Most cases of LSIL, or low-grade squamous intraepithelial lesion, are caused by human papillomavirus, but a clinically significant minority show up on Pap smears in people who test negative for HPV. When that happens, the explanation usually involves one of several overlapping factors: shifts in the vaginal microbiome, other infections, immune suppression, environmental exposures like cigarette smoke, or sometimes the limits of the diagnostic process itself. Understanding these non-HPV triggers matters because they change how follow-up is managed and how much a person should worry.
Bacterial Vaginosis and Microbiome Disruption
Bacterial vaginosis (BV) is one of the best-documented non-HPV contributors to abnormal cervical cells. BV occurs when the normally dominant Lactobacillus bacteria in the vagina are displaced by a mixed community of anaerobic organisms. In a study comparing women who were both BV-positive and HPV-negative with women who were negative for both, the BV-positive group had a higher rate of squamous abnormalities on biopsy. Among those abnormal results, the majority were low-grade lesions.1American Journal of Clinical Pathology. Association of Bacterial Vaginosis and Human Papilloma Virus Infection With Cervical Squamous Intraepithelial Lesions
The connection goes deeper than BV alone. Research into cervical microbiome profiles has found that women with abnormal cervical cytology are more likely to harbor certain bacterial community types, particularly communities not dominated by Lactobacillus species. Women with normal cytology and no high-risk HPV, by contrast, tend to have vaginal communities dominated by protective Lactobacillus species, especially L. gasseri, which was found at significantly higher levels in women without abnormalities.2PubMed Central. The vaginal microbiota, high-risk human papillomavirus infection, and cervical cytology: results from a population-based study The practical implication is that an imbalanced vaginal microbiome can create cellular changes that look like LSIL under a microscope, even when HPV is not present. Whether treating BV resolves those changes is still an open question, but the association itself is well established.
Other Sexually Transmitted Infections
Chlamydia trachomatis and Mycoplasma genitalium can independently produce abnormal-looking cervical cells. In a study of outpatients, the probability of an abnormal Pap result at or above the level of atypical squamous cells was roughly three times higher in women positive for Chlamydia and more than three and a half times higher in those positive for Mycoplasma genitalium, compared with women testing negative. The rate of STI positivity was also significantly higher among women with abnormal cytology who tested negative for high-risk HPV.3Journal of Gynecologic Oncology. Associations between sexually transmitted infections, high-risk human papillomavirus infection, and abnormal cervical Pap smear results in OB/GYN outpatients
These infections cause inflammation that can distort how cervical cells look, mimicking the classic koilocytic changes (cells with halos around their nuclei) that pathologists associate with HPV. When a Pap comes back showing LSIL but the HPV test is negative, an untreated STI is one of the first things worth investigating. Treating the underlying infection sometimes leads to normalization of the Pap on follow-up, though the timeline varies.
Immune Suppression and Autoimmune Disease
A weakened immune system makes it harder for the body to clear infections and repair damaged epithelial tissue, and that extends to the cervix. People with systemic lupus erythematosus (SLE) have a notably higher prevalence of cervical abnormalities. In one study, the prevalence of low-grade intraepithelial lesions was about six times higher in SLE patients than in controls, even though the SLE patients had fewer of the traditional risk factors for cervical disease.4Journal of Clinical Rheumatology. Is Higher Prevalence of Cervical Intraepithelial Neoplasia in Women With Lupus Due to Immunosuppression? Among the SLE patients who had been on immunosuppressive medications long-term, the prevalence was even higher.
This risk is not limited to lupus. Guidelines now recommend that immunosuppressed women without HIV follow more frequent cervical screening schedules, similar to those recommended for people living with HIV, regardless of whether they are on immunosuppressant drugs.5PubMed Central. HPV Associated Disease: Guidelines and Guideline Discussion Updated Review for Guidelines for Cervical Cancer Screening in Immunosuppressed Women Without HIV Infection The issue is partly that immunosuppression allows HPV infections to persist longer, but the chronic immune dysregulation itself also promotes the kind of low-grade cellular changes that get flagged as LSIL. Organ transplant recipients on anti-rejection drugs, people on biologic therapies for autoimmune conditions, and anyone on long-term corticosteroids fall into this higher-risk category.
Smoking and Secondhand Smoke
Cigarette smoking has a well-recognized association with cervical abnormalities, and the connection is not just about HPV persistence. In a large study of a predominantly Hispanic population, passive smoke exposure was positively associated with abnormal Pap results, with an odds ratio of about 1.7. Active smoking carried its own independent risk. Increasing hours per week of secondhand smoke exposure were specifically linked to LSIL.6PubMed Central. Passive smoke exposure and abnormal cervical cytology in a predominantly Hispanic population
Tobacco metabolites concentrate in cervical mucus, where they can directly damage epithelial DNA and suppress local immune defenses. The result is a cervix that is both more vulnerable to infection and less able to repair the kind of low-grade damage that shows up as LSIL. For people who test HPV-negative but still have abnormal Pap results, smoking status is a relevant piece of the puzzle. And unlike many of the other risk factors discussed here, it is modifiable.
Chronic Inflammation Without a Clear Infection
Persistent inflammation in the cervical tissue, even without an identifiable pathogen, can produce cellular changes that mimic LSIL. When the body sustains an inflammatory response over time, the constant cycle of tissue damage and repair increases the turnover of epithelial cells. That accelerated renewal creates more opportunities for DNA copying errors and cellular abnormalities. The reactive oxygen and nitrogen species produced during chronic inflammation can also directly damage DNA.7Spandidos Publications. Link between chronic inflammation and human papillomavirus-induced carcinogenesis (Review)
In practical terms, this means that conditions causing chronic cervical irritation, such as persistent vaginitis, chemical irritation from douching or certain spermicides, or even friction from a poorly positioned device, can occasionally produce cells that a pathologist reads as LSIL. These are not pre-cancerous in the usual sense. They represent the cervix responding to ongoing insult, and the abnormalities tend to resolve once the source of irritation is removed.
IUDs, Radiation, and Other Iatrogenic Causes
Intrauterine devices (IUDs) can alter the cervical environment in ways that generate suspicious-looking cells. A review of cervical smears from IUD users documented a range of changes including inflammatory, degenerative, and reparative atypias. About 70% of those atypias were benign. In cases where mild dysplasia was found, the cervix reverted to normal within six months of IUD removal.8PubMed. Cytopathologic changes associated with intrauterine contraceptive devices. A review of cervico-vaginal smears in 350 women This means that a Pap showing LSIL in someone with an IUD may simply reflect the device’s mechanical and biochemical effects on the cervix, not a true precancerous process.
Prior radiation therapy and chemotherapy can also produce cervical cell changes that look like LSIL. Post-irradiation dysplasia refers to abnormal cellular changes in non-cancerous epithelial cells that appear after radiation treatment. Chemotherapy produces similar changes, though typically in fewer cells.9PubMed Central. Cervical cytology: Radiation and other therapy effects If you have a history of pelvic radiation or recent chemotherapy, an LSIL reading on a Pap may be a treatment side effect rather than evidence of a new problem. Clinicians familiar with your treatment history should factor this in before recommending aggressive follow-up.
HPV Genotypes That Standard Tests Can Miss
Sometimes what looks like “HPV-negative LSIL” is actually caused by an HPV type that the screening test does not detect. Standard high-risk HPV tests are designed to pick up a panel of about 14 genotypes most strongly linked to cervical cancer. But dozens of other HPV types exist, and some of them are not harmless bystanders. A study analyzing uncommon and rare HPV genotypes found that about a quarter of infections with these less-tested types were associated with LSIL. Genotypes like HPV 68, HPV 53, HPV 66, HPV 54, and HPV 70 all showed meaningful rates of associated lesions.10PubMed Central. Uncommon and Rare Human Papillomavirus Genotypes Relating to Cervical Carcinomas
This is an important caveat when interpreting an HPV-negative LSIL result. The test may be reporting negative not because HPV is absent, but because the specific genotype responsible falls outside the test’s detection range. Clinically, this is one reason why LSIL is generally followed with repeat testing rather than being immediately dismissed when the HPV test comes back negative. The virus may still be there, just not the one the assay was looking for.
The Overdiagnosis Problem
A substantial fraction of HPV-negative LSIL may not represent a true biological abnormality at all. LSIL diagnosis has long been recognized as poorly reproducible. One study found that the agreement between pathologists on the features used to diagnose LSIL was modest at best, with nuclear features showing particularly low reproducibility. Assessment of chromatin texture, one of the key features pathologists rely on, had the least agreement among observers.11PubMed. Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC-US, ASC-H, LSIL and HSIL)
Separate research using HPV mRNA in situ hybridization as a more objective marker confirmed that LSIL diagnosis “can present considerable diagnostic challenges and is associated with poor interobserver reproducibility and overdiagnosis.”12The American Journal of Surgical Pathology. HPV E6/E7 mRNA In Situ Hybridization in the Diagnosis of Cervical Low-grade Squamous Intraepithelial Lesions (LSIL) In plain terms, some cells that one pathologist calls LSIL, another would call normal. Reactive changes from inflammation, atrophy, or even the slide preparation process can be mistaken for the nuclear irregularities that define LSIL. When the HPV test is negative and the biopsy is equivocal, overdiagnosis is a genuine possibility, and repeat testing often resolves the uncertainty.
HPV-Independent Precancers With Somatic Mutations
A recently described and genuinely distinct category of cervical abnormality has nothing to do with HPV at all. Researchers have identified HPV-independent cervical intraepithelial neoplasia driven by somatic mutations, the kind that arise spontaneously in cells rather than being introduced by a virus. These lesions harbor mutations in genes like TP53, EGFR, PIK3CA, and SMARCB1, and they resemble HPV-independent precancers already known to occur on the vulva.13The American Journal of Surgical Pathology. The Histologic and Molecular Spectrum of Highly Differentiated HPV-independent Cervical Intraepithelial Neoplasia
These are rare, but they matter because they represent a true cancer precursor pathway that exists completely outside the HPV framework. The lesions look different under the microscope from classic HPV-driven LSIL. They tend to be highly differentiated, with features like elongated epithelial rete and premature squamatization that pathologists trained to spot HPV-related changes might not immediately recognize. Their identification is relatively new, and most screening protocols are not yet designed to catch them. For now, they are mainly diagnosed after biopsy and immunohistochemical or molecular testing. Their existence is a reminder that “HPV-negative” does not always mean “no precancerous risk.”
What Happens With HPV-Negative LSIL on Follow-Up
The natural history of LSIL differs depending on whether HPV is driving it. In a large cohort of patients whose LSIL was HPV-negative, only about 3% went on to develop a high-grade lesion during follow-up, and no cases of cervical cancer were documented. Roughly 40% had persistent LSIL findings on subsequent tests.14PubMed. Follow-up outcomes in a large cohort of patients with HPV-negative LSIL cervical screening test results This is a substantially lower progression risk than what is seen in HPV-positive LSIL.
Research tracking the timeline of these outcomes adds useful context. Among women with LSIL and no HPV, about half of lesions regressed to normal or near-normal within six months. Regression took roughly the same amount of time whether the original infection was a non-oncogenic HPV type or no HPV at all, around seven to eight months on average. By contrast, women with oncogenic HPV types took significantly longer to regress.15JNCI: Journal of the National Cancer Institute. Human Papillomavirus Infection and Time to Progression and Regression of Cervical Intraepithelial Neoplasia The bottom line for anyone receiving an HPV-negative LSIL result is that the odds strongly favor resolution, and watchful waiting with repeat testing at the recommended interval is the standard approach.
When the Same Logic Applies Beyond the Cervix
LSIL is not exclusive to the cervix. The same terminology is used for low-grade squamous changes at other anogenital sites, particularly the anus. Anal LSIL shares many of the same risk factors, including HPV, immune suppression, and smoking. In one long-term study tracking anal LSIL over a median of four years, the cumulative probability of progressing to a high-grade lesion was about 14% at one year and over 44% at five years. HPV16 status and age over 44 at diagnosis were the strongest predictors of progression, while other HPV subtypes, smoking, and BMI were not significantly related to outcomes in that cohort.16PubMed Central. Anal neoplasm: Streamline follow-up of low-grade dysplasia
The progression rates for anal LSIL are considerably higher than for cervical LSIL, which underscores that the biology of these two sites is not identical even though the terminology overlaps. People at higher risk for anal LSIL, including those with HIV, men who have sex with men, and anyone with a history of anogenital HPV disease, may benefit from anal cancer screening conversations with their providers, especially as screening guidelines for anal dysplasia continue to evolve.