These data support the hypothesis that NPM IHC, together with HPV RNAScope, could be used as a predictor of transcriptionally active HPV presence and tumor progression, which is useful for therapy decisions. This study includes a small cohort of patients and, cannot report conclusive findings. Further studies with large series of patients are needed to support our hypothesis.Most oropharyngeal squamous cell carcinomas (OPSCCs) are human papillomavirus (HPV)-associated, high-risk (HR) cancers that show a better response to chemoradiotherapy and are associated with improved survival. Nucleophosmin  is a nucleolar phosphoprotein that plays different roles within the cell, such as ribosomal synthesis, cell cycle regulation, DNA damage repair and centrosome duplication. NPM is also known as an activator of inflammatory pathways. An increase in NPM expression has been observed in vitro in E6/E7 overexpressing cells and is involved in HPV assembly. In this retrospective study, we investigated the relationship between the immunohistochemical (IHC) expression of NPM and HR-HPV viral load, assayed by RNAScope in situ hybridization (ISH), in ten patients with histologically confirmed p16-positive OPSCC. Our findings show that there is a positive correlation between NPM expression and HR-HPV mRNA (Rs = 0.70,  Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are the most common types of HNSCC ,2,3. It Patients with HPV-induced OPSCC have been shown to be younger and are less likely to have a history of tobacco or alcohol use than patients with HPV-negative OPSCCs. A superior socioeconomic status and sexual behaviors have also been associated with the development of HPV-positive OPSCC . PatientThe HPV genome consists of a small double-stranded DNA and includes three major regions. The early genes (E1-E7) are expressed early in the viral infectious cycle for the regulation of transcription, plasmid replication and transformation. They encode the E6 and E7 oncoproteins, which are responsible for tumorigenesis . MoreoveThe nucleolus is known to have a central role in the interaction between the virus and the infected cell. Indeed, several viruses, including HPV, have evolved molecular strategies to take control of nucleolar functions, ultimately favoring viral replication and maturation, such as ribosomal biogenesis, cell cycle progression and apoptosis .The E7 protein has been colocalized with pRB in the nucleolus . It inte2 with concomitant expression of p16INK4a have been shown to be useful in the identification of high-risk human papillomaviruses (HR-HPV) for the development of cervical cancer [In keeping with the nucleolar role of HPV infection, changes in numbers or in mor-phometric parameters of silver-stained nucleolar organizer regions (AgNORs) have been previously proposed as useful predictors of worse prognosis in different forms of HPV- infected squamous cell carcinomas ,23,24,25l cancer .Nucleophosmin  is a nucleolar phosphoprotein that plays different roles within the cell, such as ribosomal synthesis, cell cycle regulation, repair of DNA damage and centrosome duplication .NPM interacts with different proteins from RNA and DNA viruses, including HIV, HCV, HBV and HPV, and affects the infection process . In factTherefore, NPM is required for proliferation and inhibition of differentiation in primary keratinocyte cells expressing HPV’s E6 and E7 . The hosGiven the importance of the nucleolus in HPV infection and the pivotal role of the nucleolar protein NPM in the replicative capacity of HPV-infected cells, the aim of this study is to elucidate the relationship between NPM and the HR-HPV viral load, assessed by RNAScope, in p16-positive HPV-associated OPSCC.All cases show non-keratinized  and mixed-type  SCC morphology  and posiRNAScope-ISH revealed positive staining for E6/E7 mRNA HR-HPV a,c. TherIn a similar way, we found that NPM IHC positivity was also variable in FFPE tissue. Some cases had low IHC positive staining, whereas others showed intense areas of positive staining b,d.p = 0.03)  . Moreove = 0.03) . Our datThe majority of the OPSCCs are HR-HPV-dependent and present a good clinical outcome in terms of chemoradiotherapy sensitivity compared with HPV-negative HNSCC . p16 is However, approximately 20% of p16-positive OPSCC patients may lack transcriptionally-active HR-HPV ,38,39,40In addition, a positive immunohistochemical reaction with p16 shows the presence or absence of HR-HPV infection and does not assess the amount of the viral load. Assuming that the amount of HPV viral load does establish the progression of the disease, then the p16 immunohistochemical test could not define the aggressiveness of the disease. RNAScope complementary to HPV E6/E7 mRNA is a molecular technique, which allows visualizing viral transcripts in FFPE tissue. HPV-RNAScope demonstrates high sensitivity and specificity for detection of HPV infection and has been proposed as the clinical standard for assigning a diagnosis of HPV-positive OPSCC ,42,43,44Viral proteins often target the nucleolus, nucleolar proteins are released into the nucleoplasm and cytoplasm, and in most cases, viral nucleic acids are localized in the nucleolus. On the other hand, cellular proteins can be relocated to the nucleolus and participate in the infectious process . NucleolThe expression of different biomarkers has been linked to HPV carcinogenesis. Given the importance of the nucleolus, different nucleolar proteins have been used to determine the progression of HPV infection. For example, PITC1  and nuclIn keeping with the nucleolar role of HPV infection, the numbers and the morphometric parameters of AgNORs have been proposed as useful predictors of worse prognosis in different forms of HPV-infected squamous cell carcinomas ,23,24,25Further, hyperchromasia, thickening of nuclear contour, and prominent nucleoli on Pap smear have been used as indicators of intraepithelial neoplasia grade 1 (CIN1) or greater CIN 2–3 . MoreoveSince NPM is a nucleolar protein that is frequently overexpressed in different types of tumours , we inveOur results showed a positive correlation and a linear relationship between the expression of NPM and HPV E6/E7 mRNA. These data support the hypothesis that NPM could be used as a predictor of transcriptionally active HPV presence and possibly tumor progression.NPM up-regulation is required for the inhibition of differentiation and promotion of proliferation in differentiating cells, which express E6–E7 . In addiWe previously showed that NPM intracellular protein level is increased by inflammatory cytokines and is released into the extracellular space by fibroblasts and primary keratinocytes upon cytokine stimulation associated with psoriasis .Interestingly, circulating NPM is upregulated in the plasma of psoriatic patients and positively correlated with the severity of the disease and some determinants of cardiovascular risk .Moreover, we demonstrated that NPM acts as an alarmin after genotoxic stress treatment and, when it is secreted in the extracellular space, induces inflammation by binding the Toll-Like Receptor 4 (TLR4) receptor. This interaction activates a signaling pathway that leads to the translocation of NF-kB in the nucleus with consequent induction of inflammatory genes, such as interleukin 6 (IL-6) and cyclooxygenase 2 (COX-2) .We know that an increased level of reactive oxygen and nitrogen species (ROS and RNS), produced during chronic inflammation, play a pivotal role in tumor progression, and it is established that NPM is a modulator of the cellular oxidative stress response and an activator of inflammation pathway. Given the fact that inflammation-mediated DNA damage provides a potential mechanism of HPV integration and that an excessive amount of ROS and RNS promote DNA damage , it woulTargeting a single host protein to simultaneously impact different steps of viral replication is an appealing solution. Since NPM induction increases viral replication and also cell cycle progression, targeting NPM might simultaneously impact on viral replication and on the neoplastic state that emerges from it .Different groups are currently studying the development and preclinical/clinical evaluation of NPM inhibitors. Among these, only two have been evaluated in Phase I/II cancer clinical trials, the CIGB-300 peptide in HPV-positive cervical cancers  and the Moreover, in cervical cancers treatment, 75% of the patients showed tumor reduction at colposcopy and 19% exhibited full histological regression. In addition, HPV DNA was negative in 48% of the previously positive patients. Long-term follow-up did not show adverse events or recurrences .Since we demonstrated in previous papers that NPM could be considered an extra-cellular alarmin, the circulating expression of NPM could also be exploited in future applications as a predictor of HPV viral load by salivary or blood test screening in OPSCC.In conclusion, our results show that a combination of tests, immunohistochemistry of NPM and HR-HPV RNAScope-ISH could be used in routine practice to stratify patients with HPV-positive OPSCC into a group with relatively stable disease and a group with the progression of malignancy. This finding potentially could have clinical implications because NPM inhibition could offer therapeutic benefits to these patients.This study includes a small cohort of patients and, by definition, cannot be regarded as yielding conclusive findings. Further studies with a larger cohort of patients are needed to support this hypothesis.Ten cases of OPSCC were retrieved from archives of Histopathology Division of San Carlo di Nancy Hospital and Istituto Dermopatico dell’Immacolata (IDI-IRCCS), Rome-Italy. Haematoxylin and eosin (H&E) stained slides obtained from formalin-fixed paraffin-embedded (FFPE) lesion tissue were examined.Leica Stainer Immunohistochemistry System was used for the p16 antibody . Appropriate positive and negative control tissue was used.2O2 as a substrate and samples were counterstained with hematoxylin. As a negative control, primary Abs were omitted. Stained sections were analyzed with the AxioCam digital camera coupled to the Axioplan 2 microscope . NPM staining intensity was evaluated by quantitative analysis (Image J color deconvolution) in three adjacent fields of each section by two independent observers, not aware of the status of the specimens. The mean values for each patient’s section expressed as arbitrary units (A.U.) of NPM IHC positivity area were used for correlation and regression analyses.NPM IHC was performed on FFPE tissue as previously described . BrieflyRNAscope-ISH was carried out on sections obtained from FFPE tissue. Bond III Automated. ISH for HR-HPV E6/E7 mRNA was performed using the RNAscope 2.5 VS HPV HR18 probe , which recognizes HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, genotypes. ISH was performed on an automated platform  by using the RNAscope VS Universal HRP assay , according to the manufacturer’s instructions. Positive staining has been identified as brown, punctate dots present in the nucleus and/or cytoplasm. Control probes for the bacterial gene 4-hydroxy-tetrahydrodipicolinate reductase (DapB), negative control, and for the housekeeping gene ubiquitin C (UbC), positive control for the evidence of preserved RNA, have also been included for each case.Because of the novelty of the study, whose primary objectives are mainly descriptive and exploratory, the minimum sample size has not been pre-determined.p < 0.05 was considered statistically significant.Spearman correlation analysis and linear regression analysis were carried out using GraphPad Prism . A


Participants’ mean age was 47.1 years (SD: 14.4). Most were female (74.1%), were married/partnered (51.7%), had primary education (63.8%), and identified as Kalinago (72.4%). Whereas 79.5% had heard of cervical cancer, few had heard of HPV (19.6%) or the HPV vaccine (21.8%). Among those who knew someone with cancer, 90.9% had heard of the HPV vaccine, compared with only 9.1% of those who did not know anyone with cancer (p = 0.02). Access to HPV vaccination is an immediate, cost-effective cancer prevention priority for reducing the disproportionate burden of HPV-related cancers, particularly cervical cancer, in the Caribbean. We recommend culturally targeted education interventions to improve knowledge about HPV vaccination and the link between HPV and cervical cancer.Caribbean women experience a cervical cancer incidence rate that is three times higher than that among their North American counterparts. In this study, we performed a needs assessment of the knowledge and awareness of HPV, HPV vaccination, and cervical cancer and receipt of cervical cancer screening among an indigenous Caribbean community. We purposively recruited individuals aged ≥18 from a community health care clinic ( Cervical cancer is the fourth-leading cause of cancer-related mortality among women worldwide and one of the most common malignancies among women in lower-income countries . Given iThe burden of cervical cancer in LIMCs is not evenly distributed, with some of the highest incidence occurring in indigenous communities. In general, cancer is the second-leading cause of death among indigenous people, and their survival is lower than that among non-indigenous individuals . This hiAbout 10% of the Caribbean population identifies as an indigenous race or ethnicity . HoweverTo the best of our knowledge, no studies have evaluated knowledge and awareness of HPV, HPV vaccination, or cervical cancer prevention and screening among indigenous Caribbean communities. Therefore, in this study, we developed a community partnership to assess the knowledge and awareness of preventable cancers among an Indigenous community in Dominica, West Indies. Evaluating disparities by social grouping, such as identification with an indigenous community, is a common approach to studying inequalities in health . InformeThe health belief model (HBM) provided a theoretical framework for this study. The HBM is a health behavior framework, first conceptualized by the United States Public Health Service in the 1950s, that has been widely used in public health research to describe barriers to the acceptance of health prevention and preventive screenings among adults. The HBM incorporates individual perceptions, modifying factors, and the likelihood of action to explain barriers to preventive health behaviors . The HBMThis study was a community-based participatory needs assessment of cancer prevention education. Three community partners collaborated on the development, design, and implementation of this study. The partnership consisted of local health care staff from a community clinic, including a nurse and a nursing assistant, a student-led nonprofit group consisting of first- and second-year medical students, and university research faculty and staff. The community partners met multiple times prior to the study to plan the research design, the recruitment, and the study materials . Power calculations were not conducted because the goal of this study was to establish a community partnership, and research design and sampling were based on the direction of the Kalinago tribal leadership. Nonetheless, small-sample research is highly valuable despite its limited generalizability . This coPurposive sampling occurred through a community health care clinic from 2015 to 2016 at bimonthly clinic visits. The clinic serves a population of approximately 3000 community members, the majority of whom are descendants and/or members of an indigenous tribe. A cross-sectional survey see  was admiThe surveys were completed using pen and paper by trained members of the research team based on oral responses from the participants. The survey responses were then manually entered into Excel by two members of the research team. Double data entry occurred for all surveys to validate data quality.Sociodemographic variables included sex , age , relationship status , ethnicity , education , household income (<$5000 Eastern Caribbean dollars (ECD)/<$1872 USD, ≥$5000 ECD/≥$1872 USD representing 25% of the average household income in Dominica), health insurance status , birth country , and language ). Cancer history variables included having a personal history of cancer or an immediate family member with cancer or knowing anyone with cancer .Three categories of outcomes were evaluated: cervical cancer awareness, HPV awareness and knowledge, and HPV vaccine awareness and knowledge. Cervical cancer awareness was assessed only among women with the following three questions: Have you heard of cervical cancer? . Do you know what a Pap smear is? . What do you think the likelihood is of you getting cervical cancer? . HPV awareness and knowledge were assessed in all participants with the following questions: Have you heard of human papillomavirus or HPV? HPV is not the same as HIV . HPV is able to cause cervical cancer . Most people have HPV at some point in their lives . HPV vaccine awareness and knowledge were assessed for all participants with the following questions: Before today, have you heard of the HPV vaccine ? . The HPV vaccine has 1 dose . All awareness questions were dichotomized as yes vs. no/don’t know, and all knowledge questions were dichotomized as correct vs. incorrect/don’t know. There were some missing responses, and these are noted in the tables.n = 5) tests were performed to evaluate sociodemographic and cancer history correlates with cervical cancer, HPV, and HPV vaccination awareness and knowledge outcomes. Descriptive and inferential statistics were calculated in Stata 13.1 .Descriptive statistics were calculated for sociodemographic and cancer history factors. Chi square and Fisher Exact (for cells < The mean age of the 58 participants was 47.1 years (SD: 14.4). In Whereas 79.5% of female participants had heard of cervical cancer, nearly all had heard of a Pap smear 92%, . All womp = 0.02). There was limited knowledge among participants about the contribution of HPV to cervical cancer (17%) and about the fact that most people get HPV at some point .Among all participants, few had heard of HPV (19.6%) or the HPV vaccine 23%, . A higheCervical cancer is a current public health problem among indigenous peoples in LAC. Despite the fact that the cervical cancer incidence in this region is among the highest in the world, published research in the Caribbean demonstrates a general lack of awareness of cervical cancer, HPV, and the HPV vaccine. In contrast, participants in our study had a relatively high awareness of cervical cancer, and this is likely due to the robust community health education provided at the community clinic from which our sample was drawn. While most participants had heard of cervical cancer, those who had not were in the prime age range for developing cervical cancer, suggesting that targeted efforts may be necessary among women at the ages at which they are most likely to develop cervical cancer. The possibility for reducing cervical cancer health disparities among indigenous communities in LAC is now further underscored by the availability of efficacious HPV vaccines. Our findings emphasize the need for culturally targeted HPV and HPV vaccine educational campaigns, vaccination programs, and government support.The HPV vaccine was not readily available in this community at the time of the study. Nonetheless, the cost-effectiveness of HPV vaccination far outweighs any other prevention method because treatment for cervical cancer is not readily available or easily accessed in this community; thus, individuals must travel internationally to receive it. Individuals living in this community therefore may face extreme financial hardship in trying to find the means to receive treatment for cervical cancer, even with early detection, which further emphasizes the critical need for HPV vaccine availability. Furthermore, the HPV vaccine also protects against other cancers and HPV-related morbidity. It is available for boys and girls, and the WHO recommends vaccinating both to reduce the heavy burden of HPV. Low knowledge of the HPV vaccine is an issue that needs to be addressed, but the most important next step is making the HPV vaccine readily available. To measure the effectiveness of vaccine programs, there is a need to measure national incidences of cervical cancer and other HPV-related cancers as well as the incidence of common cervical cancer screenings. We recommend future concerted efforts from community and tribal leadership, politicians, and external cancer and vaccine agencies to expand access to the HPV vaccine and implement vaccine registries in Caribbean communities.The HPV vaccine remains the most cost-effective tool for the prevention of cervical cancer, yet most participants in this study were unfamiliar with HPV and the HPV vaccine. The reason for the limited familiarity with the HPV vaccine in LAC communities, including the rural indigenous community we surveyed, may be limited access to vaccines in general and particularly to the HPV vaccine. Access to HPV vaccination is an immediate, cost-effective cancer prevention priority for reducing the disproportionate burden of HPV and cervical cancer among indigenous LAC communities. Therefore, we recommend a culturally tailored education program that is supported by local and national governing boards to help improve knowledge of HPV vaccination and the link between HPV and cervical cancer. These programs should prioritize HPV vaccination for cervical cancer prevention and may be the most cost-effective method for reducing cervical cancer health disparities in indigenous LAC communities such as the one represented by our findings . Future There are limitations that should be considered when interpreting our findings. First, the cross-sectional nature of this study precludes our ability to assess changes in the knowledge and awareness of cervical cancer, HPV, and the HPV vaccine. However, we were able to show the burden that the lack of knowledge and resources may have on this population, helping to shed light on the need for intervention. Our sample was recruited from the only health care clinic in this community. Although this means that any adult seeking care for any reason would have been identified and offered an opportunity to participate, individuals who did not seek care from the clinic were not included. The intention was to expand the survey recruitment through door-to-door recruitment by research assistants accompanied by trusted community and tribal leaders. However, these recruitment efforts were rendered impossible due to a natural disaster (hurricane), which also delayed data analysis while members of the study team were evacuated from the region. From a demographic perspective, our sample is overwhelmingly female; thus, our results may not accurately represent the knowledge and awareness of males in this community. However, given historical associations of cervical cancer and HPV with the female gender, we expect that knowledge and awareness is likely lower among males than females, further underscoring the need for community-wide culturally tailored information for both males and females. Another limitation worth noting is that our results may not be generalizable. However, as the country we recruited from is not the only LAC community impacted by HPV and cervical cancer, we strongly feel that our results may still help other populations build a foundation to conduct needs assessments to see where the gaps in their countries’ programs may lie. Our small sample size does not have the power to detect statistically significant differences in multivariable regressions; thus, we are limited in our ability to perform more complex analyses to predict associations of sociodemographic and cancer factors with our outcomes. Finally, there are limitations in the use of the HBM to examine knowledge and attitudes regarding cancer and cancer prevention among indigenous communities because it does not take into account the influence of the geographic setting and structural barriers that potentially influence likelihood of action  . This thDespite their having some of the highest incidence and mortality rates associated with cervical cancer in the world, there is a distinct gap in the literature on cervical cancer, HPV, and HPV vaccine awareness and knowledge among indigenous LAC communities. This study is a first step toward elucidating the need to improve clinical services and educational opportunities for preventing HPV infection and the development of cervical cancer, among other HPV-related diseases. Improving HPV vaccination (knowledge/awareness/receipt) among indigenous Caribbean communities is an important step toward reaching the UICC and WHO goal of equal access to preventive care to reduce premature cancer deaths by 25%.


Garcinia mangostana, has been shown to have promising anticancer properties in many types of cancer. However, the therapeutic potential of α-mangostin has been limited so far due to its poor aqueous solubility and low oral bioavailability, which limited its biopharmaceutical applications. Furthermore, α-mangostin failed to specifically reach tumors at a therapeutic concentration due and rapid elimination in vivo. We hypothesized that this drawback could be overcome by loading the drug within a delivery system conjugated to transferrin (Tf), whose receptors are overexpressed on many cancer cells and would enhance the specific delivery of α-mangostin to cancer cells, thereby enhancing its therapeutic efficacy. The objectives of this study were therefore to prepare and characterize transferrin-conjugated lipid-polymer hybrid nanoparticles (LPHN) entrapping α-mangostin, as well as to evaluate their therapeutic efficacy in vitro. We successfully prepared α-mangostin loaded LPHN using a one-step nanoprecipitation method with high drug entrapment efficiency. The conjugation of Tf to the LPHN was achieved by using the thiol–maleimide “click” reaction, leading to an increase in the particle hydrodynamic size of Tf-LPHN compared to that of unconjugated (control) LPHN (Ctrl-LPHN). Both Tf-LPHN and Ctrl-LPHN were bearing negative surface charges. Tf-LPHN and Ctrl-LPHN exhibited a sustained release of α-mangostin at pH 7.4, following an initial burst release, unlike rapid release of drug solution. The entrapment of α-mangostin in the LPHN led to an increase in α-mangostin uptake by cancer cells, and thus improved its antiproliferative activity compared to that observed with the drug solution. In conclusion, α-mangostin entrapped in the Tf-LPHN is therefore a highly promising therapeutic system that should be further optimized as therapeutic tools for cancer treatment.Alpha-mangostin, a natural xanthone mainly extracted from the pericarp of  Garcinia mangostana (commonly known as mangosteen), has a wide spectrum of pharmacological properties, for example, antiinflammation, antibacterial, antifungal, and antioxidant activities. Moreover, α-mangostin has recently gained considerable attention for its chemopreventive and therapeutic efficacies in vitro against many types of cancer, including lung, breast, liver, colon, prostate, cervical, and skin cancers [α-mangostin exerts its anticancer effect via multiple mechanisms such as enhancing the amount of reactive oxygen species, activation of focal adhesion kinase (FAK), apoptosis signal-regulating kinase 1 (ASK1)/p38, p53, poly-ADP ribose polymerase (PARP) and caspase-3, through suppression of human epidermal growth factor receptor 2 (HER2)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt), and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) as well as inhibition of folic acid synthesis (FAS) and ATP-binding cassette transporter G2 (ABCG2) [α-mangostin a very promising therapeutic molecule. However, the therapeutic potential of α-mangostin has been limited so far due to its poor solubility in water (2.03 × 10−4 mg/L at 25°C), high lipophilicity (log p 7.71), and photolytic property [α-mangostin failed to specifically reach tumors at a therapeutic concentration due to its low oral bioavailability (less than 5%) and rapid elimination in vivo, with a short biological half-life of approximately 3.5 hours [Alpha-mangostin, a natural xanthone mainly extracted from the pericarp of  cancers . It is w.5 hours , 7.α-mangostin, such as polymeric nanoparticles, liposomes, solid lipid nanoparticles, and nanomicelles [α-mangostin to cancer cells, thus improving its therapeutic efficacy.This limitation could be solved by using a drug delivery system that is able to entrap a hydrophobic drug, improve its water solubility, prolong its blood circulation time, and sustain its release over a period of time. In addition, nanocarriers can enhance drug delivery to tumors via passive targeting through the enhanced permeability and retention (EPR) effect , 9. Sevemicelles , but witmicelles , 10. On micelles , we hypoα-mangostin including liposomes and polymeric nanoparticles [α-mangostin across the blood-brain barrier in vivo [α-mangostin-loaded polymeric nanoparticles which enhanced its antiproliferative and apoptotic effects in pancreatic cancer stem cells and pancreatic cancer cell lines but had no effect on normal human pancreatic ductal epithelial cell lines [Several nanocarriers have previously been investigated to enhance the therapeutic efficacy of articles . Chen an in vivo . Verma all lines . Althougll lines , some prll lines .α-mangostin and controlling its release. The outer layer of the PLGA core was surrounded by a monolayer of heterobifunctional PEGylation phospholipid and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N--2000) (DSPE-PEG2K-MAL), which provided a stealth effect, protected the entrapped drug from freely diffusing out, and facilitated surface modification with Tf.Therefore, in this study, we selected a new combination of drug delivery systems, lipid-polymer hybrid nanoparticles, which offered advantage characteristics of both lipid-based vesicles and polymeric nanoparticles, meanwhile, overcoming the drawbacks of these nanocarriers . The bioα-mangostin and (2) to evaluate their cellular uptake and antiproliferative activity on cancer cells in vitro.The objectives of this study were therefore (1) to develop and characterize Tf-conjugated lipid-polymer hybrid nanoparticles entrapping Alpha-mangostin, Resomer® RG 503 H (acid-terminated poly (lactide-co-glycolide) (PLGA-COOH), lactide: glycolide 50 : 50, MW 24000–38000 Da, viscosity 0.32–0.44 dL/g), human holo-transferrin, 2-iminothiolane hydrochloride (Traut's reagent), 3--2, 5-diphenyl-tetrazolium bromide (MTT), and all other chemicals that are not specifically mentioned below were purchased from Sigma Aldrich, Germany. DSPE-PEG2K-MAL was obtained from Jenkem Technology, USA. Acetone and dimethyl sulfoxide (DMSO) were purchased from RCI Labscan, Thailand. Dulbecco's Modified Eagle Medium (DMEM), minimal essential medium (MEM), Roswell Park Memorial Institute 1640 (RPMI-1640), fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco, USA.α-mangostin was prepared using the one-step nanoprecipitation method. Briefly, DSPE-PEG2K-MAL (5 mg) was shaken with deionized water (5 mL) at 65°C for 1 hour. A mixture of α-mangostin (4 mg) and PLGA-COOH (25 mg) was dissolved in acetone (2.5 mL). It was subsequently added dropwise into the lipid part under moderate stirring (700 rpm). The product was stirred overnight at 25°C under a chemical fume hood to remove all acetone.The LPHN entrapping µL, 2 mg/mL in deionized water) under continuous stirring at 25°C for 2 hours. The thiolated Tf was then separated from unreacted Traut's reagent using Amicon® ultra-4 centrifugal filter units (molecular weight cut-off of 3 kDa) by centrifugation at 5,000 rpm for 30 min at 25°C. The freshly synthesized thiolated Tf was immediately conjugated to the LPHN under continuous stirring at 25°C for 2 hours.In order to conjugate Tf to LPHN, a cross-linking Traut's reagent was used to produce a thiolated Tf which can interact with the thiol-reactive maleimide group of DSPE-PEG2K-MAL. To do so, Tf  was reacted with a 10-fold molar excess of Traut's reagent  by centrifugation at 5,000 rpm for 20 min at 25°C. They were rinsed with deionized water (2 mL) before being resuspended in deionized water (1 mL). For empty nanoparticles (blank LPHN), they were prepared and conjugated with Tf in the same manner as Tf-LPHN but without α-mangostin. All nanoparticles were stored at 4°C for further experiments.The resulting Tf-LPHN and Ctrl-LPHN were purified to remove unentrapped The morphology of the LPHN was visualized by transmission electron microscopy (TEM). To do so, a drop of each LPHN was deposited on a carbon-coated copper grid (400 mesh). Dried samples on the grid were imaged at 120kv using TEM .The amount of Tf conjugated to the LPHN was quantified using the Lowry assay , as prevµL) was mixed with deionized water (990 µL) before being transferred into a folded capillary cell for measurement.The hydrodynamic size and zeta potential of the LPHN were evaluated by photon correlation spectroscopy and laser Doppler electrophoresis techniques using the Zetasizer Pro instrument . The LPHN  were mixed with methanol (990 µL) and sonicated for 15 min, followed by centrifugation at 10,000 rpm at 25°C for 10 min. The optical density of α-mangostin in the supernatant was measured at a wavelength of 317 nm. The amount of α-mangostin was calculated by correlating absorbance with the standard curve of α-mangostin. The results were expressed as a percentage of entrapment efficiency .The amount of in vitro release of α-mangostin from the LPHN. Briefly, α-mangostin either formulated as Tf-LPHN, Ctrl-LPHN, or free in solution  containing 0.5% Tween 20, pH 7.4) was placed into a SnakeSkin® dialysis tube (molecular weight cut-off of 3.5 kDa). The samples were dialyzed against 200 mL of phosphate buffer saline containing 0.5% Tween 20 (pH 7.4) and incubated at 37°C with continuous stirring (150 rpm). The release medium (1 mL) was collected in triplicates at specific time intervals  and replaced with an equal volume of fresh medium. The amount of α-mangostin was quantified by a UV-visible spectrophotometer and reported as a percentage cumulative drug release.The membrane dialysis technique was used to evaluate the 2 and subcultured every three to four days.MCF-7 human breast adenocarcinoma, A549 human lung adenocarcinoma, and B16–F10 murine melanoma cell lines were grown as monolayer cultures in either a MEM (for MCF-7 cells) or in RPMI-1640 medium (for A549 and B16–F10 cells) supplemented with 10% (v/v) FBS and 0.5% (v/v) penicillin-streptomycin. Cells were cultured at 37°C in an incubator with a humid atmosphere of 5% COα-mangostin accumulated within the cells was quantified using high-performance liquid chromatography (HPLC). Briefly, MCF-7, A549, and B16–F10 cells were seeded at a density of 2 × 105 cells/well in 6-well plates and grown at 37°C at 5% CO2 for 72 hours. They were then treated with α-mangostin , either as Tf-LPHN, Ctrl-LPHN, or free in solution. After 4 hours treatment, the cells were washed twice with cold PBS (3 mL), followed by incubation with TrypLE® Express (1 mL) for 10 min to detach the cells. The cell pellets were then collected, centrifuged at 2,000 rpm for 10 min, and washed twice with cold PBS (1 mL). After that, the cell pellets were lysed with 5% Triton-X (1 mL) and incubated overnight at 37°C. The supernatant was then separated from the cell lysates by centrifugation at 10,000 rpm for 15 min and analyzed using HPLC as follows, column: ZORBAX Eclipse XDB-C18 , mobile phase: methanol and water (95 : 5) with isocratic elution for 20 min, flow rate: 0.8 mL/min, injection volume: 50 μL, detection wavelength: 317 nm. The cellular uptake was calculated and reported as a percentage of cellular accumulation of α-mangostin.In order to measure the ability of the LPHN for cell internalization, the amount of α-mangostin either as Tf-LPHN, Ctrl-LPHN, blank LPHN, or a free drug solution. To do so, MCF-7, A549, and B16–F10 cells were seeded into 96-well microplates at a density of 3 × 103 cells/well and grown for 24 hours in an atmosphere of 37°C, 5% CO2 to allow for cell attachment. After that, the medium was then removed and replaced with fresh medium containing test samples (200 μL) at various concentrations (ranging from 0.3125–20 µg/mL). After 48 hours of treatment, the supernatant (100 µL/well) was removed and MTT solution  was added to each well, followed by incubation in an atmosphere of 37°C, 5% CO2 for 2 hours. After incubation, the medium was removed, and DMSO (100 μL) was then added to dissolve the formazan production in the cells. The optical density of the formazan solution was measured using a microplate reader at a wavelength of 570 nm. The results were calculated as the percentage cell viability compared with the untreated cells and fitted to obtain the IC50 values.The MTT assay was used to evaluate the antiproliferative activity of p values lower than 0.05.All experiments were done in triplicate and the results were expressed as mean ± standard error of mean (SEM). Statistical significance was assessed by one-way analysis of variance analysis (ANOVA) and the Tukey multiple comparison test using commercial statistical software. Differences were considered statistically significant for α-mangostin were successfully prepared using the one-step nanoprecipitation method . The average size of Tf-LPHN was also increased after being conjugated with Tf  compared to that of Ctrl-LPHN . Furthermore, the presence of Tf on the surface of nanoparticles significantly increased the net surface charge of Tf-LPHN (−43.7 ± 0.6 mV) in comparison with Ctrl-LPHN (−51.7 ± 0.2 mV).Tf-bearing and Ctrl-LPHN entrapping n method , where tn method . As showα-mangostin at pH 7.4 with initial burst release (about 16–20%) in the first hour, followed by a sustained release of α-mangostin over 72 hours , respectively. In A549 cells, it was 1.5-fold and 2.0-fold for Tf-LPHN in comparison with Ctrl-LPHN and α-mangostin solution , respectively. The highest intracellular accumulation of α-mangostin was found in B16–F10 cells treated with Tf- LPHN which was significantly higher than that observed after being incubated with Ctrl-LPHN and a free drug, respectively, by 1.6-fold and 2.5-fold .The intracellular accumulation of ll lines . As expeα-mangostin in Ctrl-LPHN led to a significant increase in its in vitro antiproliferative activity in comparison with that of the free solution, by at least 1.3-fold , while the IC50 in A549 cells was observed to be at 4.54 ± 0.18 µg/mL . However, Tf-LPHN entrapping α-mangostin indicated only limited antiproliferative activity in MCF-7 cells , probably because MCF-7 cells are more resistant to α-mangostin than the two other cell lines. By contrast, we could not determine the IC50 of the blank LPHN in any tested cell lines, demonstrating the safety of LPHN under the tested experimental conditions.The loading of 1.3-fold . The antα-mangostin for cancer treatment is hindered by a lack of tumor specificity and rapid elimination in vivo. Therefore, we developed a Tf-conjugated LPHN that could enhance tumor-selective delivery and the therapeutic efficacy of α-mangostin.The promising efficacy of The LPHN were easily prepared using the one-step nanoprecipitation method by mixing the PLGA polymer in water-miscible organic solvent (acetone) with the aqueous DSPE-PEG2K-MAL lipid dispersion in which they self-assembled. This technique is preferred  over the two-step method, where polymeric nanoparticles and lipid vesicles are separately prepared before being combined using ultrasonication and homogenization .α-mangostin loaded Tf-modified liposomes using N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling reagents [The thiol–maleimide ‘click' reaction was used to conjugate the LPHN with Tf . This threagents .α-mangostin in both Tf-LPHN and Ctrl-LPHN, ranging from 78–85%. Several types of delivery systems have previously been used to entrap α-mangostin, such as lipid-based vesicles, solid lipid nanoparticles, and polymeric nanoparticles [α-mangostin to that of what was previously reported by Chen and colleagues, who developed Tf-modified liposomes, which were able to entrap 88% of α-mangostin [α-mangostin in nanostructured lipid carriers (NLC) made from lavender essential oil and cetyl palmitate [Entrapment efficiency is one of the important parameters in the design of drug delivery systems. This parameter relies on several factors such as the types and compositions of nanocarrier, as well as the nature of drug load . Our resarticles . Our LPHangostin . A maximalmitate . Alpha-malmitate , lower t2+) in the protein, which might neutralize the negative surface charges of Tf-LPHN [In terms of particle size and morphology, although the conjugation of Tf to LPHN led to an increase in particle size, they still displayed the required size (ranging from 128 to 294 nm) that should theoretically allow their extravasation through the leaky vasculature (cut-off size approximately 400 nm) of most tumors via the EPR effect . Besides Tf-LPHN . Also, i Tf-LPHN .α-mangostin could be efficiently sustained released (up to 45% at 72 hours) from the LPHN with initial burst release, unlike a rapid release of free α-mangostin. Thus, the initial burst release observed in our LPHN formulations may attribute to the presence of PEGylated lipid, which facilitates water absorption and accelerates the diffusion of α-mangostin entrapped in the outer layer of the polymer core through a water-filled pore, the most common drug release mechanism of polymer-based nanoparticles [α-mangostin, MW of 410.5 g/mol) also have a high propensity for burst release due to osmotic pressure [α-mangostin [α-mangostin was also sustained released (24.27% over 24 hours at pH 7.4) from polymeric nanoparticles made from poly (ethylene glycol)-poly (propylene glycol)-poly (ethylene glycol) (PEG-PCL-PEG) triblock copolymer, then its cumulative release increased constantly to reach a high level of 90.47% after 96 hours. In our study, Tf-LPHN exhibited a slower release of α-mangostin than their Ctrl-LPHN. This may be due to the ability of Tf to conjugate to the surface of nanoparticles to reduce water absorption of the nanoparticles, thus, slowing the rate of α-mangostin diffusion through water-filled pores. Similar to previous studies, the release of plumbagin from Tf-targeted hybrid nanoparticles  was significantly slower than nontargeted nanoparticles [The drug release proﬁle is another parameter of controlled release systems, which might contribute to the concentration of the drug at the targeted sites as well as its therapeutic efficacy upon administration . Our exparticles . In addipressure . Yu and angostin . In theiarticles .α-mangostin into the LPHN significantly increased its accumulation within the three tested cell lines. The conjugation of Tf to LPHN further enhanced the uptake of α-mangostin (up to 2.5-fold) when compared to a free drug solution. This improvement might be explained by the fact that Tf-modified LPHN entrapping α-mangostin are taken up by clathrin-mediated endocytosis, a highly speciﬁc internalization mechanism, unlike low molecular weight drugs or hydrophobic drugs, like α-mangostin, are able to enter the cells by nonspecific passive diffusion [α-mangostin in bEnd3 cells was increased about 1.3-fold and 4-fold in comparison with unconjugated vesicles and free drug solution, respectively [α-mangostin after treatment with Tf-LPHN. This might be explained by the different expression levels of Tf receptors on the target cell surface [Cellular uptake experiments revealed that loading iffusion , 35. Theiffusion . A similectively . Guo andectively . Among t surface , where Bα-mangostin within the LPHN also increased its antiproliferative activity compared with a free drug in all tested cell lines. Furthermore, the conjugation of Tf to LPHN further improved the IC50 values, showing approximately up to 2.5-fold increase. These findings were consistent with an increase in cellular uptake of α-mangostin after being treated with LPHN and were in accordance with previous reports demonstrating that the therapeutic efficacy of α-mangostin was improved by entrapment in drug delivery systems. For example, liposomes containing α-mangostin improved its antiproliferative activity on HepG2 human hepatocyte carcinoma cells with IC50 of 1.9 µM, as compared with 4.6 µM for α-mangostin solution [β-cyclodextrin-coated α-mangostin polymeric nanoparticles on A549 lung cancer cells was enhanced by 2.1-fold in comparison with free α-mangostin [α-mangostin chitosan-loaded kappa carrageenan nanoparticles (IC50 of 4.7 µg/mL) exhibited a higher antiproliferative activity on MCF-7 cells than drug solution (IC50 of 8.2 µg/mL) [The entrapment of solution . In anotangostin . Wathoni2 µg/mL) , in linein vitro results confirmed the advantage of using drug delivery systems combined with transferrin targeting ligand, which signiﬁcantly increased the cellular accumulation and antiproliferative activity of α-mangostin in cancer cells overexpressing Tf receptors. However, despite these promising results, the in vivo experiment of Tf-LPHN entrapping α-mangostin is required and should be further investigated for its therapeutic efficacy, biodistribution, and toxicity.These α-mangostin, with high percentage of drug entrapment and high Tf conjugation efficiency. The nanocarriers had small particle sizes with negative charges and also displayed a sustained drug release profiles. In vitro evaluation showed that the cellular accumulation of α-mangostin was enhanced when loaded in Tf-LPHN, compared to that of nontargeted formulations and drug solutions, resulting in an improvement in its antiproliferative activity in all the tested cell lines.We successfully developed Tf-bearing LPHN entrapping α-mangostin in Tf-bearing lipid-polymer hybrid nanoparticles is therefore a highly promising strategy for cancer treatment and should be further investigated and optimized to improve its therapeutic efficacy.The entrapment of


Few studies have explored the prognosis of fetuses with congenital anomalies. This study aimed to investigate the prevalence and prognosis of congenital anomalies in IVF/ICSI pregnancies, and to analyze the influencing factors contributing to poor prognosis.Conceptions following In this multicenter retrospective cohort study, we followed 405,473 embryo transfer cycles at 15 reproductive centers between January 2010 and December 2019 and enrolled 2,006 intrauterine pregnancies with congenital anomalies. The relatively positive prognosis group with one or more live births and neonatal survival for more than 7 days was compared with the poor prognosis group with poorer outcomes.p < 0.001). Multivariate logistic regression analysis revealed no significant differences in the relatively positive prognosis rate among the various IVF/ICSI protocols. The relatively positive prognosis rate of fertilization failure cycles was 0.180 times that of normal fertilization cycles.Among the 168,270 ongoing intrauterine pregnancy cycles, the prevalence of congenital anomalies was 1.19%, wherein the malformation rates of cycles with late abortion and delivery were 2.37%  and 0.93% , respectively. Among all IVF/ICSI cycles with congenital anomalies, the relatively positive prognosis rate was 61.39%. Moreover, the fertilization failure rate (2 pro-nuclei rate < 25%) in the poor prognosis group was significantly higher than that in the relatively positive prognosis group (10.89% vs. 5.09%, Poor fertilization rates during IVF/ICSI treatments are more likely to have poor prognosis in fetuses or neonates with congenital anomalies, and obstetric management should be strengthened in pregnant women, with which pregnant women should be recommended to strengthen obstetric management. In European countries, approximately 2% of infants are born annually through ART , an increasing number of infertile couples have conceived by undergoing ough ART . In BeijCongenital anomalies are a leading cause of fetal or neonatal death in the perinatal period. Their treatment is complicated, resulting in great psychological and financial burden on the child and his family. Recently, several studies have demonstrated that pregnancies conceived with IVF/ICSI have an increased risk of congenital anomalies compared to spontaneous conceptions –7, and sThe underlying mechanisms explaining the association between the risk of congenital anomalies and ART remain unclear, including infertility itself  and the However, few studies have assessed the clinical pregnancy outcomes of fetuses or infants with congenital anomalies born after IVF/ICSI. This study aimed to evaluate the prevalence and prognosis of congenital anomalies among pregnancies conceived through IVF/ICSI treatments from 2010 to 2019, and to explore the factors contributing to poor prognosis.This retrospective, multicenter, cohort study collected infertile patients undergoing IVF/ICSI cycles at 15 reproductive centers in China. This study was approved by the Medical Science Research Ethics Committee of Peking University Third Hospital (IRB00006761-M2019487). All data collection and analysis procedures conducted in this trial were performed in accordance with the Declaration of Helsinki. Individual informed consent was waived, as it is a retrospective study.From January 2010 to December 2019, we followed up 199,591 IVF-fresh cycles and 114,816 IVF-frozen cycles from 15 reproductive centers. Then, a total of 405,473 embryo transfers were performed. Finally, 174,639 women obtained clinical pregnancy, including 6,369 heterotopic pregnancy or early miscarriage and 168,270 intrauterine pregnancy of more than 12 weeks Figure 1We collected and recorded detailed information from all centers in this study, including parental basic characteristics , IVF/ICSI indications, IVF/ICSI specific techniques , and IVF/ICSI outcomes .Clinical pregnancy outcomes were primarily follow-up data recorded at the end of the pregnancy. Pregnancies ≥28 weeks and <28 weeks but with a live birth were considered deliveries. Pregnancy loss referred to late abortions between 12 and 28 weeks, including spontaneous abortions, embryo damage, and induced abortions for various reasons, divided into miscarriages <20 and ≥20 weeks.After confirming intrauterine pregnancy, women were scheduled for a systematic prenatal examination in the obstetrics department to identify the presence of deformities or chromosomal abnormalities. In addition to the malformations detected during pregnancy, any structural, functional, and genetic anomalies diagnosed after late abortion or delivery were defined as congenital anomalies , which wIVF/ICSI cycles with congenital anomalies were grouped into relatively positive prognosis and poor prognosis based on the outcomes of fetuses or neonates. Based on the Manual of Maternal and Child Health Surveillance of China , hospitaTo explore the potential factors influencing the prognosis of offspring with congenital anomalies, we made a comparison on the details collected between the relatively positive prognosis group and the poor prognosis group.Between the two groups, we also compared the rate of fertilization failure, when the number of 2PN embryos accounted for was less than 25% of the number of oocytes collected in one IVF/ICSI cycle, and their pregnancy outcomes, including pregnancy loss rate, delivery rate, live birth rate, birth weight, and birth height.t-test or the chi-squared test, as appropriate. Given that many factors may affect the prognosis of pregnancies with congenital anomalies, multivariate logistic regression analysis was performed to analyze the potential influencing factors contributing to poor prognosis, with using the odds ratios (ORs) and corresponding 95% confidence intervals (CIs). Statistical significance was defined as a two-sided p < 0.05.All statistical analyses were conducted using SPSS software, version 26.0 for Windows . Continuous variables were reported as mean ± standard deviation (SD). The characteristics of two groups were compared using an independent sample Over the study period, we followed up a total of 168,270 IVF/ICSI cycles with ongoing intrauterine pregnancies and finally enrolled 2,006 pregnancies with congenital anomalies. The overall prevalence of congenital anomalies in the study population was 1.19%, wherein the prevalence of cycles with late pregnancy loss and delivery was 2.37%  and 0.93% , respectively. Based on ICD-10 codes, the incidence of congenital anomalies in specific organ systems is shown in p < 0.001); the trend continued for the fathers . Furthermore, they were more likely to have primary infertility  and be nulliparous  than were those in the poor prognosis group. No statistically significant differences were found in mean body mass index (BMI) of the couples, infertility duration, or IVF/ICSI indications . Mothers in the relatively positive prognosis group tended to be younger . In cycles treated with ICSI, the rates of mature oocytes  and 2PN embryos  were lower in pregnancies with poor prognosis. Furthermore, the rate of fertilization failure (2PN rate <25%) in the poor prognosis group was significantly higher than in the relatively positive prognosis group   Table 3.p = 0.001) and birth height .The clinical outcomes of the study populations were demonstrated in p = 0.002) for patients with a relatively positive prognosis.Multivariate logistic regression analysis showed that pluriparity, early preterm birth (delivery at 28–34 weeks), and fertilization failure were factors for poor prognosis in IVF/ICSI pregnancies with congenital anomalies after adjusting for all confounding variables Table 5.In this 2010–2019 multicenter comprehensive follow-up study in China, the overall prevalence of congenital anomalies among intrauterine pregnancies ≥12 weeks conceived through IVF/ICSI was 1.19%. Nearly two-thirds could obtain a relatively positive prognosis with one or more live births surviving more than 7 days. Moreover, patients with fertilization failure were more likely to have a poor prognosis for fetuses with congenital anomalies.The prevalence of congenital anomalies in the present study was similar to the rate of 1.23% observed in a multicenter study conducted from 2004 to 2008 . AnotherConsistent with previous studies , 15, theTo date, several studies have indicated that ART use is associated with an increased risk of congenital anomalies , 20, 21.p < 0.001).Few studies have explored the prognosis of fetuses with congenital anomalies. Our study showed that the relatively positive prognosis rate was 61.39% 1,258/2,006). Previously, Zhang et al. recorded the outcomes of fetuses with congenital heart disease  between the two prognosis groups. Older women have a lower quality of oocytes and an increased risk of chromosomal abnormalities in their offspring , resultip = 0.011), there was no substantial difference in the effect on prognosis whether patients were treated with ICSI (p = 0.161) after excluding other confounding factors. When comparing fresh and frozen-thawed embryo transfers, our study showed a similar rate of relatively positive prognosis (p = 0.808). The consensus is that the transfer of a single embryo has better perinatal outcomes for both mothers and offspring (p = 0.931).It has been proposed that specific ART procedures, such as fresh or frozen embryo transfer after IVF/ICSI or ICSI, have little impact on the prevalence of congenital anomalies , 28. A 2ffspring , 30. In More importantly, pregnancies with a poor prognosis had a lower rate of mature oocytes and a higher fertilization failure rate (2PN rate < 25%). Multivariate logistic regression analysis showed that the relatively positive prognosis rate of fertilization failure cycles was 0.180 times that of normal fertilization cycles. Epigenetic remodeling, including DNA methylation, chromatin accessibility, and histone modifications, occurs primarily during human gametogenesis and early embryonic development . ART proSeveral limitations exist in our study. First, there are no clear guidelines for defining favorable or unfavorable prognosis. In this study, we defined a relatively positive prognosis as having one or more live births surviving for more than 7 days. However, quite a few babies suffering from congenital anomalies die within the first month of life. At present, in majority of reproductive centers in China, the routine follow-up endpoint for ongoing pregnancies conceived by IVF/ICSI is 1–2 weeks after the expected date of confinement. The terminal point of the Chinese hospital-based birth defect surveillance system is 7 days after birth . As a muIn conclusion, this is the first multicenter study on the prognosis of pregnancies with congenital anomalies after IVF/ICSI. Moreover, our study suggests that poor fertilization rates during IVF treatment are associated with a poor prognosis in fetuses or neonates with congenital anomalies. Hence, couples experiencing fertilization failure should be recommended to strengthen obstetric management and active prenatal diagnosis.The datasets presented in this article are not readily available because:According to the requirements of this research funding, the original data are uniformly managed by the database of the Information Center for the National Health Commission of the People’s Republic of China and cannot be freely shared.Requests to access the datasets should be directed to:Wang Yuanyuan, yyuanwang@163.com.The studies involving human participants were reviewed and approved by Peking University Third Hospital Medical Science Research Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.HC, YW, and JQ contributed to conception and design of the study. YH, HW, XH, CL, and XJ were in charge of data collection. JB and LC performed the statistical analysis and wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. All authors contributed to the article and approved the submitted version.This work was supported by the National Key Research and Development Program of China (No. 2019YFC1005106).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The study aims to identify the correlation between human papillomavirus (HPV) infection and sociodemographic and sexual reproductive health factors in Ecuadorian women from March to August 2019.120 women were randomly selected from two gynecological clinics to complete a questionnaire and provide a biospecimen. PCR-Hybridization was used to genotype 37 HPV serotypes in samples obtained by endo-cervical brushing for liquid-based cytology. Sociodemographic and sexual health data were collected through a validated questionnaire during a medical consultation. Mathematical modeling of HPV infection was done using bivariate logistic regression.65.0% of the women sampled had an HPV infection; 74.3% of these women had co-infections with other HPV genotypes. Out of the women who were HPV positive, 75.6% were diagnosed with high-risk genotypes from HPV strains 18, 35, 52, and 66. Parity, immunosuppression, and use of oral contraception/intrauterine devices (IUDs) were identified as associated variables. The explanatory model had a sensitivity of 89.5% and a specificity of 73.8%.The predominant strains of HPV among Ecuadorian women are diverse. The risk of HPV infection is a complex phenomenon where biological and psychosocial variables are integrated into a model. In populations with limited access to health services, low socioeconomic status, and negative sociocultural beliefs about sexually transmitted infections (STIs), surveys can be used as a pre-screening step for HPV infections. The diagnostic value of the model should be tested in multicenter studies that include women from all over the country. Human papillomavirus, the most common STI worldwide , is assoThere are a variety of risk factors associated with recurring HPV infections. Among these, early intercourse, number of sexual partners, and infrequent condom use are the most prevalent . DiffereChronic infection of HPV strains 16 and 18 have been implicated in the majority of cervical cancer cases; a statistic that led to the creation of the HPV vaccinations, such as Gardasil and Cervarix . In EcuaTo reduce this incidence rate and premature mortality, this research aims to associate HPV infection in Ecuadorian women from Cuenca with sociodemographic, sexual-reproductive health and biological variables. Using this data, we aim to create a mathematical model that will aid in HPV pre-screening, contributing to Ecuador’s goal to reduce 25% of mortality from cancer by 2025 .This study includes a quantitative cross-sectional evaluation of women who attended two gynecology clinics in the city of Cuenca, Ecuador from March 2019 to August 2019.The potential sample size was calculated using the sample size estimation formula for proportions in known populations plus 10% of estimated losses. The participating population size was estimated according to the volume of work activity of previous months in the gynecology service in that period. The rate of known HPV infection in the region was retrieved from previous studies.N = 175, significance level ∝ = 0.05, precision d = 5%, lost samples = 10%, HPV infection probability p = 40%).The formula used is as follows:Using random sampling, the patients were assigned sequential numbers upon arrival at the healthcare facility during the research period. Upon initial consultation, the medical professional assessed whether the participant met the following inclusion/exclusion criteria:Inclusion criteria: current treatment at the respective healthcare facility; sexually active life; age between 18–65 years old; female.Exclusion criteria: pregnancy; previous diagnosis of HPV infection or cervical cancer; treatment with immunosuppressive drugs.The final sample in the study was 120 women.A patient questionnaire was used for the collection of sociodemographic and sexual-reproductive health data and was applied with reliability  and acceptable content validity (Aiken’s V of 0.82) . The queThe collection of biospecimens was carried out according to the protocols issued by the Ministry of Public Health (MSP) of Ecuador . EndocerFrom the liquid-based sample, an aliquot was made in a laminar flow hood for the identification of HPV, by broad spectrum genotyping, using the 37 HPV GenoArray Diagnostic Kit . The kit detected the presence of 37 different HPV genotypes: 15 high , 6 low ) and 16 undetermined risk . HPV DNA was extracted from cervical cells using an alkaline lysis method. Samples were centrifuged for 10 min at 14000 rpm at 20°C, resuspended in 100 μL elution buffer, and stored at −20°C until PCR. The concentration and purity of DNA (OD260/OD280 1.6–1.8) was determined by Nanodrop 2000 . This was followed by amplification, according to manufacturer recommendations, through conventional PCR and utilization of biotinylated PGMY primers . The PCR mix was prepared according to the manufacturer’s instructions to obtain a final reaction volume of 25 μL . Amplification was performed with an initial denaturation at 95°C for 5 min (min), followed by 40 cycles of denaturation at 95°C for 20 s (s), annealing at 55°C for 30 s, and elongation at 72°C for 30 s, then a final elongation at 72°C for 5 min.After amplification, the genetic material was denatured and subjected to membrane hybridization for 20 min using specific probes. All assays included a positive and a negative PCR control in addition to the hybridization control itself. The hybridization process was carried out using the HibriMax (Hybribio) kit according to the manufacturer instructions. This was conducted by washing, addition of streptavidin–horseradish peroxidase conjugate, membrane blocking, and addition of developer substrate (nitroblue tetrazolium-5-bromo-4-chloro3-indolyl phosphate). The membranes contained the immobilized probes of the genotypes of interest that hybridize with biotinylated amplified PCR products. Streptavidin–horseradish peroxidase conjugate was added to bind to the biotinylated PCR products. The direct visualization of the breakdown product (purple precipitate) from the addition of substrate nitroblue tetrazolium-5-bromo-4-chloro3-indolyl phosphate, was interpreted as positive for the corresponding HPV DNA type as indicated in the schematic diagram .U test for determining whether sets are from the same distribution. Differences in proportions and bivariate association were estimated using cross tables . The risk estimation used odds ratio (OR) with confidence intervals from bivariate analysis and confirmed by binary logistic regression using the progressive Wald method. The significance level of all tests was less than or equal to 0.05. The equation that describes HPV infection probability derives from a mathematical model of binomial logistic regression.Data was stored in an electronic database and processed using IBM SPSS 23.0 statistical software. Analysis of frequency and measures of central tendency position  and dispersion  were used. Differences between groups were established using the Mann–Whitney or Kruskall-Wallis The research protocol ensured informed consent and good practice established in the Declaration of Helsinki. Prior consent from all subjects involved was obtained by signature after the research objectives were presented orally and in writing. The anonymity and the will of the respondents and interviewees regarding the disclosure of information was respected. The research was reviewed and approved by the Catholic University of Cuenca Human Research Ethics Committee with resolution number CEISH 2018–09-017 R.Sociodemographic results of the survey are shown in 2X = 73.876; p = 0.000). The place of residence was predominantly urban (60.8%) over rural areas (39.2%) . Must participants had a secondary or university level of education . Additional demographics revealed that 78.3% of the sample had incomes below $875 USD  64.2% consumed alcohol regularly , 23.3% of participants had a current or past consumption of tobacco , and only 5.8% of participants have consumed hard drugs .The average age of participants was 35.6 years old (+/− 10.9). Most patients were between 31–45 years (41.7%), and 18–30 years (40.0% of patients) followed by ages 46–55 (11.7%) and 56–65 (6.6%) . For a majority of the sample, the first instance of coitarche occurred at age 19 years or older (64.2%) . The number of sexual partners per month varied throughout the participants with 77.5% indicating they have had 1–2 sexual partners, 16.7% between 3–4 partners, and 5.8% 5 or more partners . 59.1% of sample patients reported a frequency of 1–4 sexual relations per month .Most participants (52.5%) had a partner . The remaining 47.5% were either single (35.0%) or divorced (12.50%) . 42.5% of the participants were currently using oral contraceptives or intrauterine devices and 38.3% did not use any contraceptive method . The majority of women had given birth to one or no children (56.7%) and 43.3% were multiparous . 69.2% of participants reported having a history of STIs (besides HPV) as opposed to 7.5% never having contracted an STI. It should be noted that 23.3% were unaware of their STI history .Frequency of condom use in sexual intercourse was in low proportion (19.2%) compared to women that never or sometimes use condoms in coital (80.8%) . Clinical immunosuppression was found in 70.8% of the women .The access to gynecological health care had high occurrence with 85.0% of the sample population having at least one appointment per year, and the remaining participants (15.0%) having less than one appointment per year . Background knowledge on HPV was higher for the group with high school-college education compared to elementary-no education. No differences in knowledge were found due to age  or marital status .The sample population had an average level of knowledge on HPV of 13.6 +/− 2.0 (95% CI 13.2–13.9) on a scale of 20. There were significant differences in knowledge according to the level of education (The frequency of infection according to HPV strain is shown in p > 0.050). There was no association between age groups and HPV infection .Even though the groups of women aged 56–65 and 31–45 years old had a higher proportion of HPV infections , the Z t2X = 7.234; p = 0.041). HPV 16 had a low frequency of infection in the group studied.The frequency distribution of the hrHPV strains is shown in The correlation analysis showed a moderate association between HPV infection and certain biological, sociodemographic, sexual and reproductive health variables. 2X = 5.852; p = 0.026; V Cramer = − 0.293; p = 0.016). For married women, this association was not found .While overall no association was found between HPV infection and condom use, when accounting for marital status, participants who were single or in common law unions, condom use was associated with lower infectivity , knowledge about HPV , gynecological medical care , place of residence , education level , alcohol consumption , age groups , previous STIs , frequency of sexual intercourse , and marital status . These were not associated with HPV infection in the sample studied.The remaining variables included: the age at which the participant became sexually active (p > 0.050) or low predictive power with a non-significant OR. No confounding or interaction variables were found. The characteristics of the model obtained by binary logistic regression are shown in Binary logistic regression confirmed an association between parity, immunosuppression symptoms, use of oral contraceptives/IUDs, and positive HPV infection. The rest of the variables were excluded due to their low statistical significance , diagnostic power of 81.7%, a high sensitivity of 89.5% and an acceptable specificity of 73.8%. This model makes it possible to explain up to 42.9% of the variance of the infection with HPV according to the Nagelkerke coefficient of Determination (R2 = 0.429). Even though the model is not comprehensively adequate, select variables have been viewed as possible explanations for HPV infection. This model attempts to rule out tobacco use and number of sexual partners as significant explanations of infection according to their respective OR values.The model has significant values of Hosmer Lemeshow test  reports more than 1 million new sexually transmitted infections each day . The freIn Ecuador, there is no epidemiological study about HPV infection within the country in its entirety. For instance, ENSANUT’s 2019 report  does not contain information about HPV infection. Neither exists a national program to test HPV infection, even when deaths for UCC increased to 902 women in the country in 2019  which suggest differences in susceptibility to HPV infection. Biological characteristics of the host, such as immune status, trophic predisposition to the virus, and favorable vaginal biome could promote a greater viral infection.Although infection with HPV is a high-frequency phenomenon, viral clearance occurs in 80–90% of infected women . One of The substantial differences in the circulating genotypes in the south highlands of Ecuador and a possible change in the patterns of infection with HPV suggest a specific approach in diagnostic research, prevention, and treatment , 34. NonU” shape view like in several Latin-American countries   . In thesd Chile) , or a poThe role of risky sexual behavior as a determinant of STIs has been validated by numerous investigations . Unlike There is also a lack of overall correlation between condom use and HPV infection. This finding may be related to the inconsistent use of condoms in married women who are infected by marital and extramarital sex, which is identified by Calatrava et al. in other populations of women infected with STIs .Although tobacco use potentiates the appearance of cervical cancer in women infected with hrHPV , the effHormonal contraception and HPV infection have been found to induce the progression of UCC . EstrogePrevious research has corroborated an association between the use of estrogenic oral contraceptives and hrHPV infection. The influence of female hormones on HPV infection is not well understood, but the effect has recently been suggested. Estrogens induce the expression of the polysaccharide layer present in the basement membrane of the cervical epithelium of animal models , 48. ResAlthough previous epidemiological studies documented parity as a risk factor for cervical cancer, the reported strength of association is variable and inconsistent. Several investigations revealed a connection between parity, HPV infection, and CCU . These sInterpretations of the discussed findings should consider the tested demographic in relation to sampling method and sociocultural characteristics. The extrapolation of results must be assessed carefully in different contexts. While utilizing the random selection method, there was potential for bias in sampling. Participants who came for healthcare services at the facility expressed interest in research participation, and this may introduce potential bias with its convenience.Sample size and cultural diversity of participants was considered a limitation of this study. We acknowledge that the results represent a small-scale sample of the sexually active female population of the Ecuadorian highlands. Further studies should be carried out to increase external validity. We suggest continuing with different multicentric studies to substantiate diagnostic value within a large and diverse group of women in regions of Ecuador.The survey method can also be considered a limitation based on the participant’s ability to answer accurately. To achieve reliable data, a comfortable environment must be established to maintain confidence in doctor-patient disclosure. Although a relationship was found between HPV and immunosuppression symptoms, it is inaccurate to draw a correlation based on our study due to the reliability of the survey method and absence of molecular testing. These clinical implications could also be confounded by participants, resulting in a source of perception bias. We suggest that our method should be validated further through pilot testing in populations under study.The predominant strains of HPV among Ecuadorian women are diverse. The risk of HPV infection is a complex phenomenon where biological and psychosocial variables are integrated into a model. HPV pre-screening in populations with limited access to health services, low socioeconomic status, and negative sociocultural beliefs about STIs could use surveys as a step before screening for HPV infections. The diagnostic value of the model should be tested further in multicenter studies all over Ecuador.https://ucacueedu-my.sharepoint.com/:f:/g/personal/jbaculima_ucacue_edu_ec/ElO4GinuTYpIqSv39NWCDI8B4d_LBdfiiruxKz4W2mU1pw?e=5KZNNhAccesionkey:Investigacion.BF.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The studies involving human participants were reviewed and approved by Human Research Ethics Committee UCACUE. The patients/participants provided their written informed consent to participate in this study.CR: research design, survey design and validation, biospecimen processing, and data processing and statistical analysis. YH: research design, survey design and validation, and data processing and statistical analysis. DA: research design and data processing and statistical analysis. ZS and LM: data processing and statistical analysis, biospecimen collection, and sociodemographic data collection. EC, LG, and MM: survey design and validation, biospecimen processing, and sociodemographic data collection. MS: biospecimen collection and sociodemographic data collection. JB: data processing and statistical analysis. All authors contributed to the article and approved the submitted version.Funding was received from UCACUE (Grant UCACUE-DIPVP-2018-165-OF).CR and JB were employed by Diagnostic Department, MEDsan, Inc., Saint Petersburg, FL, United States.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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This systematic review highlights the impact of HPV diagnosis and the electrosurgical excision procedure (LEEP) on mental health and sexual functioning. Indeed, our results showed that there is a correlation between abnormal cytology results/HPV infection and subsequent treatment interventions (LEEP/LEETZ) and women’s mental health and sexual life. The conclusions of our review underline the importance of sex education and knowledge about sexually transmitted diseases. An improvement in this area could prevent such high transmission of oncogenic HPV. Furthermore, the number of studies evaluating the impact of the procedure (LEEP/LEETZ) and the number of female participants have been very small, thus further research in this direction could be very beneficial. Additionally, psychological support should be provided to any patient who needs it. In this respect, such care should be part of the routine management of patients receiving an HPV diagnosis. Finally, the diagnosis and information about the current state of health should be communicated in an appropriate manner by gynecologists. Proper communication could significantly reduce the patient’s stress and anxiety.The impact of HPV diagnosis and subsequent treatment with the electrosurgical excision procedure (LEEP) on anxiety, depression, psychosocial quality of life, and sexual functioning has not been thoroughly investigated. The aim of this review was to systematically summarize the available knowledge on this topic, according to PRISMA guidelines. Data from observational and intervention studies were analyzed. A total of 60 records were included, of which 50 papers addressed the impact of HPV diagnosis on patients’ psychosocial status, while 10 studies addressed the impact of the implemented LEEP procedure on patients’ mental health and sexual functioning. The results indicated a negative impact of HPV diagnosis on the occurrence of depressive and anxiety symptoms, poorer quality of life, as well as on the sexual functioning of the affected women. The results of the studies to date have not confirmed the negative impact of the LEEP procedure on mental health and sexual life, although more research is needed in this area. It is necessary to implement additional procedures to minimize anxiety and distress in patients receiving a diagnosis of HPV or abnormal cytology and to improve awareness of sexually transmitted pathogens. Human papillomavirus (HPV) is a micro-sized, envelope-free deoxyribonucleic acid (DNA) virus that infects the cells of the deep layers  of the skin or mucosa . HPV infOncogenic types of HPV called high-risk HPV (HR HPV), mainly HPV 16 and 18, tend to cause most precancerous conditions and cancers of the cervix, as well as of the vulva, vagina, anus, penis, oral cavity, and throat. Persistent HR HPV infection is a major risk factor for the development of HPV-associated cancers of the anogenital region, as well as the head and neck area. Infection with non-oncogenic types of HPV called low-risk HPV (LR HPV), mainly HPV 6 and 11, can cause genital warts, including condylomata, and recurrent respiratory papillomatosis. However, they are not risk factors for cancers of viral etiology .Methods that reduce the risk of developing cervical cancer include primary prevention, such as HPV vaccination, and health education through the use of condoms, reducing the number of sexual partners, and general health behaviors, such as smoking cessation; as well as secondary prevention with screening tests .One can venture to say that in Poland sex education is poor and does not meet contemporary expectations or the needs of adolescents and young adults. The elderly were in a comparable situation at the time of sexual initiation and later. As a result, there is no widespread public awareness of the health and emotional consequences of sexual contact without physical protection (condom) and, when possible, immunological protection (HPV vaccination). The only information about the consequences of sexual imprudence concerns the potential risk of unplanned pregnancy. What is missing is information on the risk of sexually transmitted infections, such as HIV, syphilis, hepatitis B, hepatitis C, chlamydiosis and parasitic diseases , and HPV infection . NeverthPersistent HPV HR infection in the cervix can lead to the formation of high-grade squamous intraepithelial lesions (HSIL) or cervical intraepithelial neoplasia (HSIL CIN-2/CIN-3) lesions that precede the onset of cervical cancer. Women with histologically confirmed high-grade lesions (CIN2+) in the cervical mucosa are typically recommended to undergo surgical treatment, which involves the removal of the affected area using a procedure known as the loop electrosurgical excision procedure (LEEP) or large loop excision of transformation zone (LLETZ). The stress of being diagnosed with a disease closely related to a woman’s sexual intercourse and her partners, the prospect of surgery, the altered anatomical relations within the cervix after surgery, and the change in the location of nerve endings can all take a toll on patients’ overall well-being, mental state, and perception of sexual satisfaction.Studies have indicated that emotional reactions to a HPV infection test among women in the general population are also influenced by socioeconomic factors. Women with a lower education level, unemployed, younger age, unmarried, or not cohabiting may be more likely to experience adverse psychological reactions  associated with a positive HPV infection test. Information regarding a positive HPV test result in Poland is usually preceded by an abnormal gynecological cytology result. This is often accompanied by anxiety, fear of cancer, feelings of harm and injustice, and a search for the culprit of HPV infection. Currently, various reports related to the impact of a positive HPV test result on patients’ emotional reactions are available. Studies indicated that women receiving such a diagnosis are at risk of anxiety and depression ,7,8,9,10At present, HPV-related genital precancerous lesions appear frequently. While LSIL (low-grade) lesions are likely to resolve within a few years, HSIL (high-grade) lesions can cause a well-founded fear of progression and tend to recur, especially during the first few years after excitotoxic treatment. Consequently, they may also raise concerns about the consequences on sexual health. Referral for in-depth diagnostics, including colposcopic evaluation with histopathological verification is an integral part of providing medical services for the diagnosis and treatment of precancerous conditions of the cervix. The psychosocial impact of a positive HPV test can result, not only in psychological stress, but also significantly affect treatment outcomes through compliance. The World Health Organization (WHO) recommends counseling as an interpersonal communication strategy between a health professional and the female patient, to learn more about HPV and cervical cancer prevention and to give room for conversations about intimate topics, such as sexuality, disease, and death, and thus to encourage women to take preventive measures . Thus, mGiven the relevance of the topic of HPV infection and its health consequences, we aimed to systematically review the literature and summarize the conclusions from the available studies.The aim of this study was to review the available literature in terms of (1) the impact of abnormal cytology results/HPV infection on women’s mental health and sexual life; and the impact of (2) subsequent treatment interventions (LEEP/LEETZ) on women’s mental health and sexual life.On 26 September 2022, a search for articles was conducted in the MEDLINE, Embase, PsycINFO, and BIOSIS databases, with the aim of reviewing the impact of abnormal cytology results/HPV infection on women’s mental health and sexual life, and the impact of subsequent treatment interventions (LEEP/LEETZ) on women’s mental health and sexual life. Terms targeting anxiety, mood disorders, and sexual dysfunction related to a HPV-positive test or LEEP procedure were used, employing medical subject headings (MeSH), text words, and keywords. The following combinations of words were used:  AND . The findings were organized in accordance with the recommended format of the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines see . In accoOut of the 1351 records initially retrieved, 374 were found to be ineligible based on the inclusion criteria. After removing 374 duplicate articles, 609 articles were deemed eligible. Upon reviewing the titles and abstracts, an additional 1291 publications were excluded for the following reasons: involving animal models, being a review paper, or having a prospective observational design format. Other exclusion criteria included studies that did not describe patients with HPV diagnosis or an abnormal cytology result, related medical interventions, and psychological impact on mental health and sexual life. However, 60 articles met the inclusion criteria, of which two concerned the same clinical trial. The results are shown in We reviewed 609 publications on the impact of an abnormal cytology result, HPV infection, and subsequent medical interventions on women’s mental health and sexual life. Sixty records met the inclusion criteria . Fifty-fThe participants involved in the research had an abnormal cytology result or had received an HPV-positive test result. In order to understand the impact of an abnormal cytology result, HPV infection, and subsequent medical interventions on women’s mental health and sexual life, participants were screened and examined according to Diagnostic and Statistical Manual of Mental Disorders (DSM) criteria, using the Hospital Anxiety and Depression Scale (HADS) ,26,27,28This review includes 59 studies, where authors investigated the effect of HPV diagnosis on anxiety, depression, and psychosocial quality of life , while iThe observational studies were classified using the ROBINS-E tool. Fourteen of the fifty-six included studies were rated as ‘low risk of bias’ ,40,47,48Of the 49 studies assessing mental and sexual health among women with HPV infection, 45 were observational studies, of which 30 used validated tools to assess anxiety, depressive symptoms, quality of life and sexual function, and 15 were qualitative studies using unvalidated questionnaires, surveys, or interviews .Forty-six studies assessed the impact of the HPV diagnosis on depressive or anxiety symptoms: nine on quality of life, and 15 on sexual function .Nineteen studies simultaneously assessed more than one of the functional aspects mentioned above, and two concerned well-being in relation to the way medical personnel conveyed information. The following groups were considered: women positive for HPV (n = 22); women with genital lesions linked to HPV (n = 11); women with abnormal cervical cytology (n = 10); women with low-grade cytological abnormalities (n = 7), women diagnosed with high-risk HPV (n = 3); women diagnosed with a borderline smear (n = 3); and women undergoing a Pap smear, viral typing, or colposcopy (n = 2). Of the 44 studies assessing the impact of HPV infection on anxiety and depressive symptoms, 41 (93%) confirmed a negative impact of the diagnosis on these symptoms . Patients also reported feelings of fear and worry ,58,62,63The impact of the loop electrosurgical procedure on anxiety, depression, quality of life, and sexual functions was assessed in 10 studies . Seven oThis review summarizes the existing literature on the impact of an abnormal cytology result, HPV infection, and subsequent medical interventions on women’s mental health and sexual life. To the best of our knowledge, this is the first systematic review covering this topic. The available literature suggests that there was a broad diversity in the methodology used in the conducted studies. Most of the studies performed to date focused on the overall psychosocial impact, including anxiety, anger, disappointment, depression, fear, feelings of shame and guilt, and sexual functioning.The fear and anxiety caused by abnormal Pap smear results or positive HPV tests are largely due to the screening program itself . WithoutDespite such varied methodologies, it can be summarized that receiving information about abnormal cytology results or an active HPV infection has a negative impact on women’s mental health and sexual life. Among studies focused on measuring the impact of HPV diagnosis on anxiety or depression, as many as 89% confirmed such a negative impact . The impIt is important to emphasize that the women participating in the studies described shame, especially when they were informed in advance that HPV infection occurs mainly through unprotected sexual intercourse. Since sexual intercourse is still considered a taboo topic for most of society, in particular accidental intercourse, the women felt humiliation. McCaffery et al. found negative social and psychological implications associated with a positive HPV test result. Women were found to be stressed about disclosing their results to others . In addiAs regards the impact of the LEEP procedure on anxiety or depression and quality of life, we identified only four studies addressing this issue. These studies did not indicate any particular negative impact of the LEEP procedure on mental health. However, more research is needed, as studies addressing this issue are lacking. In contrast, more work has been done on the impact of the LEEP procedure on the quality of sexual life. We found fairly divergent results, with a predominance of no adverse effects of the LEEP procedure on sexual function, as indicated by 60% of the analyzed papers. The study conducted by Gaurav et al. indicated an overall improvement in sexual satisfaction, by minimizing the occurrence of persistent dyspareunia symptoms and postcoital bleeding in female patients . The benAn additional important issue that is worth addressing is the education and socio-economic factors of those receiving a positive HPV test result. Women with lower education and lower socio-economic levels showed significantly higher levels of stress and anxiety. This fact may be related to the better education, understanding, and knowledge of the causes and consequences of the virus infection in women classified with a higher socio-economic level ,41,59. SOverall, the analysis of the studies indicated a huge deficit in women’s knowledge about sexually transmitted diseases, especially about HPV and its oncogenicity. This highlights the need for an increase in sex education in society, especially concerning knowledge about these diseases and routes of transmission. In addition, information on HPV vaccination could reduce the risk of infection and thus the risk of cervical cancer. It can be assumed that such knowledge would result in a reduced incidence of unprotected intercourse or accidental intercourse. Moreover, full information about the available and emerging new treatments, both surgical and non-surgical  , would aTraining midwives in this area could be a prudent step. Moreover, providing informational and educational brochures to patients, allowing them to learn about the topic in the comfort of their homes, could also be a valuable solution. Furthermore, sexological care can be important for women, who may experience changes in their sexual life or relationships after diagnosis and treatment. This type of care can provide women with information and resources to address sexual concerns and improve their sexual function and satisfaction.Due to the differences in the outcome of mental health and sexual functioning measures, it was not possible to conduct a meta-analysis. Given that there are disparities between countries associated with health and welfare systems, we cannot conclude with certainty that the insufficient number of studies from other parts of the world did not affect the results of this study. In addition, we only included studies written in English.The results of this systematic review show that receiving a positive HPV test or abnormal cytology result has a negative impact on women’s mental health and sexual function. Moreover, although studies focused on sexual well-being and functioning after the LEEP procedure showed divergent results, the majority of studies did not confirm negative consequences of the procedure. Nevertheless, more randomized trials are needed to investigate this topic further.In addition, the studies included in this review provide insights into patients’ perceptions of their situation. There is still much work to be done in sexual education, including providing information about sexually transmitted diseases. It seems crucial to provide patients with professional information about their condition or planned medical procedures, which could significantly reduce their stress and anxiety.It is crucial to recognize that each woman’s experience with HPV and the LEEP procedure may differ, and their requirements for psychological and sexological care may vary. Thus, healthcare providers should offer personalized care that meets each woman’s unique needs and preferences, to ensure optimal outcomes.This review highlighted the need to implement additional procedures to minimize anxiety and distress in patients receiving a diagnosis of HPV or abnormal cytology, and to improve awareness of sexually transmitted pathogens. The impact of the LEEP procedure on mental health and sexual life has not been confirmed by the current studies, and further research in this area, on a larger group of women undergoing this procedure, is necessary. In addition, it would be worth expanding the study to a larger group of people, after implementing the aforementioned broad psychological and sexological support, along with extending information and educational activities. Studies focusing on concerns about getting pregnant in women with a positive HR HPV test could also be valuable.