Cervical cancer is the third primary cancer in females worldwide. It is mainly caused by persistent infections with high-risk human papillomavirus (HR-HPV) or sometimes low-risk HPV (LR-HPV), which tend to develop from pre-cancerous lesions into an invasive form of cancer.

In most cases, these viral infections are transitory and heal on their own (around 80-90% of HPV infections disappear spontaneously within 24 months after their detection).¹ Both the regression and the clearance of the infection have been associated with the composition of the cervicovaginal microenvironment, defined by the cervicovaginal microbiome (CVM) and the subject's immune system.²

An estimated of 20-59% of women are known to have been infected with one or several types of human papillomavirus (HPV). The increased number of multiple infections post-menopause is believed to be a consequence of a diminished immunity response.

The clinical relevance of multiple infections with HPV is controversial. Some studies have shown that this clinical condition leads to a higher risk of cervical lesions as opposed to patients who have a single HPV infection. Moreover, other trials have shown that multiple infections with HR-HPV do not increase the risk of cervical cancer, but further analysis is required.³

HPV is widely deemed one of the most common sexually transmitted diseases⁴, and the main factor causing cervical intraepithelial neoplasia (CIN) or cervical intraepithelial lesion, and cervical cancer.⁵

The risk of suffering from an HPV infection is higher in the age group of 20 to 24, which is added to the risk of acquiring infections by a plurality of genotypes. This trend diminishes gradually with age, until a second peak is noted in women from 50 to 59 years old, which has led to a reassessment of the times when a timely screening is required to discard cervical cancer⁶, since we are looking at a population of subjects who are aging and at risk of developing malignant cervical neoplasia that goes under the radar.

Squamous intraepithelial lesions (SIL), also known as CIN, are atypical squamous changes occurring in a specific cervix area. Changes can be mild (low-grade intraepithelial lesions, or CIN 1 if they involve only a third of the epithelium), moderate (if they involve two thirds of the epithelium), or severe (if they include changes in the total tickness of the epithelium) depending on their depth (HSIL or CIN 2 or CIN 3).⁷

Only 1 out of 10 females having low-grade intraepithelial cervical lesions will develop a high-grade intraepithelial neoplasia⁸; nonetheless, the biological processes differentiating a lesion that progresses as such from a lesion that will go away on its own are still unknown.

It should be highlighted that many LSIL carriers are not subjected to a viral genotyping process, and even fewer go through epigenetic regulator screenings such as MicroRNA (miRNA), which have been proven useful to detect the progression of LSIL.⁸

The risk factors need to be assessed during the follow-up stage where, unfortunately, a vast number of patients are lost. This situation does not justify overtreatment but demands improving health strategies.

In this article (the first part of a series of two), we will review the factors that may impact on viral persistence and cell transformation of low-grade intraepithelial lesions (LSIL) with high-risk human papillomavirus and its management options.

The cervix is covered by two epithelium layers: an exocervical tissue (stratified epithelial layer) and an endocervical tissue (cylindrical type) in which glandular cells (columnar epithelium) are found that cover the endocervix, although a protrusion towards the exocervix (physiological ectropion) is considered a normal variant. These features expose these columnar cells to the vaginal microenvironment, with the meeting area of both epithelia meet —the transition zone— offering wider access to the base site of both the target reserve cells and the oncogenic agents. These exchanges are mainly observed during the reproduction stages.⁹

Although only some women develop infection persistence upon the onset of a SIL of any grade, several factors (including genetic and epigenetic) have been the subject of research, leading to the hypothesis that homeostasis of the vaginal microbiome plays a crucial role in the outcome of this infectious process.

The vagina is usually colonized by different types of Lactobacillus sp., which provide a rich microenvironment that sustains the normal vaginal microbiome and protects against pathogenic agents, including the HPV and other bacteria with intracellular capabilities. That shutts down the response to overgrowths and triggers the adequate immunity response in order to commence an effective reconstruction of the epithelium.

This process is essential to prevent traumas that would otherwise open the doors to invading agents in the stroma or the base layers.

A persistent infection by HPV is the main factor that stimulates cervical carcinogenesis^10,11, so studying the causes that promote the acquisition of this virus, cervical biomarkers secreted locally, and the changes involved in the vaginal microbiota account for the need to carry out more thorough analyses and to assess the chances with different therapies that may have an impact on the host-virus relation and the vaginal microbiota.

Hormonal Factors

Several trials have identified a synergic combination between the HPV infection status and a repeatedly increased level of endogenous and exogenous estrogens —such as in repeated gravida and the use of oral hormones— which augments the probability of developing cervical cancer.¹²

Viral oncogenes express oncoproteins E6 and E7, which attach to p53 and pRB, respectively, overcoming the carrier's immune system. Several theories suggest that estrogens may stimulate these viral processes and promote the transformation that eventually results in cervical cancer.¹² This is proposed because the cervix is highly sensitive to estrogens, especially the transformation area where tumors begin to grow.⁶

Users of oral birth control methods are at a higher risk of developing high-grade squamous intraepithelial lesions (HSIL) compared to users of hormonal intrauterine devices (IUD). This increased risk is due to mTOR, which plays a key role in the interaction between the virus and the host's cells through the oncoproteins E6 and E7. These oncoproteins overexpress kinase B, resulting in the hyperactivation of mTOR, which increases the translation of E7 and modulates cell growth by inducing the activation of the estrogen receptor (ER) independently of its ligand. This process promotes a persistent HPV infection, which is a fundamental factor in the development of cervical intraepithelial lesions.¹²

E6 degrades p53 through ubiquitination, and E7 binds to pRB, releasing the E2F factor and subsequently activating AKT. E6 can also activate mTOR through the mTORC1 complex, and AKT is overexpressed.¹³ In this case, HPV oncoproteins E6 and E7 attach to p53 and pRB tumor suppressors, respectively, which results in the degradation of p53 and pRB by ubiquitination.

It has been demonstrated that WALP, a protein that is key to cervical oncogenesis, increases the sensitivity of ESR1 to estrogen by activating MACROD1, which in turn activates RAC1 and ERK1/2, both having an essential involvement in cell proliferation and oncogenic mechanisms.

Moreover, sexual behavior patterns among users of hormonal birth control methods and non-users should be highlighted, together with the type of birth control method being used and the factors that have an impact on the development of lesions associated with HPV.^14,15

Hence, the conclusion reached by the authors is that not only does WAPL increase the sensitivity to estrogens, but it also produces chromosomic instability through SALL 4 in cervical carcinogenesis.^7,8

Further research focused on the use of hormonal birth control methods, sexual behavior patterns, and HPV infection persistence (among others) is still required, to find if the devices also have an impact on the incidence of precancerous lesions.¹⁵

Microbiome

The equilibrium or disruption of the vaginal microbiome (VM) is crucial to the impact on HPV infection persistence. Furthermore, a healthy VM includes low microbial diversity and prevalence of Lactobacillus sp., which prevent the overpopulation of other pathogenic bacteria by producing lactic acid, bacteriocin, and hydrogen peroxyde, thus preserving the integrity of the mucus barriers that defend against virus and other opportunistic bacteria.⁵

Cervicovaginal mucosa is made of layered squamous epithelium covered by mucus and lubricated by cervicovaginal fluid that helps support the CVM.

The CVM is a series of microbial communities that form a symbiotic relationship with the carrier and are frequently categorized by different species of Lactobacillus, which secrete lactic acid and help maintain an acidic pH. Specific Lactobacillus can be divided into five different community states types (CST). CST I, II, III, and V are mainly L. crispatus, L gasseri, L. iners, and L. jensenii. The L. crispatus, L. gasseri, and L. jensenii can produce lactic acid and H2O2, acidify the vaginal environment to a pH <4.5, and inhibit the growth of other viruses and bacteria. CST IV (in which L. iners is predominant) is considered an excessive state of Lactobacillus.

The Lactobacillus species adhere to the epithelium surface to prevent pathogenic bacteria from becoming attached to it. They modulate the immune response by stimulating the host's defense mechanisms against pathogens: macrophages, dendritic cells, and T cells.

The cervicovaginal ecosystem also includes innate immune cells, adaptive cells, macrophages, NK cells, neutrophils, dendritic cells, Langerhans cells, T-cells, lymphocytes made of a series of elements secreted by other vaginal cells and bacteria, including mucins and antimicrobial molecules, ß defenses, Elafin, lipocalin, secretory leukocyte protease inhibitors and immunoglobulins IgA and IgG, all of which act as the line of defense against vaginal pathogenic agents.²

VM is affected in one way or another by different factors, including ethnicity, hormone changes —particularly estrogens and progesterone— throughout the menstrual cycle or menopause, use of birth control hormones, sexual activity, hygiene practices, nutrition, and history of infections.

In the microbiome, Lactobacillus (the predominant genus, and specifically species crispatus, gasseri, iners and jensenni) is associated with the fastest clearance rate of HPV infections.

CST IV is associated with persistent HR-HPV infections and is characterized by a low concentration of Lactobacillus as well as by the presence of anaerobic microorganisms that cause vaginal infections, including Gardnerella vaginalis, Megasphera, Prevotella, and others.

The most prevalent species in groups I, II, III, and V of vaginal microbiome profiles are Lactobacillus crispatus, L. inners, L. jensenii, and L. gasseri. Their presence is associated with a faster clearance of HPV infection.

Group IV, however, is commonly associated with HR-HPV persistent infections. Not surprisingly, this group is characterized by a low concentration of Lactobacillus and a higher presence of anaerobic microorganisms known to cause vaginal infections, such as Gardnerella vaginalis, Megasphera, and Prevotella, among others.

Bacterial vaginosis (BV) is typically characterized by a lower concentration of Lactobacillus and an increased concentration of anaerobic bacteria, with a type IV microbial environment (CST IV), which conveys a higher susceptibility to sexually transmitted infections (STI), including HPV.^5,16,17

An increase in the diversity of VM and a decreased count of Lactobacillus sp. are both associated with the progression of the intraepithelial lesions in terms of their severity (SIL or CIN), as well as viral persistence.¹⁷

The secondary coadjuvants to these changes may be associated with a decreased production of protective lactic acid and bacteriocin, increased oxidative stress, and alterations of the integrity of the mucus induced by dysbiosis, which in turn may promote the access of the virus to the cells. In fact, species such as Snethia spp have a probable pathologic role in the acquisition and persistence of HPV infections.⁵

An HPV infection on its own may promote changes in the vaginal microbiota by inducing an immune response to the carrier's mucus, in addition to genital inflammation.

Furthermore, factors such as elevated genital inflammation, vaginal pH tending to alkalinity, and VM with decreased Lactobacillus counts have been associated with HPV persistence favoring progression to cervical malignancy.¹¹

Therefore, the stability of VM must be considered as one of the main factors contributing to HPV infections and the development of precancerous lesions in the cervix. A theory has been proposed stating that manipulation and modification of the VM microbiome with the use of pre- and pro-biotics as well as symbiotics or even a transplant of normal vaginal microbiota organisms may have the potential to constitute an innovative therapy.¹⁰

Infections Promoting Prevalence and Persistence

Most HPV infections are transient. An approximate 70-80% thereof is self-restricted within two years. However, several factors, including genetic predisposition, immune response, environment, and microbiome, may all impact the eradication or persistence of the virus.

Twenty to thirty percent of all HPV infections are persistent and only 1-2% thereof evolve into cervical cancer, with genotypes 16 and 18 identified as the causes of 70% of cancerous lesions associated with HPV worldwide while in the larynx and cutaneous warts, 90% are types 6 and 11.¹⁶

Studies have suggested identifying specific genotypes of HPV and a normal viral load. Still, the presence of multiple HPV genotypes has not been given a specific weight. However, such genotypes may act as biomarkers to identify women who are at a higher risk of developing cervical cancer.¹⁸

Persistence is defined as an HPV-positive infection present at two moments separated by a minimum period of four months and up to 5-7 years, according to the provisions of the International Agency for Research on Cancer.¹²

The persistence of an HR-HPV infection has a significant role, as the permanence of this virus in the cervical epithelium promotes the onset of low-grade and high-grade (precancerous) intraepithelial lesions or cancerous ones, depending on the immunologic status of the patient as well as on other risk factors, including the presence of simultaneous infections.

Among said simultaneous infections, Chlamydia trachomatis contributes to the permanence of the virus within the tissue, as it affects the MMR path. The MMR path detects the cells that have mutations in specific genes and participates in the correction of such mutations when certain flaws occur during the process of copying the DNA from one cell to another, i.e., cells having flaws in their self-repair mechanisms that can lead to cancer by inhibiting the expression of suppressor genes. When abnormal conditions prevail in the MMR path, genomic stability is not achieved due to the invasion of C. trachomatis, which provokes proteasomal degradation of E2, thus creating a mutation that results in oncogenesis.¹⁹

A trial conducted in China in 2020 assessed the relationship between simultaneous bacterial vaginal infections and HPV infection. This trial found that bacterial infections or microbiome dysbiosis do not necessarily promote the persistence of HPV and the progression toward LSIL or HSIL.

In fact, this trial found evidence to the fact that the HPV infection on its own has the potential to provoke an imbalance and increased growth of such agents, thereby disrupting the vaginal microbiome, thus creating a vicious loop between the infection caused by HPV and bacterial vaginosis, inducing permanent damage in the cell structure of the epithelium.⁵

Human papillomavirus

The clearance of LSIL, from a vigilance or a conservative approach, is approximately 59% since most go through clearance within two years following diagnosis.

The probability of developing an HSIL within a 5-year term is 12.7%, and most clinicians remain wondering which patients will show regression and which ones will show progression.^20,21

Various prospective cohort studies of women with a positive DNA test for HPV (types 16 or 18) and also normal cytology have shown that such patients are at an 18-21% risk of developing a high-grade intraepithelial lesion within ten years²⁰; that is why vigilance is necessary, but also, why DNA screening would be a more reliable diagnostic tool for such purpose.

Cervical cancer screening programs have the purpose of detecting and eliminating precancerous lesions associated with HPV in a timely fashion.

Cervical cancer is more prevalent in countries classified as low- and mid-level income countries according to several indicators of human development indexes, considering a low per-capita income, low level of education, and lower life expectancy, in contrast to countries having a higher human development index. This situation results in a low number of women receiving smears/screenings and poor access to vaccines against HPV.²

When a human being becomes infected with HPV, three different sequences may occur: a latent infection, a subclinical infection, or a clinical manifestation of the disease.²²

Full viral genome integration within the host genome is required for an oncogenic transformation.²³ However, further risk factors also play a role, including consumption of tobacco, having several sexual partners (more than 3), high gravida number, and long-term oral hormone consumption (over five years).²

There are more than 150 HPV genotypes capable of infecting both mucous and skin epithelium. In this group, there are 14 genotypes belonging to the group of alpha papillomavirus defined as HR-HPV²⁴, having an elevated transformation capacity by inhibiting tumor-suppressor genes such as TP53 and RB in cervical epithelial cells¹⁸, thus favoring the development and growth of cervical carcinoma.

Since they are present in 99% of malignant tumors in the cervix²⁴, this factor demonstrates that infection by HR-HPV is a contributing cause —but not enough in itself— for developing cervical carcinoma.²⁴ Not every HPV has the same clinical significance to determine their malignancy potential, but the presence of HPV 16, 18, 31, or 45 conveys a higher risk of progression.³

Women infected by multiple genotypes of HPV have shown to be at a greater risk of developing LSIL, in contrast to women with single-genotype infections, with the statistical relationship between multiple infections and invasive cancer still under study.³ However, a pattern can be identified among carriers of multiple HPV infections, with one peak among young women and a second peak among older females (perimenopause).¹⁸

In infections caused by HR-HPV, an evasion of local immunity response has been determined that minimize the production of antigens and trigger the expression of viral oncoproteins E5, E6, and E7, which are easily translocated into the cell nucleus.

During the early stages of the infection, these viruses express a low presence of oncoproteins E5, E6, and E7. In contrast, during later stages, HR-HPV promotes the expression of capsid proteins, which are then secreted into the epithelium, where only a low number of antigen-presenting cells is found, promoting a decline of immunity response. This decline in HPV-infected cells also affects their capacity to activate local immunity cells, thus promoting a global immunosuppression environment that makes room for carcinogenesis.²

In a study, it has been suggested that cancer screening in the cervix should be suspended by the recommended age (65 years old, although the controversy remains) if such smears have been performed regularly, or at any age if life expectancy is limited, or if a given patient has undergone a hysterectomy with no precancerous or cancerous lesions in the cervix. Negative HPV tests are associated with a lower risk of cervical cancer for around 10-15 years, while negative cytology tests are associated with 5-7 years of reduced risk if the screening is performed appropriately.⁶

New molecular-level strategies have been proven valid to be performed alongside tissue biopsies and clinical diagnosis tests to determine if LSIL in a patient has a high carcinogenic potential due to HPV infections made by viral variants such as 16 and 18. MicroRNAs (miRNA) —small non-coding RNAs with approximately 22 nucleotides— have been identified as some of the most promising markers. MicroRNAs are present both in blood and in tissues, making their detection and analysis a safe and effective approach.²⁵

A trial was performed among Mexican patients, analyzing the expression through real-time PCR of serum microRNAs miR-15b, miR-34a, and miR-218 in patients having LSIL or cervical cancer and patients who had no HPV infections or cancer.²⁵

The relevance of this study is that it validates that the miRNA levels in serum are similar to the ones present in biopsies, which facilitates obtaining blood samples from patients, using less invasive methods, and performing an adequate treatment follow-up.

The study also showed that miR-34 and miR-218 —both antineoplastic transcription regulators— are highly expressed in patients screened for LSIL.

Similarly, miR-15b —a molecular regulator present in carcinogenic processes— is expressed at a much lower ratio. The expression pattern is inverted when LSIL evolves into cervical cancer, thereby increasing the levels of miR-15b in the analyzed tumorigenic tissue.

Additionally, abnormal expression of genes associated with carcinogenesis was determined, including Bcl-2 (target gene of miR-15b, miR-34a, and miR-218), cyclins D1 and E1 (target genes of miR-34a and miR-218) and DNA mismatch repair gene Msh2 (target gene of miR-15b), in samples from patients with a positive cervical cancer screening.^25,26

The expression pattern proposed herein may be crucial to assist in the detection of LSIL that may derive in cancer, thus allowing to carry out a closer follow-up on the patients having cervical tissue damaged by a history of HPV infection.

Immunohistochemical markers, particularly p16INK4a and Ki-67, have emerged as quite valuable tools in differentiating LSIL of the cervix.^27-29

The overexpression of p16INK4a, associated with the cell cycle disruption by HR-HPV, facilitates neoplastic progression. This marker has improved diagnostic accuracy by distinguishing between benign changes and precursor lesions. Additionally, Ki-67 is used to assess cell proliferation, providing an additional evaluation of the progression risk.^27-29

Combining these markers is crucial for the stratification risk and clinical management of LSIL.