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Structure and histology of the cervical TZ. A. Schematic representation of the cervix showing the TZ between ectocervix and endocervix. B. Histology of the cervical TZ showing the stratified squamous epithelium and underlying Nabothian cysts. C. Schematic showing the surface features of ectocervix, endocervix and TZ that aid in tissue dissection. The ectocervix is easily identified because the surface is smooth, white, and shiny with no mucous. The endocervix surface is rough, red in color, and covered with mucous. The TZ contains Nabothian cysts (swollen glands due to occlusion of ducts by squamous metaplasia). These large cysts are easily visible and diagnostic for the TZ. D. Photograph of a typical cervical specimen showing each region. https://doi.org/10.1371/journal.pone.0199761.g001
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Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for cervical cancer. Greater than 90% of these cancers originate in the cervical transformation zone (TZ), a narrow region of metaplastic squamous epithelium that develops at the squamocolumnar junction between the ectocervix and endocervix. It is unclear why the...
Citations
... The metaplastic epithelium of the TZ, including the SCJ, is the prime target of HPV infection and cervical carcinogenesis [29]. The susceptibility of this region to HPV infection is possibly due to decreased immune responsiveness and increased estrogen responsiveness associated with an increased level of estrogen receptors [30]. ...
One pillar of the predictive, preventive, and personalized medicine framework strategies is the female health. The evaluation of women’s lifestyle and dietary habits in context with genetic and modifiable risk factors may reflect the prevention of cervical cancer before the occurrence of clinical symptoms and prediction of cervical lesion behavior. The main aim of this review is to analyze publications in the field of precision medicine that allow the use of research knowledge of cervical microbiome, epigenetic modifications, and inflammation in potential application in clinical practice. Personalized approach in evaluating patient’s risk of future development of cervical abnormality should consider the biomarkers of the local microenvironment characterized by the microbial composition, epigenetic pattern of cervical epithelium, and presence of chronic inflammation. Novel sequencing techniques enable a more detailed characterization of actual state in cervical epithelium. Better understanding of all changes in multiomics level enables a better assessment of disease prognosis and selects the eligible targeted therapy in personalized medicine. Restoring of healthy vaginal microflora and reversing the outbreak of cervical abnormality can be also achieved by dietary habits as well as uptake of prebiotics, probiotics, synbiotics, microbial transplantation, and others.
... These include cells from the human ectocervix zone, endocervix zone cultured in vitro, and the human papillomavirus (HPVs, involving HPV16 E6 and E7) immortalized human cervical transformation zone cells. The cell differentiation was induced in the medium with 1.4 mM calcium chloride to induce differentiation [25]. Under the induction, multiple cell morphologies were shown to vary in size and shape, and cell debris was easily spotted. ...
Cell segmentation is a critical step for image-based experimental analysis. Existing cell segmentation methods are neither entirely automated nor perform well under basic laboratory microscopy. This study proposes an efficient and automated cell segmentation method involving morphological operations to automatically achieve cell segmentation for phase-contrast microscopes. Manual/visual counting of cell segmentation serves as the control group (156 images as ground truth) to evaluate the proposed method’s performance. The proposed technology’s adaptive performance is assessed at varying conditions, including artificial blurriness, illumination, and image size. Compared to the Trainable Weka Segmentation method, the Empirical Gradient Threshold method, and the ilastik segmentation software, the proposed method achieved better segmentation accuracy (dice coefficient: 90.07, IoU: 82.16%, and 6.51% as the average relative error on measuring cell area). The proposed method also has good reliability, even under unfavored imaging conditions at which manual labeling or human intervention is inefficient. Additionally, similar degrees of segmentation accuracy were confirmed when the ground truth data and the generated data from the proposed method were applied individually to train modified U-Net models (16848 images). These results demonstrated good accuracy and high practicality of the proposed cell segmentation method with phase-contrast microscopy image data.
... Cell culture system Tissue Model Reference a 2D Ovaries Tissue-derived ovarian surface epithelial cultures [7][8][9] Immortalized ovarian surface epithelial cultures [10][11][12] Fallopian tubes Tissue-derived epithelial cultures [13] Immortalized epithelial cultures [14][15][16] Endometrium Tissue-derived epithelial cultures [17][18][19] Tissue-derived stromal cultures [18,20] Tissue-derived endothelial cultures [21][22][23] iPSC-derived stromal cultures [24] Carcinoma-derived cell lines (HEC-1, Ishikawa, RL95-2, St-1b, ECC-1) [25][26][27][28][29] Cervix Tissue-derived epithelial cultures [30-32, 34, 35] HPV16-immortalized epithelial cell line [36] Carcinoma-derived cell lines (HeLa,SiHa,C33a, CaSki,ME-180) [33,37] 3D Ovaries Tissue-derived epithelial spheroid cultures [38,39] Fallopian tubes Tissue-derived epithelial spheroid cultures [40] iPSC-derived epithelial cell 3D cultures [41] Endometrium Tissue-derived epithelial cell 3D cultures [42] Organotypic cultures from endometrial tissue [43][44][45][46] Mesenchymal-derived epithelial-like cell 3D cultures [47] Spheroids derived from endometrial adenocarcinoma cell lines [48] Spheroids of mesenchymal stem cells derived from menstrual blood/endometrial fragments [49] Cervix Organotypic cultures from primary cervical epithelial cells [50,51] Organotypic cultures from HPV16immortalized cells [50] Tissue explants Ovaries Ovarian tissue explants [52] Fallopian tubes Fimbria explants [53,54] Endometrium Non-pregnant endometrium explants [55,56] 1st trimester decidua parietalis explants [57,58] Cervix Cervical explants [59] A summary of the different in vitro tools available for studying the biology of human FRT (excluding the recently derived organoid models). The models have been grouped according to the culture type: 2D monolayer cultures, 3D models and tissue explants. ...
Both the proper functioning of the female reproductive tract (FRT) and normal placental development are essential for women’s health, wellbeing, and pregnancy outcome. The study of the FRT in humans has been challenging due to limitations in the in vitro and in vivo tools available. Recent developments in 3D organoid technology that model the different regions of the FRT include organoids of the ovaries, fallopian tubes, endometrium and cervix, as well as placental trophoblast. These models are opening up new avenues to investigate the normal biology and pathology of the FRT. In this review, we discuss the advances, potential, and limitations of organoid cultures of the human FRT.
... Primary cervical cells (both keratinocytes and fibroblasts from the uterine cervix) are now commercially available. The decision on which cells to use may be relevant due to tissue region susceptibility differences [56], tissue microenvironments, and variations in signalling affecting differentiation and possibly the viral life cycle and immune environment. Components of the dermal equivalent (typically collagen and fibroblasts), making up the ECM may also be relevant factors. ...
Persistent infection with oncogenic human papillomavirus (HPV) may lead to cancer in mucosal and skin tissue. Consequently, HPV must have developed strategies to escape host immune surveillance. Nevertheless, most HPV infections are cleared by the infected host. Our laboratory investigates Langerhans cells (LCs), acting at the interface between innate and adaptive immunity. We hypothesize that this first line of defence is vital for potential HPV elimination. As an alternative to animal models, we use smaller-scale epithelial organoids grown from human primary keratinocytes derived from various anatomical sites. This approach is amenable to large sample sizes—an essential aspect for scientific rigour and statistical power. To evaluate LCs phenotypically and molecularly during the viral life cycle and onset of carcinogenesis, we have included an engineered myeloid cell line with the ability to acquire an LC phenotype. This model is accurately tailored for the crucial time-window of early virus elimination in a complex organism and will shed more light on our long-standing research question of how naturally occurring HPV variants influence disease development. It may also be applied to other microorganism–host interaction research or enquiries of epithelium immunobiology. Finally, our continuously updated pathogen–host analysis tool enables state-of-the-art bioinformatics analyses of next-generation sequencing data.
This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’.
... Betagal staining kit (Invitrogen, Carlsbad, CA) was used to test the existence of beta-galactosidase in senescent cells. Contamination with HPV was assessed using PCR (Deng, Hillpot, Yeboah, Mondal, & Woodworth, 2018). A step-by-step protocol for isolation and growth of cervical cells is presented in the appendix of this manuscript. ...
... Collagen rafts were then transferred to 100 mm cell culture dishes and 1 × 10 6 normal cervical epithelial cells or HPV-16 immortalized cervical cells (Deng, Hillpot, Yeboah et al., 2018) were seeded on the surface of each raft. The epithelial cells were allowed to attach to rafts at 37°C for 2 hr, then 15 ml of raft culture medium (McCance, Kopan, Fuchs, & Laimins, 1988) was added to submerge the rafts as described (Woodworth et al., 2005). ...
Cervical cancer is a major public health problem and research using cell culture models has improved understanding of this disease. The human cervix contains three anatomic regions; ectocervix with stratified squamous epithelium, endocervix with secretory epithelium, and transformation zone (TZ) with metaplastic cells. Most cervical cancers originate within the TZ. However, little is known about the biology of TZ cells or why they are highly susceptible to carcinogenesis. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, TZ or endocervix. We examined the effects of different serum‐free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. We also optimized conditions for organotypic culture of cervical epithelial cells using collagen rafts with human cervical stromal cells. Finally, we present a step‐by‐step protocol for culturing cells from each region of human cervix.
... HPV16-immortalized epithelial cells from TZ and endocervix become more dysplastic than ectocervical cells. We tested a series of HPV16-immortalized cell lines derived from ectocervix, TZ, and endocervix of eight different patients 19 . We used each cell line at approximately 80 to 95 population doublings or 25 to 30 passages. ...
... HPV16-immortalized cell lines used for this work have been characterized previously 19 . Most were derived by infection with retroviruses encoding HPV16 E6/E7 genes (18 lines from 6 patients) and contain integrated rather than episomal genomes. ...
... Recent studies describe a discrete population of squamocolumnar junction cells (SC) that have a specific gene expression profile and that may be progenitors for cervical cancer 32 . We detected one of these proteins (MMP-7) in 9 to 13% of cells cultured from TZ or endocervix 19 , but MMP-7 was also expressed at a low level in cells from ectocervix. Cytokeratin 17 (K17) and p63 are two additional markers for reserve cells of TZ and endocervix 21 . ...
A major risk factor for cervical cancer is persistent infection with high-risk human papillomaviruses (HPV) which can cause cervical intraepithelial neoplasia. Greater than 90% of cervical cancers develop in the transformation zone (TZ), a small region of metaplastic squamous epithelium at the squamocolumnar junction between endocervix and ectocervix. However, it is unclear why this region is highly susceptible to malignant progression. We hypothesized that cells from TZ were more susceptible to dysplastic differentiation, a precursor to cervical cancer. We used three-dimensional organotypic culture to compare differentiation of HPV16-immortalized epithelial cell lines derived from ectocervix, TZ, and endocervix. We show that immortal cells from TZ or endocervix form epithelia that are more dysplastic than immortal cells from ectocervix. A higher percentage of immortal cells from TZ and endocervix express the proliferation marker Ki-67 and are positive for phospho-Akt. Immortal cells from TZ and endocervix invade collagen rafts and express increased levels of matrix metalloproteinase-1. Inhibition of MMP-1 or Akt activity blocks invasion. We conclude that HPV16-immortalized cells cultured from TZ or endocervix are more susceptible to dysplastic differentiation, and this might enhance their susceptibility to cervical cancer.
If not eliminated by the immune system and persisting over years, oropharyngeal high-risk HPV infection can lead to cancer development in the oropharynx. HPV infection is very commonly found in the genital region and can serve as an HPV reservoir. In this study, we investigate whether women with a genital HPV infection are at a higher risk of harboring an undetected oropharyngeal HPV infection via genital–oropharyngeal transmission.
Women presenting for routine gynecological checkups were included in this study. All participants received an HPV brush test from the genital region as well as from the oropharynx. Additionally, probable risk factors for an HPV infection were assessed in a structured questionnaire.
142 women were included in this study. The rate of oropharyngeal HPV infection was low with 2/142 (1,4%) women positive for a low-risk HPV genotype. In the genital brush test, 54/142 (38%) women were tested HPV positive of which 41/142 (29%) were positive for a high-risk HPV genotype.
The rate of an oropharyngeal HPV detection in our population was low with 2/142 women harboring a low-risk HPV infection.
The maladies affecting the female reproductive tract (FRT) range from infections to endometriosis to carcinomas. In vitro models of the FRT play an increasingly important role in both basic and translational research, since the anatomy and physiology of the FRT of humans and other primates differ significantly from most of the commonly used animal models, including rodents. Using organoid culture to study the FRT has overcome the longstanding hurdle of maintaining epithelial phenotype in culture. Both ECM-derived and engineered materials have proved critical for maintaining a physiological phenotype of FRT cells in vitro by providing the requisite 3D environment, ligands, and architecture. Advanced materials have also enabled the systematic study of factors contributing to the invasive metastatic processes. Meanwhile, microphysiological devices make it possible to incorporate physical signals such as flow and cyclic exposure to hormones. Going forward, advanced materials compatible with hormones and optimised to support FRT-derived cells' long-term growth, will play a key role in addressing the diverse array of FRT pathologies and lead to impactful new treatments that support the improvement of women's health.
Statement of Significance
: The female reproductive system is a crucial component of the female anatomy. In addition to enabling reproduction, it has wide ranging influence on tissues throughout the body via endocrine signalling. This intrinsic role in regulating normal female biology makes it susceptible to a variety of female-specific diseases. However, the complexity and human-specific features of the reproductive system make it challenging to study. This has spurred the development of human-relevant in vitro models for helping to decipher the complex issues that can affect the reproductive system, including endometriosis, infection, and cancer. In this Review, we cover the current state of in vitro models for studying the female reproductive system, and the key role biomaterials play in enabling their development.
