Diagnosis of non-neoplastic endocervical diseases

Corina Grigoriu1,2, Alice Negru2, Gina Calinescu2, Andra Magdalena Balan2, Lucica Elena Eddan-Visan2, Nicolae Bacalbasa1, Irina Balescu3, Consuela-Madalina Gheorghe4, Tiberiu Augustin Georgescu5,6, Emilia Maria Vladareanu7, Roxana Elena Bohiltea1,8 1Department of Gynecology and Obstetrics, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 2Department of Gynecology and Obstetrics, Emergency University Hospital, Bucharest, Romania 3Department of Visceral Surgery, Ponderas Academic Hospital, Bucharest, Romania 4Department of Marketing and Medical Technology, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 5Department of Pathology, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 6“Alessandrescu-Rusescu” National Institute for Mother and Child Health, Bucharest, Romania 7Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 8Department of Gynecology and Obstetrics, Filantropia Clinical Hospital, Bucharest, Romania


INTRODUCTION
Endocervical pathology is commonly encountered in daily outpatient gynecological practice and is apparently simple to diagnose. Remote resonance (both in time and anatomically in space) of untreated endocervical pathologies, however, indicates difficulties in detection and perhaps even in treatment.
The cervix is a large part of the female genital tract from the paramesonephric ducts (Mullerian ducts). It is divided into exocervix and endocervix; the latter practically represent the inside of the cer-vix or the cervical canal, which is spindle-shaped, delimited by the external os towards the vagina and by the internal os towards the uterine cavity. The endocervix consists of a single-celled layer of epithelium, made up of tall columnar cells. Cells, which have round-oval nuclei located basally, are mostly secretory cells (apocrine and merocrine). There are also a small number of ciliated cells, the cilia can be observed only by electron microscopy, along with mucin droplets and secretory granules of varying sizes. Recent research shows that only cytokeratin 16 is expressed in the endocervical epithelium (different from the stratified exocervical epithelium, in which several cytokeratins are expressed). The endocervical epithelium is in fact a pseudoglandular epithelium: it does not actually contain glands, but epithelial invaginations occur, on a depth of 5-8 mm, forming crypts.
The role of the endocervix is that of a boundary between the lower genital tract (vulva and vagina) and the upper genital tract, that is between an environment rich in microorganisms (vaginal microenvironment) and the sterile endometrium and endosalpinx. At this level there are various influences, from the fluctuations of the hormonal secretion of the different phases of the menstrual cycle and the presence of the commensal flora (pathogenic condition), to the aggression of the germs with sexual transmission, etc. Also, the vaginal and exocervix squamous epithelia may be aggressed by chemical agents or physical changes. The endocervix is the gateway for germs such as Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma etc., which have a tropism for the columnar epithelium.
The major barrier role belongs to the cervical mucus. The main constituents of cervical mucus are mucins (glycosylated proteins, so far 19 have been identified). Mucins can be membrane-forming or soluble (small molecule) gel-forming. Another important component is antimicrobial peptides, capable of destroying bacterial membranes. It is interesting to note that microorganisms selectively adhere to certain mucins, produce proteases that can degrade mucins as well as other mucus defense factors.
At the gynecological examination, a white-yellowish, yellowish or yellow-green leukorrhea can be observed. Endocervical mucus can be opaque or even purulent. When a swab is inserted, its yellowish coloration is observed. A suggestive sign may be bleeding when touching the cervix (during the Pap smear).
The etiological factors of endocervicitis are, in the order of frequency: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma and Ureaplasma (1). The focus will be on Mycoplasma genitalium, whose role in genital pathology is increasingly better defined. It is a microorganism of the genus My-coplasma, class Mollicute (without its own wall, it cannot be identified by Gram staining), being the smallest prokaryote that can multiply (2). It has a parasitic lifestyle, taking most of the nutrients from the host (which it cannot synthesize). It has its own motility, attaches, and adheres to cells and internalizes, similar to Chlamydia (different from other mycoplasmas). Upon entry into the cell, it evades the host's immune system (lymphocyte suppression and up-regulation of cytokine expression). It can destroy the host cell by secreting a toxin and/ or releasing hydrogen peroxide. It has a slow growth on culture media, detectable by nucleic acid amplification tests (NAATs). It is certainly different from Mycoplasma hominis and Ureaplasma urealyticum. There are still debates if it belongs to the germs that cause sexually transmitted diseases (STDs), although its transmission is demonstrated by identifying an identical genome in partners. It is located on the ciliated epithelia of the urinary and genital tract, and the environment rich in progesterone promotes colonization. It has been isolated in the respiratory tract and synovial fluid, but appears to be pathogenic only at the urogenital level. The resulting clinical picture is the consequence of the host organism's response to cell invasion by mycoplasmas. It seems to persist for years in the infected body, many specialists suggesting a possible involvement in the triggering of autoimmune diseases (3).
Risk factors for endocervical infection are: age under 20 years, non-white race, lower level of education, smoking, increased number of sexual partners (over 2 in the last 12 months), vaginosis, its prevalence being 1% in the sexually active adult population (between Gonococcus 0.4% and Chlamydia trachomatis 2.4%).
Mycoplasma endocervicitis is considered to have a similar incidence as Chlamydia infection in women considered "at risk" -early onset of sexual life, large number of sexual partners (6% Mycoplasmas, 10% Chlamydia); it is often asymptomatic or with symptoms that if carefully sought, suggest cervicitis, urethritis ("urethral syndrome" -dysuria, discomfort during and after urination, pollakiuria), endometritis, pelvic inflammatory disease, most often chronic, with reactivations.
The possible association with bacterial vaginosis should be mentioned (clue cells are observed on the smear collected from the vagina, but paradoxically also an inflammatory reaction). Long-term effects (male and female infertility) and probable obstetric complications (premature birth, less the risk of miscarriage) should not be forgotten. Finally, the hematogenous dissemination at the site of the primary infection should be noted, with manifestations at the level of the joints (reactive arthritis) and of the ocular conjunctiva -conjunctivitis (Reiter's disease).
The diagnosis is on the one hand clinical ( Figure  1) (of exclusion -cervicitis, non-gonococcal and non-chlamydial pelvic inflammatory disease), but, when possible, it should be confirmed in the laboratory, by performing the following: nucleic acid amplification tests (NAATs) and serological tests. Indirectly, the presence of over 30 polymorphonuclear leukocytes per microscopic field -the new criterion for cervicitis -confirms the presence of Mycoplasma genitalium in most cases (4).

FIGURE 1. Endocervicitis (personal collection of Corina Grigoriu)
The careful observation of the ultrasound aspect of the cervix may suggest endocervicitis: irregular endocervical canal is observed (hyperechoic line becomes irregular or disappears), with possible poorly defined hypoechoic areas, numerous Naboth cysts (proof of chronic inflammation). Color Doppler test shows increased vascularity at this level (5).
What should be mentioned is the continuous lesion, with the possible evolution from endocervicitis to endometritis, endosalpingitis and pelvic inflammatory disease. For this reason, it is important to promptly recognize endocervicitis, in order to limit the progression of the disease. Major risk factors are genital Chlamydia trachomatis infection, young age, more than two sexual partners, and associated risk factors: mycoplasma genitalium infection, bacterial vaginosis, history of leukorrhea and/ or chronic pelvic pain, and lower level of education (6). The clinical picture is dominated by moderate-severe pelvic pain, diffuse abdominal pain, pain in the right hypochondrium, deep secondary dyspareunia, leukorrhea, and vaginal bleeding.

ENDOCERVICAL POLYPS
Endocervical polyps are proliferations of glandular tissue that protrude inside or at the surface of the cervix. They are the most common benign proliferation in the female genital tract and of postmenopausal vaginal bleeding ( Figure 2).

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Endocervical polyps have a maximum incidence around the age of 40. In their etiology, a possible role belongs to chronic cervical inflammation, but the intervention of a hormonal factor is also possible, because they can coexist with endometrial hyperplasia. They can be single or multiple, with a short or long, narrow, or wide pedicle. Most often, the endocervical polyps have a long and thin pedicle. Its length varies between a few millimeters and a few centimeters.
They may be symptomatic, manifesting by bleeding (often intermenstrual or postcoital) and/ or leukorrhea. They degenerate very rarely (about one in 200 cases), so they should be checked histopathologically. Six types of polyps have been described: adenomatous (80%), cystic, fibrous, vascular, inflammatory and fibromyomatous. Their surface consists of glandular or squamous tissue (depending on the origin and the degree of possible squamous metaplasia, which can cover the tip of the polyp). The pedicle is a vascular axis with a variable amount of connective tissue around it.
Polyps can sometimes be observed on clinical examination (examination of the cervix with valves), can be externalized after performing a cytological test, but are often seen when performing imaging tests (transvaginal ultrasound of the cervix) (Figures 3, 4).
At the transvaginal ultrasound, it is recommended to apply additional pressure with the transducer. The presence of a hyper or hypoechoic mobile formation can be observed in the endocervical canal, which "slides" during the examination. The presence of a blade of fluid (mucus) in the endocervical canal makes the examination much easier. Doppler examination allows the identification of the vascular pedicle and especially its origin (thus differentiating a formation with cervical origin from an endometrial polyp).

NABOTH CYSTS
Naboth cysts are retention cysts present in the endocervical epithelium. They are formed by cover-ing the holes of the "glands" (crypts) at this level, as a spontaneous defense response of the cervix (usually post-infectious, inflammatory or after interventions on the cervix) (7). This defense process consists in stimulating the squamous metaplasia, with the consequent obstruction of the glandular orifice and the progressive accumulation of mucus. They can be single or multiple and do not exceed an average size of one centimeter (8).
They can be observed both on the surface of the exocervix (by direct or colposcopic examination), but also at the cervical canal (by transvaginal ultrasound -single or multiple anechoic or hypoechoic images in the endocervical mucosa, some with expansion on stromal cells, with negative Doppler signal) ( Figure 5). Very rarely, an agglomeration of Naboth cysts can be seen on ultrasound, which appears as a complex multicystic formation (9).
They are not considered pathological, because they do not present any annoying symptoms.

CERVICAL FIBROIDS
Cervical fibroids are rare (less than 10% of all uterine fibroids). Their origin is probably the isthmic area, because the cervix is almost devoid of muscle fibers. Depending on the direction of growth of these smooth muscle formations, the neighboring symptoms appear (dysuria, pollakiuria, ureteral obstruction, dyspareunia, or obstruction of the endocervical canal). Sometimes, a submucosal uterine fibroid can protrude into the endocervix or even outside, into the vagina.
They are diagnosed during the examination with valves, at the digital vaginal examination or most frequently on imaging examination, by transvaginal ultrasound.
Transvaginal ultrasound shows a hypoechoic, regular, well-defined formation, located in the en-docervical stroma. Depending on its structure (cystic, fatty degeneration, or calcification) the ultrasound appearance may be more complex (Figure 6). Doppler examination can show the uterine origin of fibroids by visualizing the vascular pedicle.

SECONDARY STENOSES OF THE UTERINE CERVIX
Secondary stenoses of the uterine cervix are obstructions in the internal os and may be consecutive to infections, surgery in the area, post-irradiation, and due to postmenopausal or neoplastic atrophy (10). The most common are iatrogenic, occurring after diathermy loop excisions, conization or electrocoagulation of the cervix (11,12).
The symptoms vary depending on the degree of stenosis (complete or incomplete) and the presence or absence of menstruation. Women of reproductive age may have symptoms such as pelvic pain, dysmenorrhea, abnormal bleeding, amenorrhea, or infertility (in the context of secondary endometriosis), while in postmenopause, patients are asymptomatic for a long period of time, then developing hematometra, hydrometra or even pyometra. The diagnosis is established based on clinical suspicion.
Ultrasound examination can detect collections in the uterine cavity. If the stenosis occurred at the level of the "new" external os postoperatively, accumulation of mucus and/ or blood may appear in the endocervical canal (inhomogeneous appearance on ultrasound examination, with negative Doppler signal). Stenosis is demonstrated by the inability to penetrate the cervical canal with a Hegar dilator 1 mm or 2 mm.

CERVICAL ENDOMETRIOSIS
Cervical endometriosis is one of the rarest locations of the disease (0.15-2.5% of cases of endome-triosis). The location can be shallow, on the exocervix, or deeper.
Patients may be asymptomatic or present with pelvic pain, vaginal bleeding, or superficial dyspareunia. In cases of deep, infiltrative endometriosis with evolution towards the cervix, the symptoms can become important, even disabling (severe dysmenorrhea, pelvic pain, deep dyspareunia).
Superficial endometriosis of the uterine cervix is found on examination of the cervix or on colposcopy (bluish-violet lesion, much more obvious during menstruation). There are endometriotic lesions that develop in the depth of the stroma, but also deep, infiltrative lesions that extend from the pelvic cavity to the back of the cervix, pushing it backward or deforming it, which can be suspected at the gynecological examination.
Ultrasound examination describes a formation with a complex structure, quite difficult to differentiate from a polyp or myoma (hypoechoic images, worse delimited (ill-defined)). Association with other endometriotic lesions in the pelvis may aid in establishing the diagnosis (Figure 7).

GLANDULAR PRENEOPLASTIC CERVICAL LESIONS
Diagnosing glandular intraepithelial preneoplastic lesions remains a challenge for any clinician. Severe glandular atypia can coexist in over 50% of cases with squamous lesions (so-called mixed lesions). It should also be emphasized that glandular lesions may not be detected on cytology tests, representing surprises of the pathological diagnosis on the excision sample. These diagnostic difficulties are determined on the one hand by the fact that the exfoliative cytology (Pap test) is superior in diagnosing squamous lesions compared to glandular ones, and on colposcopic examination the lesions may not be visible (located high in the endocervical canal) or, even if they are visible, they are difficult to recognize (13).
Risk factors for high-grade cervical lesions are: immunosuppression (concomitant HIV infection or immunosuppressive medication), smoking (nicotine degradation products present in the cervical mucus decrease local immunity), concomitant genital infectious diseases (Mycoplasma genitalium) and familial factors (familial aggregations are described, without the identification of a specific risk factor).
The main oncogenic factor is persistent high-risk strains HPV infection. Among the known strains, strains 16,18,45,52,35,31, 33 are frequently found in endocervical glandular lesions. Although HPV infection underlies cervical oncogenesis, a viral infection is detected in less than 45% of patients with glandular lesions (14).
For the diagnosis of intraepithelial glandular lesions, the following can be used: cervical cytology by Pap smear, HPV testing, colposcopic examination, confirmation of lesions is mandatory by histopathological examination (the examined samples can be taken by endocervical biopsy curettage or cone biopsy).
It is recommended to complete the examination with a transvaginal ultrasound (thorough examination of the cervix and endometrium), and in patients over 35 years old it is mandatory to perform endometrial cavity curettage, with histopathological examination. Young patients, less than 35 years old, with atypical glandular cytological result and risk factors for chronic anovulation also benefit from examination of the endometrium (15).

INTERPRETATION OF PAP SMEAR RESULTS WITH GLANDULAR AFFECTION
Atypical glandular cells are found in a few Pap smear results (0.4-0.6%), being more common in women over 40 years old. In most cases, they reveal only intraepithelial lesions, but in about 20% of cases, a neoplastic lesion (endocervix, endometrium or endosalpinx) is confirmed, which must be sought and should not be missed under any circumstances.
According to current Bethesda terminology, abnormal cytology results in glandular cells can be the following: atypical glandular cells (AGC) (endocervical cells not otherwise specified (NOS); endometrial cells -NOS; glandular cells -NOS); atypical glandular cells -endocervical cells, in favor of neoplasia (cells have marked changes, but still insufficient to be interpreted as adenocarcinoma); glandular cells, in favor of neoplasia; endocervical adenocarcinoma in situ; adenocarcinoma: endocervical; endometrial; extrauterine; not otherwise specificed (NOS).
From the cytologist's point of view, AGC is a difficult cytological diagnosis. An endometrial cell/ reac-tive endocervical cell/ tubal metaplasia/ cervical endometriosis may mimic adenocarcinoma in situ (AIS). When squamous cell changes coexist, the main substrate is most likely a squamous lesion. If AGC is detected in a patient over 50 years old, endometrial cancer is most commonly diagnosed. If AGC is detected in women under 40 years old, a major squamous lesion (more than CIN 2) can be expected. In patients with repeated AGC NOS, the most likely substrate is endometrial cancer (16).
In addition to intraepithelial or neoplastic lesions, atypical glandular cells may be caused by glandular and squamous reactive and regenerative changes, Arias Stella reaction, cervical polyps, tubal or serous metaplasia, cervical endometriosis, microglandular hyperplasia, or changes associated with an intrauterine device ( Figure 8) (17).  4. Tests that identify the cellular markers of the infection -identification of the double cytological marking p16 and Ki 67 (which allows the identification of cells with disrupted cellular activity, by integrating the viral genome into the host genome -immunocytological test CIN 2+ CINtec PLUS). Sampling is done by exo-and endocervical brushing, with an endocervical brush or spatula. The collected sample is discharged into special containers, which contain the liquid transport medium.
HPV tests detect viral DNA replication. There is a 1-5% rate of false-negative results, explained by smaller lesions. They are also associated with unsatisfactory colposcopies and are more common in women over 40 years old. A negative detection test means no replication of a strain with an increased oncogenic risk. A positive genotyping identifies the type, further allowing the assessment of the risk of developing cervical intraepithelial neoplasia. A HPV test can be negative if the patient has a latent infection, but which, against a background of decreased immunity, can be activated at any time.

COLPOSCOPIC EXAMINATION
Colposcopic examination is often difficult, even technically limited, due to the non-visualization of the squamocolumnar junction (SCJ). It cannot make the difference between AIS and adenocarcinoma. Glandular lesions can mimic any other lesion, there are often associations with squamous lesions. The point of reference is that the AIS lesions are in the vicinity of the SCJ. The suggestive colposcopic aspects are: disappearance of the villous structure, flattening, whitening -very similar to immature metaplasia; mottled appearance, "white alternating with red" after the application of acetic acid -similar to an area of immature transformation; intense acetowhite, elevated appearance, at distance from SCJ, somehow isolated.
The following can also be observed: papillary growths, confluent papillae, irregular papillary projections, vascular axis visible in the papilla, with the appearance of a loop (sagittal) or thick spots, papillae that converge in dense acetowhite areas. Vascular changes are quite characteristic: suggestive aspects of "thread heads" (vessels that come from nowhere and go nowhere), hanging plants tendrils, roots (sometimes with more dilated, tuberous areas), hieroglyphs, small and many dots (the tips of the vascular loops in the flattened papillae). Extremely difficult to diagnose are lesions from the depths of the glandular crypts. In mixed lesions, the squamous lesion or the metaplastic epithelium may cover the glandular lesion in the depth of the crypt, and frequently these lesions may have neither cytological nor colposcopic expression (15).

ADENOCARCINOMA IN SITU
It is considered a precursor of cervical adenocarcinoma (premalignant glandular lesion). The average age at which it is diagnosed is 36.9 years, declining much in the last decade. Its incidence is increasing and is probably due to the increased incidence of persistent HPV infection with strain 16 and/ or 18. The indirect role of combined oral contraception is possible but still debatable. It rarely starts with vaginal bleeding (18).
The diagnostic methods are cytology, HPV testing, colposcopic examination, the certainty diagnosis is histopathology on the biopsy sample (fragmentary biopsy, cervical conization, or endocervical curettage).
AIS is most often revealed by cytology (AGC 50-70%, atypical squamous cells (25-30%, mixed 15%), but there are also adenocarcinomas in situ with negative cytology (5%) (19). The lesion may originate anywhere in the transformation zone, extending to the endocervix; 10-15% of patients have multifocal lesions with normal epithelial areas frequently exceeding 2 mm or lesions may be located high in the endocervical canal and may be missed on cytological examination (deep in the endocervical crypts).
Cervical conization is indicated primarily for diagnostic purposes, in the following situations: AIS cytology or adenocarcinoma, with biopsy and negative endocervical curettage; AGC cytology, in favor of neoplasia, with biopsy, negative endocervical and endometrial curettage; persistent AGC cytology, with negative biopsy results, endocervical and endometrial curettage.
Cervical conization should be performed classically, with the scalpel, so as not to have any type of electrical artefact. This way, there is a possibility of excising a single conical sample for the pathologist. On the other hand, it can sometimes be a surprise diagnosis after a wide diathermy loop excision. The procedure must always be completed with curettage of the remaining endocervix, and, in patients over 35 years old, also with endometrial curettage.
If the edges of the conical sample are negative, there is still a risk of 20% of residual AIS and a risk of 1% of adenocarcinoma, which require an extremely rigorous supervision of patients after conization (20). Cytology and endocervical brushing should be repeated every 6 months, given that colposcopy often becomes technically unsatisfactory.

CONCLUSIONS
Endocervical pathology is varied. It can be correctly diagnosed starting from the clinical picture, completed with laboratory investigations, bacteriological examinations, Pap smear test, molecular tests for the diagnosis of HPV infection, colposcopy, but also by thorough ultrasound examinations. Ultrasound examination of the cervix should be part of the routine examination, because its systematic evaluation can significantly contribute to refining the diagnosis.