together with validation that the preparation preserves native morphology, would substantially strengthen confidence in the organ-scale conclusions.

(2) Several of the central morphological claims are supported primarily only by representative reconstructions. This includes the proposed flattening/creasing dynamics, alternating stretched and shrunken regions, persistence of abnormal folding in the mutant uterus, and the extent of structural rescue following progesterone supplementation. The authors could extract objective measures from the reconstructed luminal surface and provide more statistical analysis to demonstrate the reproducibility of the results.

(3) The manuscript appears to over-reach in concluding that luminal remodeling zones embryos before attachment from day 4 to 5. As presented, the data support a correlation between luminal architecture and embryo position, but do not discriminate between (i) luminal remodeling directing embryo positioning, (ii) embryos locally shaping the lumen, or (iii) parallel regulation of both. The evidence is based on observations of the uterus and the inside blastocysts at certain time points around implantation. Without the time-lapse analysis within the uterus, the dynamic interactions between embryos and the uterus couldn't be determined.

(4) The key conclusion of the manuscript is that uterine p38α regulates luminal epithelial remodeling required for embryo attachment, as shown in the title. Against this background, the finding that epithelial-restricted loss of p38α does not overtly impair fertility is notable, as it suggests that the major function of p38α may not be epithelial cell-autonomous but instead may arise through other uterine compartments that secondarily influence the epithelium. At present, however, this conclusion remains insufficiently supported: the epithelial-specific model is not characterized in sufficient depth during the peri-implantation period, and the transcriptomic evidence for altered epithelial-stromal communication does not by itself explain the phenotypic difference between uterine-wide and epithelial-specific deletion. If stromal p38α is proposed as the critical upstream regulator, more direct testing, such as stromal-specific deletion, would be needed.

Reviewer #2 (Public review):

Summary:

In this study, the authors aimed to characterize the architectural reorganization of the uterine luminal epithelium during the implantation period. Using 3D histological reconstruction, single-cell RNA sequencing, and spatial transcriptomics, the authors characterize luminal remodeling during the peri-implantation period and employ a mouse model to explore the role of p38α in regulating luminal flattening.

Strengths:

This study clearly described the changes in luminal architecture during implantation. Moreover, they also used integration of multiple advanced techniques, including 3D tissue reconstruction, single-cell transcriptomics, and spatial transcriptomics, which together provide a detailed description of the molecular characteristics of the uterine architecture during implantation.

Weaknesses:

The authors used PR-Cre to generate uterine p38α knockout mice. This Cre driver deletes p38α not only in epithelial cells but also in stromal compartments. Therefore, it remains unclear whether the observed phenotype arises from epithelial cells, stromal cells, or a combination of both. Previous studies have shown that p38α regulates epithelial polarity, cytoskeletal organization, and E-cadherin localization. However, the current study does not examine changes in cell adhesion or epithelial junction integrity. Previous studies have reported that uterine fluid absorption during implantation is closely associated with luminal closure and remodeling. It would be important to determine whether epithelial transport-related genes are altered in the mutant uterus. Could dysregulated fluid homeostasis contribute to the implantation defects observed in the p38α-deficient mice?