Microbial-derived D-lactate and LPS shape growth and inflammatory signalling in endometrial glandular epithelium

This study demonstrates that endometrial epithelial organoids serve as a robust model to reveal how microbial-derived D-lactate and LPS differentially modulate epithelial growth and inflammatory signaling, respectively, without significantly disrupting key markers of uterine receptivity.

The Gist

The Big Picture: The Uterus as a "Hotel" for a Baby

Imagine the uterus is a high-end hotel, and the endometrium (the inner lining) is the lobby and the rooms. For a baby (the embryo) to check in and stay, the hotel needs to be in a very specific state of readiness called "receptivity."

For a long time, scientists thought this hotel lobby was a sterile, germ-free zone. But new technology has shown that there are actually tiny "guests" (bacteria) living there, or at least their footprints (chemical signals) are there.

This study asks: Do these bacterial guests change how the hotel staff (the uterine cells) behave? Specifically, they looked at two very different types of "guests":

1. The Good Neighbor: Represented by D-Lactate. This is a chemical produced by Lactobacillus, the "good" bacteria that usually keep the reproductive tract healthy.
2. The Troublemaker: Represented by LPS. This is a piece of the outer shell of "bad" bacteria (like E. coli) that causes inflammation.

The Experiment: A 3D "Mini-Hotel" in a Dish

To test this without using human volunteers, the scientists grew Endometrial Epithelial Organoids (EEOs).

• The Analogy: Think of these as tiny, 3D Lego castles built from the woman's own uterine cells. They aren't just flat cells on a plate; they curl up into little spheres that look and act just like the real uterine lining.
• The Setup: They took these mini-castles and treated them with hormones to make them "ready for check-in" (mimicking the time of the month when a baby could implant). Then, they added the "Good Neighbor" chemicals (D-Lactate) or the "Troublemaker" chemicals (LPS) to see what happened.

What They Found

1. No Cytotoxicity (The Hotel Didn't Burn Down)

First, they checked if these chemicals killed the cells.

• Result: No. Whether they added a little or a lot of the "Good Neighbor" or the "Troublemaker," the cells stayed alive and healthy. The hotel didn't catch fire; the staff just started talking differently.

2. The "Good Neighbor" (D-Lactate) Helps the Hotel Get Ready

When the cells were exposed to D-Lactate (from the good bacteria) while they were being prepped for the baby:

• The Effect: It tweaked the genes responsible for growth and structure.
• The Metaphor: Imagine the hotel staff receiving a memo from the "Good Neighbor" saying, "Hey, the lobby looks great, but let's polish the floors and make sure the doors open smoothly."
• The Science: D-Lactate seemed to encourage genes that help the tissue mature and organize itself. It didn't cause chaos; it helped the cells fine-tune their "receptive" state, potentially making it easier for a baby to attach.

3. The "Troublemaker" (LPS) Starts a Security Alarm

When the cells were exposed to LPS (from the bad bacteria):

• The Effect: It triggered inflammatory and immune response genes.
• The Metaphor: Imagine the "Troublemaker" walks in and sets off the fire alarm. The hotel staff stops polishing the floors and immediately grabs their fire extinguishers and security radios. They are now in "defense mode" rather than "welcome mode."
• The Science: The cells started shouting, "We are under attack!" They activated genes related to fighting infection. While this is good for stopping a real infection, it might be bad for a baby trying to implant because the environment becomes too chaotic and hostile.

4. The Hormone Factor: The "Manager" Must Be Present

Here is the most interesting twist: The chemicals didn't do much if the hormones weren't there.

• The Analogy: The uterine cells are like employees who only listen to the "Microbe Memo" if the "Hotel Manager" (Hormones like Estrogen and Progesterone) is in the office.
• The Result: If the cells weren't being prepped by hormones, the D-Lactate and LPS barely changed anything. But once the hormones were added, the cells suddenly became very sensitive to the bacterial signals. The hormones "woke up" the cells' ability to hear the bacteria.

Why Does This Matter?

This study helps us understand why some women have trouble getting pregnant (implantation failure) while others don't.

• The "Good" Scenario: If your uterine environment is dominated by Lactobacillus (producing D-Lactate), it might be gently whispering to the uterine cells, "Get ready, we are safe, let's welcome the baby."
• The "Bad" Scenario: If your environment has too many "bad" bacteria (producing LPS), it might be screaming "DANGER!" at the uterine cells. Even if the hormones say "Get ready," the cells are too busy fighting a fake fire to let the baby in.

The Bottom Line

The uterus isn't just a passive waiting room; it's a dynamic environment that listens to its bacterial neighbors.

• Good bacteria (via D-Lactate) seem to help the uterus get organized and ready for a baby.
• Bad bacteria (via LPS) seem to put the uterus on high alert, which might accidentally push the baby away.

This research uses these tiny 3D "Lego hotels" to prove that the balance of bacteria in the womb is a crucial, silent conversation that determines whether a pregnancy can start.

Technical Summary

1. Problem Statement

The uterine environment was historically considered sterile, but recent sequencing technologies have revealed the presence of microbial signals in the endometrium. While a Lactobacillus-dominated microbiome is associated with successful implantation, and Gram-negative bacteria (producing Lipopolysaccharides or LPS) are linked to inflammation and implantation failure, the specific molecular mechanisms by which bacterial metabolites influence endometrial receptivity remain unclear.

• Key Gap: It is unknown how specific microbial metabolites, such as D-lactate (D-lac) (a biomarker of Lactobacillus activity) and LPS (a marker of Gram-negative bacteria), modulate the transcriptomic profile of the endometrial epithelium during the critical window of implantation.
• Objective: To elucidate the effects of D-lac and LPS on the gene expression, metabolic activity, and receptivity markers of endometrial epithelial cells under hormonally induced receptive conditions.

2. Methodology

The study utilized Endometrial Epithelial Organoids (EEOs) derived from fertile donors to create a physiologically relevant 3D in vitro model.

• Sample Collection: Endometrial biopsies were obtained from three fertile women (mean age 31.3) during the secretory phase of the menstrual cycle.
• Organoid Culture: Epithelial glands were isolated, dissociated, and cultured in Matrigel to form 3D organoids. Organoids were expanded and then used between passages 2–10.
• Experimental Design:
  • Hormonal Stimulation: Organoids were treated with Estradiol (E2), Progesterone (P4), and 8-Br-cAMP (EPC) to mimic the mid-secretory (receptive) phase.
  • Treatments: EEOs were exposed to varying concentrations of:
    • D-lactate: 2.5, 5, and 10 mM.
    • LPS (from E. coli O111:B4): 2.5, 5, and 10 ng/mL.
  • Control Groups: Non-hormonally stimulated (Non-EPC) and hormonally stimulated (EPC) controls without bacterial compounds.
• Assays Performed:
  • Metabolic Activity: Resazurin-based assays (absorbance and fluorescence) to assess cytotoxicity and cell viability.
  • Immunofluorescence: Staining for KRT (epithelial identity) and Ki-67 (proliferation).
  • Transcriptomics: Bulk RNA sequencing (RNA-seq) followed by differential gene expression (DGE) analysis using DESeq2.
  • Bioinformatics: Gene Ontology (GO) enrichment analysis and comparison against the beREADY model (a validated set of 68 endometrial receptivity markers).

3. Key Contributions

• Model Validation: Demonstrated that EEOs are a robust platform for studying host-microbe interactions, successfully recapitulating the hormonal responsiveness and transcriptomic profile of the receptive endometrium in vitro.
• Hormonal Context Dependency: Established that the endometrial epithelium's response to microbial signals is strictly hormonally dependent. Neither D-lac nor LPS induced significant transcriptional changes in the absence of hormonal stimulation (EPC).
• Distinct Mechanisms of Action: Differentiated the molecular impact of "protective" (D-lac) vs. "pathogenic" (LPS) signals:
  • D-lac modulates genes related to epithelial development, tissue remodeling, and growth regulation.
  • LPS triggers inflammatory signaling and immune response pathways.
• Specific Marker Identification: Identified unique gene expression changes (e.g., DYNLT3 upregulation by D-lac; MAP3K5 upregulation by LPS) that are not observed with hormonal stimulation alone, suggesting specific microbial modulation of receptivity.

4. Results

A. Metabolic Activity and Viability

• Exposure to D-lac (up to 10 mM) and LPS (up to 10 ng/mL) for 6 days did not significantly alter the relative metabolic activity or viability of EEOs compared to controls. This confirmed the treatments were non-cytotoxic at the tested concentrations.

B. Hormonal Stimulation Effects

• Hormonal stimulation (EPC) alone induced 289 differentially expressed genes (DEGs) (198 upregulated, 91 downregulated) compared to non-stimulated controls.
• Key receptivity markers such as PAEP (glycodelin), SPP1 (osteopontin), LIF, and AQP3 were significantly upregulated, confirming the organoids achieved a receptive phenotype.

C. Effects of D-lactate (D-lac)

• Non-Hormonal Context: No significant gene expression changes were observed.
• Hormonal Context (EPC + D-lac):
  • 10 mM D-lac: Induced significant DEGs (COL1A2, MRC2, SMOC1) and enriched pathways for epithelial development and growth regulation (genes: KRT6A, IGFBP5, WNT2B, IHH).
  • 2.5 mM D-lac: Enriched pathways for cell death regulation and proliferation control.
  • Receptivity Markers: D-lac exposure uniquely upregulated DYNLT3 (an epithelial-specific receptivity marker) at 2.5 mM, suggesting D-lac may reinforce molecular programs supporting implantation.

D. Effects of Lipopolysaccharide (LPS)

• Non-Hormonal Context: No significant gene expression changes.
• Hormonal Context (EPC + LPS):
  • 2.5 ng/mL: Upregulated genes linked to defense response to symbionts and cytokine response (OAS3, IFIT3, TNFRSF1B).
  • 5 ng/mL: Enriched pathways for chronic inflammatory response and cell development (upregulation of OLFM4, a proliferative marker, suggesting LPS may interfere with the transition to the secretory phase).
  • 10 ng/mL: Enriched ERBB2 signaling and EGF receptor pathways (AREG, HBEGF), which are associated with cell proliferation and can oppose apoptosis.
  • Receptivity Markers: LPS uniquely upregulated MAP3K5 (a kinase involved in inflammation and stress), indicating an inflammatory response rather than a supportive receptive state.

5. Significance and Conclusion

• Microbial Modulation of Receptivity: The study provides evidence that the endometrial epithelium integrates microbial signals specifically during the hormonally receptive window.
• Dual Role of Microbiome:
  • D-lactate (from Lactobacillus) appears to support the receptive state by promoting epithelial maturation and upregulating specific receptivity markers (DYNLT3).
  • LPS (from Gram-negative bacteria) triggers inflammatory pathways and proliferation signals that may disrupt the delicate balance required for embryo implantation, potentially contributing to recurrent implantation failure.
• Clinical Implications: These findings suggest that the composition of the uterine microbiome (specifically the ratio of D-lac producers vs. LPS producers) could be a critical factor in reproductive success.
• Limitations: The study used a pure epithelial organoid model, lacking stromal and immune cells, and tested individual compounds rather than complex microbial communities. Future work should incorporate multicellular models to fully recapitulate in vivo interactions.

In summary, this research demonstrates that D-lactate and LPS exert distinct, hormonally mediated modulatory effects on the endometrial epithelium, with D-lac potentially enhancing receptivity and LPS inducing inflammatory responses that may compromise implantation.