Avalilação PREreview de Distinct HPO axis responses and ovarian aging trajectories to chronic unpredictable mild stress in reproductively young versus middle-aged female mice

Major Issues

1. Lack of estrous cycle stratification

The study does not account for the estrous cycle stage during hormonal, histological, or transcriptomic analyses. Given the marked physiological fluctuations in ovarian hormones across proestrus and diestrus, the observed differences between control and CUMS groups may partially reflect cycle-stage variability rather than the direct effects of chronic stress. Stage-specific analyses would substantially strengthen the conclusions.

2. Limited mechanistic interpretation of transcriptomic data

Although bulk RNA-seq identified stress-associated transcriptional changes, it does not reveal the cellular populations driving these alterations. Incorporating cell-type-specific analyses (e.g., computational deconvolution, immunostaining, or single-cell transcriptomics) would help determine whether astrocytes, microglia, oligodendrocytes/OPCs, excitatory neurons, inhibitory neurons, or dentate granule neurons are primarily affected by CUMS.

3. Lack of cellular validation

The study reports transcriptomic alterations without validating changes in specific neural cell populations. Confirmation using established cell identity markers would provide stronger mechanistic evidence linking chronic stress to cell-type-specific responses within the brain.

4. Lack of brain-specific molecular characterization

The study measures endocrine alterations primarily in serum but does not investigate corresponding molecular changes within the brain. Quantifying stress- and reproduction-related hormones (where technically appropriate) or their downstream signaling in relevant brain regions (e.g., hippocampus and hypothalamus) would help establish a mechanistic link between peripheral endocrine alterations and central nervous system dysfunction.

5. Lack of assessment of synaptic integrity

Although the study reports endocrine and transcriptomic alterations following CUMS, it does not evaluate whether these changes are accompanied by alterations in synaptic structure. Assessing synaptic density using established pre- and postsynaptic markers (e.g., Synaptophysin, PSD-95, Bassoon, Homer1) would provide important mechanistic insight into how chronic stress affects neuronal connectivity.

Minor Issues

1. Comparison of transcriptomic variability across estrous stages

Including transcriptomic or epigenetic comparisons between proestrus and diestrus, together with comparisons to male animals, would help determine whether biological variability across the estrous cycle is comparable to or smaller than sex-dependent variability.

2. Integration of endocrine and molecular findings

Correlating corticosterone, reproductive hormone levels, behavioral outcomes, and transcriptomic alterations at the individual-animal level would strengthen the biological interpretation of the CUMS model.

3. Future evaluation of stage-specific therapeutic responses

Future studies should investigate whether therapeutic interventions produce different outcomes when administered during proestrus versus diestrus, as hormonal status may influence treatment efficacy and translational relevance.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they used generative AI to come up with new ideas for their review.