Inflammasome-Related Markers and Long Non-Coding RNAs in Seminal Plasma: Associations with Sperm DNA Fragmentation and Male Infertility

High sperm DNA fragmentation correlated with elevated NLRP3, caspase-1, IL-1β, and IL-18 — but the protein data came from only 5 men per group

Journal: Journal of Reproductive Immunology | Published: 2026-05-06 | Type: Journal Article | PMID: 42128473 Authors: Ejehi MA, Mirfakhraie R (Shahid Beheshti University of Medical Sciences); Tavalaee M, Naderi N, Izadi T, Nasr-Esfahani MH (Royan Institute for Reproductive Biomedicine, Tehran) Funding/COI: Funding not disclosed. Authors declare no competing interests.

Summary

This Iranian cross-sectional study sorted 60 infertile men into low-SDF (<30%) and high-SDF (>30%) groups using sperm chromatin structure assay, then looked for inflammasome proteins and long non-coding RNAs (lncRNAs) in semen that track with fragmentation. Men with high SDF had worse sperm motility, viability, and concentration, and the high-SDF group showed higher levels of NLRP3-pathway proteins and elevated MALAT1 lncRNA. The authors are candid that their inflammasome protein data is exploratory at best — those measurements came from a five-man-per-group subset.

Claims

• Men with high SDF (>30%) had lower sperm concentration, motility, and viability compared to low-SDF men; semen volume and total sperm count did not differ
• High residual histone content (a marker of incomplete sperm chromatin remodeling) was elevated in the high-SDF group
• In a 10-man ELISA subset (n=5 per group), seminal plasma concentrations of NLRP3, caspase-1, IL-1β, and IL-18 were all higher in the high-SDF group
• MALAT1 lncRNA and NLRP3 mRNA expression tended to be higher in the high-SDF group's whole semen pellet; ANRIL and MEG3 expression did not differ between groups
• Sperm motility was inversely correlated with both MALAT1 and NLRP3 expression
• SDF showed a positive correlation with NLRP3 protein levels
• ANRIL expression correlated positively with sperm count; MEG3 correlated positively with ANRIL

Study Quality

The RNA side of this study (n=30 per group) is the sturdier half. Sperm DNA fragmentation was measured with SCSA, a validated flow-cytometry assay, and multiple lncRNA targets were profiled simultaneously. The design is cross-sectional and the sample is small, but the correlations between NLRP3/MALAT1 expression and sperm parameters are at least based on a full complement of 60 participants.

The inflammasome protein data is another story. The ELISA measurements — the part that actually implicates the NLRP3 inflammasome pathway rather than just its transcripts — were run on five men per group. That is not a sample size; it is a pilot experiment nested inside a small study. No power calculation is reported for this subset, and the paper itself labels these findings "exploratory." There is an additional methodological wrinkle throughout: RNA was extracted from unpurified whole semen cell pellets, not from isolated sperm. That means lncRNA signals may originate from leukocytes, epithelial cells, or other non-sperm seminal components, which is a meaningful confound when interpreting "sperm" inflammation.

Red Flags

• ELISA subset of n=5 per group is too small to draw any inference; this is a hypothesis generator, not a finding
• RNA from unpurified whole semen pellets conflates sperm-derived transcripts with leukocyte and somatic cell signals — the paper acknowledges this but does not quantify the contamination
• Inflammasome activity was not directly measured; NLRP3 protein presence and pathway activation are not the same thing
• Cross-sectional design cannot establish whether inflammation precedes DNA damage or results from it
• No funding source disclosed, which limits assessment of institutional bias
• "Tended to show" appears multiple times in place of statistical significance language — implies some comparisons did not reach significance

Strengths

• SCSA is a validated, standardized method for SDF quantification; the 30% cutoff is a conventional clinical threshold
• Royan Institute is one of the better-regarded reproductive biomedicine centers in the region; the group has a track record in sperm biology
• Measuring multiple inflammasome components simultaneously (NLRP3, caspase-1, IL-1β, IL-18) rather than a single proxy adds mechanistic texture
• Authors are unusually transparent about limitations, flagging unpurified RNA and the absence of direct inflammasome activation data explicitly in the abstract
• The lncRNA targets (MALAT1, ANRIL, MEG3) are biologically plausible — each has established roles in inflammation or epigenetic regulation

Verdict

The idea is sound: chronic low-grade inflammasome activation in semen might destabilize sperm chromatin, and MALAT1 plus NLRP3 transcripts could serve as non-invasive markers. The RNA correlations in 60 men are at least nominally interesting. But the headline finding — that inflammasome proteins are actually elevated in high-SDF seminal plasma — rests on five men per group, a number that belongs in a methods section, not a conclusion. Until the ELISA data is replicated in a properly powered cohort with isolated sperm fractions and direct inflammasome activation assays, this paper establishes a question, not an answer.