We found that OSTE+ showed cytotoxicity, but OSTE+H2O did not. Extracts were analyzed using LC-MS and GC-MS in order to identify leaked chemicals.The flexibility of implantable neural probes has increased during the last 10 years, starting with stiff materials such as silicone to more flexible materials like polyimide. We have developed a novel polymer based on Off-Stoichiometry Thiol-Enes + Epoxy , which is up to 100 times more flexible than polyimide. Since a flexible neural probe should be more biocompatible than a stiff probe, an OSTE+ probe should be more biocompatible than one composed of a more rigid material. We have investigated the toxicity of OSTE+ as well as of OSTE+ that had been incubated in water for a week (OSTE+H2O extracts. The detected levels of each chemical found in the LC-MS and the GC-MS analysis were below the toxicity level when compared to MTT assays of all the individual chemicals, except for one of the initiators that had an IC50 value close to the detected levels.Most constituents were found in extracts of OSTE+, whereas only initiators were found in OSTE+Hin vivo studies.Our notion is that the toxicity of OSTE+ was caused by one of the initiators, by impurities in the constituents or by synergistic effects of low doses of leaked chemicals. However, our conclusion is that if OSTE+ is incubated for one week in water, OSTE+ is not cytotoxic and suitable for further  The current research on neural probes for deep brain stimulation has created new possibilities to treat conditions such as chronic pain and motor symptoms associated with Parkinson’s disease, in addition to enabling control of prosthetic limbs –4. DurinOff-Stoichiometry Thiol-Enes + Epoxy (OSTE+) is a new polymer that was developed to be used in microfluidic devices  instead There are several studies showing the biocompatibility of Parylene C, PDMS and polyimide –23. Howein vitro cytotoxicity assays (MTT assays) together with microscopy techniques and the analysis of possible constituents leaching from the materials was performed with liquid chromatography - mass spectroscopy (LC-MS) and gas chromatography – mass spectroscopy (GC-MS). This is an initial study of the cytotoxicity of OSTE+ and the results show that OSTE+, if handled correctly, renders low toxicity to cells, this warrants further studies in compliance with ISO 10993–1 in order to completely deduce the biocompatibility.In this study, our goals were to determine the biocompatibility of OSTE+ as well as investigate if there was leaching of constituents from the final polymer. The biocompatibility of various preparations of OSTE+ was tested using 2O throughout this paper. All samples were then incubated in water at 37 °C for 72 h (in compliance with ISO standard 10993–12) and the extractions were used in two different assay systems: chemical analysis and cytotoxicity analysis (in compliance with ISO standard 10993–5). The whole process is shown schematically in Fig. The manufacturing of the OSTE+ samples started with a molding step where the samples were cured in a PDMS mold using UV-light, followed by a post exposure bake at 65 °C. Half of the OSTE+ samples were then incubated in MilliQ water  at room temperature for 7 days to allow non-bound constituents to leach out. These samples are called OSTE+HThe cytotoxicity tests were performed using MTT assays in compliance with ISO standard 10993–5, and all samples were extracted using ISO standard 10993–12.OSTE+ is produced in a two-step polymerization reaction as previously described . First, In the first polymerization step, UV light was used o activate Lucirin® TPO-L  that initiated cross-linking between the thiol, tris[2-(3-mercaptopropionyloxy)ethyl] isocyanurate , and the two allyls, trimethylolpropane diallyl ether  and 2,4,6-triallyloxy-1,3,5-triazine . In the second polymerization step, 1,5-diazabicyclo[4.3.0]non-5-ene  was used as the initiator for cross-linking between the thiol and the epoxy resin, D.E.N 341 Epoxy Novolac resin .All ingredients were weighed according to a stoichiometric ratio of monomers of 1.5 : 0.47 : 0.53 : 0.5 for thiol, diallyl, triallyl and the epoxy resin, respectively. TPO-L and DBN were used at 0.2 %. Finally, 3 % acetone (Sigma Aldrich) was added to the mixture. All ingredients were mixed and degassed under vacuum for 5 min. The mixture was then poured into a pre-fabricated PDMS mold. The mold containing the mixture was then covered with a polycarbonate film and exposed to UV-light for 40 min using a Karl Suss MA4 mask aligner. The polycarbonate film was removed after the exposure and the OSTE+ pieces were hard-baked  in an oven at 65 °C overnight. The ratio of the different constituents results in an OSTE+ with a glass transition temperature of 39 °C .Polyimide was selected as a reference material since it is a material used in neural probes today , 20, 22.A sheet of HDPE  was cut into pieces using a milling machine. The pieces were cleaned with acetone (Sigma Aldrich) in an ultrasonic bath followed by a rinse in ethanol  and MilliQ water.2O, polyimide or HDPE samples and the water was 3 cm2/ml. The samples were washed in MilliQ water before extraction. The vials were gently shaken at 37 °C for 72 h. When the extraction solution was used in cytotoxicity tests, the polymer samples were first sterilized using an autoclave at 121 °C and the extractions were performed with sterile water.Extractions of polymer samples were obtained according to ISO standard 10993–12. The extraction was performed in cleaned borosilicate glass vials and water  was used as the extraction medium. The ratio of the surface area of the OSTE+, OSTE+H2O as well as blank samples, were analyzed using LC-MS.To obtain standards for the LC-MS analysis, an exact amount of each chemical used for the production of OSTE+ was dissolved in methanol (Sigma Aldrich) overnight (except acetone and D.E.N 431). The following day the solutions were diluted to a concentration of 500 μg/ml using 50 % MilliQ water and 50 % methanol. This was followed by a second dilution step to 50 μg/ml using MilliQ water. Acetone was not tested since it is not detectable in the LC-MS system used; D.E.N 431 was not tested since it is not water-soluble. To simulate the UV-lithography process of the fabrication of OSTE+, all standards were exposed to UV-light using a Karl Suss MA4 mask aligner for 40 min. All standards, extractions (described above) of OSTE+, extractions of OSTE+HA LC-MS system  was used with an Acquity CHS C18 1.7 μm 2.1x50 mm column and a sample volume of 5 μl. The mass spectrometer was configured to scan 120–1000 Da at 2 scans/s. Acetonitrile  and MilliQ water were used at a flow rate of 250 μl/min. The concentration of acetonitrile in the mobile phase was: starting at 5 % for 1 min, ramp to 95 % over 3 min, hold for 2 min, ramp down to 5 % in 0.1 min.2O extractions (described above) was pipetted to a new vial containing 1 ml of toluene. The toluene extraction samples were mixed using a vortex for 40 h, followed by centrifugation for 1 min. To ensure that all chemicals in the extracts were transferred into the organic phase, 2 samples of each extraction were made acidic by adding 50 μl of 0.1 M HCl (VWR) and 2 samples of each extraction were made basic by adding 50 μl of 0.1 M NaOH (VWR) just before the addition of toluene to each extraction. The extracts spiked with HCl are referred to as OSTE+/HCl and OSTE+H2O/HCl throughout the paper and the extracts spiked with NaOH are referred to as OSTE+/NaOH and OSTE+H2O/NaOH throughout the paper. All samples and extracts together with a sample of pure toluene were analyzed using GC-MS. Acetone was not tested since it is used as a cleaning step in the GC-MS analysis.To obtain standards for the GC-MS analysis, an exact amount of all constituents (except acetone) was dissolved in toluene (Sigma Aldrich and VWR). To simulate the UV-lithography process of the fabrication of OSTE+, all samples were exposed to UV-light for 40 min using a Karl Suss MA4 mask aligner. 400 μl of the OSTE+ extractions or OSTE+HAll analyses were performed on a gas chromatograph (Agilent 6890) equipped with an injector in split mode at 250 °C, equipped with an auto sampler (sample volume of 5 μl) and a mass spectrometry detector (Agilent 5973-N). The column used in the system was a HP-5 ms fused silica capillary column (5 % phenyl-methylpolysiloxane) of 30 m × 0.25 mm with a phase thickness of 0.25 μm. The temperature program of the column was: 50 °C, hold for 1 min, ramp to 300 °C over 10 min, hold for 15 min. The carrier gas used was helium at a flow rate of 1.2 ml/min. The mass spectrometer was run in either scan mode (30–600 Da with 2.6 scan/s) or in selected ion monitoring (SIM) mode, using mass to charge ratios (m/z) of 70, 81, 82, 99, 123, 125, 147, 187, 197, 254, 321 together with a dwell time of 20 ms.Four OSTE+ pieces were cut into two halves. One half was put into MilliQ water for 7 days and then each pair was studied by scanning electron microscopy  to see if 7 days of incubation in water affected the surface of OSTE+.2O, polyimide and HDPE. Polyimide was selected since it is regarded as biocompatible and there are several studies that can be used for comparison. HDPE is a biocompatible material that sometimes is used as a negative control in different cell viability assays [Four different materials were tested in the MTT assay: OSTE+, OSTE+Hy assays , 25, 26.i.e. RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine, at 37 °C in 5 % CO2 in air. The cells were sub-cultured twice every week at a density of 12 500 cells/cm2.The mouse fibroblast cell line L929 was purchased from American Type Culture Collection  and was grown as a monolayer culture in complete cell culture medium, i.e. a final extract concentration of 50 % in 1x concentrated RPMI-1640 containing 10 % FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine, before addition to cell cultures Further dilutions of the 50 % concentrated polymer extracts were done in 1x concentrated RPMI-1640 medium supplemented with 10 % FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine.The extracts were obtained as described above and kept at 4 °C for a maximum time of 24 h before use in the cytotoxicity assay with L929 cells. The extracts were diluted 1:2 in 2x concentrated RPMI-1640 medium supplemented with 20 % FBS, 200 U/ml penicillin, 200 μg/ml streptomycin and 2 mmol/l L-glutamine, 2 in humidified air for 24 h. The medium was then removed and changed to medium containing the different extracts at concentrations ranging from 6.25–50 % medium containing 2.5 % dimethyl sulfoxide (DMSO: positive control) or control medium (complete cell culture medium with 0 % extract). The cells were then kept at 37 °C in 5 % CO2 in humidified air for 72 h before addition of MTT to a final concentration of 0.5 mg/ml. The 96-well plates were incubated with MTT at 37 °C for 1 h before removal of the medium, and the cells with blue formazan crystals were then dissolved in DMSO. The following spectrometry reading at 540 nm was done using a SPECTRAmax M2 instrument  and analyzed using SoftMax® Pro v. 4.6 (Molecular Devices). A number of 3–6 biological replicates  were used for all the treatments of the L929 cells and a number of 6 technical replicates (i.e. number of n) from each extract were used for each biological replicate. After correcting all absorbance values for background, the percent of control was calculated as absorbance units in the presence of test compound as percentage of that in control. The results of the MTT assay are resumed to reflect the cell number, thus, 50 % percent MTT reduction of control implies that there were 50 % less cells at that treatment concentration [L929 cells were seeded in complete cell culture medium in 96-well plates (2000 cells/well in 180 μl medium) and were then incubated at 37 °C in 5 % COntration .2 incubator for 24 h before addition of the chemicals. Indicated concentrations of the chemicals were added to the wells and the cells were incubated for 72 h at 37 °C in 5 % CO2 in humidified air. The dose response was evaluated using MTT as described above.Each chemical used in the fabrication (except acetone), 1 or 0.1 mg, was added to a sterile flask and 1000 μl sterile water was added. Further dilutions were made using sterile water. D.E.N 431 was not soluble in water and after shaking the sample and letting it stand for a while, only the water solution was used in the test. L929 cells were seeded in complete medium in 96-well plates (2000–3000 cells/well in 180 μl medium) and the plates were incubated in a CO2 in humidified air for 24 h. The growth medium was then removed and changed to growth medium without extract or containing the different polymer extracts . The cells were then incubated for 72 h before they were photographed with an Olympus CKX41 inverted phase contrast microscope  equipped with an Olympus SC30 camera (Olympus Corporation) then analyzed using the software cellSense Standard version 1.4 (Olympus Corporation). The cells were then fixed in 3.7 % paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS). The fixed cells were stained with Alexa Fluor 647-conjugated phalloidin in PBS containing 1 % bovine serum albumin and 1 % Tween-20 for 1 h and in PBS containing 1 μg/ml bisbenzimide for 1.5 min to visualize the cytoskeleton and nuclei, respectively. The glass coverslips were mounted on glass slides using Mowiol 4–88 and left in a refrigerator overnight. The samples were imaged with a LSM510 confocal laser scanning microscope  equipped with a Hamamatsu R6357 photomultiplier . A 405 nm diode-pumped solid-state laser was used to excite the bisbenzimide and a band-pass filter of 420–480 nm was used for the emission. The Alexa Fluor 647-conjugated phalloidin was excited with a 633 nm HeNe ion laser and a 650 nm long-pass filter was used for the emission. The cells were visualized by z-stacks composed of single optical planes in high magnification and the images were analyzed with the software ZEN 2012 (black edition) version 8.0 (Carl Zeiss Microscopy GmbH).L929 cells were seeded in 12-well plates containing sterile glass coverslips, whereupon they were incubated at 37 °C in 5 % CO2O and blank samples were analyzed using LC-MS.Standards of all constituents (except acetone and D.E.N 431), extracts of OSTE+, extracts of OSTE+Href is the concentration of the standard, href is the peak height of the standard in the TIC and h is the peak height detected in the sample for that specific mass to charge ratio that corresponds to the chemical in question.From the total ion current (TIC) chromatogram together with the molecular structure of each chemical and the mass to charge ratio the lower limit of detection (LOD) for each chemical could be calculated as well as the detection level for each chemical in the samples Table . The LOD2O extracts and blank samples are shown in Fig. 2O extracts only DBN and TPO-L were detected. It should be noted that each peak shown in Fig. 2O. Unidentified contributors to the peak heights imply that there were impurities in the chemicals or that they are derived from the chromatographic set up. When comparing the peak height of all the different chemicals in the OSTE+ and OSTE+H2O extractions, the detected levels of each chemical are either lower or below LOD for OSTE+H2O. Acetone and D.E.N 431 were not analyzed since they are not water-soluble and standard curves could not be made.TIC chromatograms from the LC-MS analysis of OSTE+ extracts, OSTE+H2O and blank samples were analyzed using GC-MS. The standards were analyzed using the GC-MS in scan mode. Peaks and different mass to charge ratios for all chemicals were obtained from this analysis. In order to increase the sensitivity of the GC-MS, all mass to charge ratios found when running the GC-MS in scan mode were used to run the GC-MS in SIM mode. Analysis of data from running the GC-MS in SIM mode allowed for the calculation of the lower limit of detection of all chemicals  whereas the cells photographed in the confocal microscope were fixed and stained with phalloidin and bisbenzimide to visualize the cytoskeleton (actin filaments) and the nuclei, respectively  and of the individual chemicals that were used in the OSTE+ fabrication process. The morphology of cells treated with extracts was investigated using phase contrast and confocal microscopy. GC-MC and LC-MS were used to analyze chemicals found in extracts of OSTE+ and extracts of OSTE+H2O. SEM was used to investigate the surface structure of OSTE+ and OSTE+H2O.OSTE+ is a new polymer that is more flexible then other polymers used for neural probes. This should allow probes constructed in OSTE+ to follow the micro motions of the brain, hence increasing the biocompatibility of the neural probe . Our groThe results of the cytotoxicity test Fig.  imply th2O extracts, which is expected since they are only used as initiators in the process and are not immobilized in the final polymer network. Because the detected levels of most constituents were close to the respective LOD, some may still have leached out into the OSTE+H2O extracts. The IC50 value found for DBN  showed that the water-soluble chemicals DBN, diallyl, triallyl and TPO-L were found in the extracts made from OSTE+. But only DBN and TPO-L were detected in the OSTE+HBN Table  is closels Table  the IC50in cells , 31. Thu2O samples. The nano-sized cracks were somewhat wider in the OSTE+H2O sample. In our previous work we did not use acetone in the mixture and we did not see any cracks when using that formula [Since oxygen will inhibit the UV-initiated polymerization step and PDMS has a high oxygen permeability , 33, the formula . We therin vivo tests have to be conducted.OSTE+ is a very promising material to use for neural probes because of its superior flexibility compared to other polymers used in neural probes today. The toxicity of OSTE+ extracts was presumably derived from DBN, unknown impurities in the constituents or from low dose synergetic effects of the constituents. However, by pre-incubating the final polymer in water for 7 days, OSTE+ was shown to be nontoxic to cells. The use of high-purity chemicals in the fabrication process, removal of acetone from the fabrication, the addition of a cleaning step in water during fabrication and the use of a silicon wafer as the substrate instead of PDMS should minimize the toxicity rendering implants made from OSTE+ nontoxic. In order to ensure the biocompatibility of OSTE+ for neural implants both short and long term


HPV is the most prevalent sexually transmitted infection and its effect in cancer induction is well documented. HPV infections are mostly asymptomatic, but it is unclear whether HPV infections can result in alterations of reproductive health.To determine the relationship between human papillomavirus infections and reproductive health in both men and women.A systematic literature review was performed in PubMed and ScienceDirect data bases from January 1994 through August 2014.HPV infections are shown to be significantly associated to many adverse effects in the reproductive function. These adverse effects were reported in different levels from cells production to pregnancy and may be related to the infecting genotype.It appears from this study that HPV detection and genotyping could be of great value in infertility diagnosis at least in idiopathic infertility cases. Like for the risk of carcinogenesis, another classification of HPV regarding the risk of fertility alteration may be considered after deep investigations. Human infertility is defined as the inability for a couple to conceive and produce offspring after at least twelve consecutive months of unprotected sexual intercourse. This definition seems insufficient because it does not take into consideration all the cases. In effect, for many couples, this inability can be overcome by a treatment or a medically assisted procreation procedure after diagnosis . InfertiInfertility results from multiple factors that are responsible for impairments in the reproductive function in men and/or women. These factors include congenital and hormonal disorders, lifestyle, environmental hazards and psychological state. All these factors can lead to impairments in the function of genital organs, the production of reproductive cells, semen quality, sperm cells transport to oocyte, fertilization and embryo implantation steps –6. In thNeisseria gonorrheae, Treponema pallidum and Chlamydia trachomatis can lead to fertility alterations ..13].The literature search retrieved a total of 1021 hits from which 114 records were selected for screening. Duplicated hits and articles that were not written in English or French were excluded. There remained 62 records that underwent the abstract screening. After abstract screening and full text reading, a total of 21 articles met our inclusion criteria. The whole search data flow diagram is presented in in vitro fertilization or pregnancy outcomes. Authors mostly discuss the relationship between HPV and fertility parameters without giving special consideration to HPV persistence. Selected articles were pooled into three different groups according to the reproductive health aspect they are focused on:A group of nine (9) articles that studied the effect of HPV on semen parameters –22.in vitro fertilization outcomes [A group of five (5) articles that are focused on the effect of HPV on outcomes –27.And a group of seven (7) articles that studied the effect of HPV infection on pregnancy outcomes –34.It appears from this investigation that many research teams were interested in the possible interactions between HPV infection and the human reproductive health. Data from diverse studies about the relationship between HPV and reproductive health are now available and concern different aspects of the reproductive function. In effect, articles that met inclusion criteria were mainly focused on HPV infection and its relationship with semen parameters, in-vitro experimental studies show that exposure to HPV DNA induces DNA fragmentation in sperm cells. As in somatic cells, DNA fragmentation in spermatozoa is a signal that leads to apoptosis. It is noteworthy to consider that the effect of HPV DNA on semen could be genotype dependent. When exposure to DNA from HPV 16 or 31 leads to DNA fragmentation in sperm cells, nothing occurs when these cells are exposed to DNA from HPV types 18, 33 or 6 /11. In the same studies, it was also shown that DNA from HPV 16 or HPV 6/11 reduce more significantly the amplitude of sperm cells lateral head displacement compared to DNA from HPV types 18, 31 and 33 [It is now well established that HPV infections in men result in semen contamination. This contamination was suggested to be responsible for many alterations in semen quality . In effe1 and 33 ,15. In a1 and 33  recordedSeveral epidemiological studies revealed that HPV is more prevalent in infertile men or those with leukocytospermia compared to fertile ones , 18, 22.in vitro experiments.From the selected studies, an Italian cross-sectional study failed to find association between HPV and semen parameters . This coIn vitro fertilization (IVF) and artificial insemination are medically assisted solutions for conception in many infertile couples. These approaches are proposed to couples for which semen parameters are altered or infertility is due to cervical hostility to spermatozoa. Considering the fact that HPV is associated with altered semen parameters, in vitro fertilization and embryo transfer could be considered a viable alternative in cases of reduced fertility associated with semen HPV infection.It's well known that sperm cells adsorb HPV particles preferentially in two distinct sites along the equatorial region. This adsorption does not abrogate the fertilization potency of sperm cells but, it is unclear whether HPV infection has effect on steps following fecundation . TherefoTwo distinct studies revealed a significant association between HPV infection and IVF failure. In the Italian study , the ratConsidering that HPV does not interfere in oocyte fertilization, the adverse effects of HPV in IVF may be explained by: i) cervical HPV-induced cytological lesions or ii) eventual alterations of the fetal health. The first hypothesis was explored in two different studies. Netherland study shows a significant association (p< 0.02) between HPV-induced cervical lesions and fertility reduction, the rate of high grade cervical lesions is two times higher in sub-fertile women eligible for IVF in comparison to the general population . HoweverIn comparison to studies related to HPV oncogenic effect in the female genital tract, there is a lack of studies on HPV effects in female fertility parameters such as hormonal balance or oocyte production. The most explored subject is the relationship that could exist between HPV infection and pregnancy outcomes. Most studies explored the subject epidemiologically but in vitro experimentations have been made to understand the effect of the virus in the fetal health. To highlight the effects of HPV on the pregnancy outcomes, we compared the results of studies that investigated the linkage between HPV infection and miscarriages or premature rupture of the membrane during pregnancy.In vitro study of Gomez et al. [z et al.  proved tCase-control epidemiological studies were performed comparing the HPV prevalence of women who miscarried and that of controls. The results of the included studies are reported in When cervical HPV infection is considered instead of placental or abortive tissue infection, significant association is found between HPV positivity and premature rupture of membrane . If HPV An Italian case-control study explored the possible role of HPV in recurrent miscarriages, comparing HPV prevalence in women with history of multiple miscarriages and in women with at least one pregnancy at term and no history of spontaneous miscarriages. The results show that HPV prevalence was higher in the group of women without a history of recurrent miscarriages. To explain this observation the authors hypothesized that: i) the immune reaction is potentially responsible for recurrent miscarriages and could be protective against cervical HPV infection ii) women with recurrent miscarriages were effectively HPV positive during their difficult pregnancies but the infection cleared more rapidly than women without recurrent miscarriages .Thus, HPV infections can lead to adverse effects such as premature rupture of membrane or miscarriages. In fact, HPV trophoblasts infection corrupts the embryo’s health and its ability to invade the uterine wall. Even if cervical HPV infections can remain silent without cellular lesions, infected women can bear a healthy embryo but they should be paid more attention because of virus transmission to babies, a membrane premature rupture or miscarriage risks.in vitro studies have been made to explore this subject.HPV infection is well known for its ability to induce malignancy in the ano-genital and aero-digestive regions. The other aspect of HPV infection that deserves more attention is its association to damages that lead to reduced fertility or sterility. Both epidemiological and It appears from this study that HPV can be associated to i) apoptosis in sperm cells; ii) alterations of semen quality through cell count decrease, amplitude of lateral head displacement reduction, mobility reduction and increase of anti-sperm antibodies level; iii) apoptosis in embryonic cells; iv) miscarriages or premature rupture of membrane.If HPV is not the main cause, it must be considered as a risk factor of reduced fertility or infertility. Effectively, HPV detection in pregnant women or on their partners can be considered as a risk of preterm birth, miscarriages and virus transmission to the newborn. Thus, it will be useful to consider HPV detection in both men and women for infertility diagnosis and before IVF procedures.Overall, some questions remain unanswered and many studies are required to clarify the mechanisms underlining the effects of HPV in both female and male reproductive system and to determine the role of HPV persistence in fertility alterations. In effect, we don't know if reduced fertility or infertility associated to HPV reflects transient effects and if total fertility will be restored after HPV clearance. Finally, as for cytological lesions induction, HPV genotype classification according to the risk of sterility and fertility reduction may be envisaged.S1 Checklist(DOCX)Click here for additional data file.


Military as family explored how many veterans experienced the military environment as a “family” that took care of them and provided structure. Normal is alien encompassed many veterans experiences of disconnection from people at home, lack of support from institutions, lack of structure, and loss of purpose upon return to civilian life. Searching for a new normal included strategies and supports veterans found to reconnect in the face of these challenges. A veteran who had successfully transitioned and provided support and advice as a peer navigator was frequently discussed as a key resource. A minority of respondents—those who were mistreated by the military system, women veterans, and veterans recovering from substance abuse problems—were less able to access peer support. Other reconnection strategies included becoming an ambassador to the military experience, and knowing transition challenges would ease with time. Results were consistent with and are discussed in the context of homecoming theory and social climate theory. Social support is known to be protective for veterans, but our findings add the nuance of substantial obstacles veterans face in locating and accessing support, due to disconnection and unsupportive institutions. Larger scale work is needed to better understand how to foster peer connection, build reconnection with family, and engage the broader community to understand and support veterans; interventions to support reconnection for veterans should be developed.Afghanistan and Iraq veterans experienced traumas during deployment, and disrupted connections with friends and family. In this context, it is critical to understand the nature of veterans’ transition to civilian life, the challenges navigated, and approaches to reconnection. We investigated these issues in a qualitative study, framed by homecoming theory, that comprised in-depth interviews with 24 veterans. Using an inductive thematic analysis approach, we developed three overarching themes.  United States (US) service members who served in the wars in Afghanistan and Iraq faced long and often multiple deployments and a constant risk of injury and death ,2. In adThe high levels of psychological, substance use, and physical health problems in Afghanistan and Iraq veterans are well documented ,10–13. VHomecoming theory—developed after World War II—serves as a valuable framework for understanding the challenges in the transition from military service . HomecomHomecoming theory emphasizes the theme of disconnection or alienation as part of the challenge faced by returning veterans. Yet little is known about how veterans who served in the wars in Iraq and Afghanistan experience the transition—i.e. the challenges of return to civilian life, key differences between military and civilian environments, how veterans navigate challenges and approach reconnection, as well as what resources mitigate transition difficulties. Gaining insight into the transition experience is critical to understand the difficulties faced by returned veterans as well as to inform interventions to support successful readjustment. To address these issues, we conducted a qualitative investigation of Afghanistan and Iraq veterans’ transition from military to civilian life.We conducted in-depth interviews with 24 Afghanistan and Iraq veterans in 2009–2011 in California, US. Veterans were recruited purposively to include both women and men and a range of ages, race/ethnicities, and military service branches. The study was advertised through organizations and events that served a wide array of veterans. Veterans were not recruited based on care seeking or referral for psychiatric or other health problems. Interested veterans contacted the Study Director (MW) and were informed that the study concerned veterans’ experiences upon return from the wars in Iraq and Afghanistan. Veterans were given the option to complete the interview in person or by telephone, and were offered a $50 gift card in compensation for their time. Written informed consent was obtained for in-person interviews, and oral informed consent was obtained for telephone interviews. Written informed consent was not possible for telephone interviews because the respondent and Study Director were in different locations. In instances of oral informed consent, the Study Director printed the respondent’s name, and signed and dated the form to indicate she received consent. All study procedures, including all consent procedures and forms, were reviewed and approved by the Committee for the Protection of Human Subjects at the University of California, Berkeley.The semi-structured interview guide focused on what was most helpful during the transition to civilian life and what challenges made transition more difficult. Veterans were asked to narrate their experiences, with probes for experiences with family, friends, other veterans, and the general community. See military as family underwent revision) and added new themes (unsupportive institutions was added at this stage). When JA and JM coded different themes, they discussed and came to agreement upon the theme(s) that best fit each interview section. For presentation in this paper, selected quotes are the most evocative and illustrative of a theme, considering also the need for brevity.We applied a thematic analysis approach to the interview data ,19. The The 24 veterans represented a wide range of demographic and military characteristics see . Ages ramilitary as family, normal is alien, and searching for a new normal.Three overarching themes were developed and are elaborated below: Many veterans experienced challenging conditions and traumatic events while in the military. These included combat and improvised explosive devices (IEDs), as well as environmental challenges such as sand and wind, and close quarters residence on Naval vessels. Respondents indicated the uniqueness of these experiences with phrases such as “only we know what we’ve been through.” Despite the challenges of service, the military environment itself was experienced as a “family” that took care of service members and provided a structured set of expectations. The overarching theme of military as family contains two subsidiary themes: a) caretaker and b) structure.I went into West Point at 18… [and in the past it was] my parents taking care of all of that, and then [the military] taking care of all that… as someone who had major medical work done and was going to need more done it was frightening. I didn’t understand how [civilian health care] worked, and honestly I barely understand how it works now… I was used to being in a system where everything just made sense.Nearly half (10 of 24) of the veterans viewed the military as an institution that took care of its members. Multiple participants described the military as family, e.g. “the guys that I served with, they were my family over there. So, we have that connection—that bond. And there's always somebody that you can talk to.” Others described the environment in terms reminiscent of the care a family would provide to a child such as “holding their hand,” “safety net,” and “comfort.” One female veteran described how the military fulfilled a parental role related to medical care after she left home:A career Reservist who had a leadership position described taking on a parental role in helping calm distressed soldiers in challenging situations by giving them concrete and simple tasks to complete.A small minority of veterans thought that the military had turned against them during service. They expressed a profound feeling of betrayal, analogous to the experience of being betrayed by family. As one female veteran who had been sexually assaulted in the military explained, “I mean, Iraq—you expect people to shoot at you, you expect people to die, you expect people to be killed. You don't expect your fellow soldiers to turn on you.”Nearly half of veterans (11 of 24) noted that the structure in the military environment provided clarity and simplicity to decisions and procedures. They described the structure of the system as something to “hold onto” in the chaos of a war zone, and also as a framework that provided the opportunity to excel. The environment was described as “black and white”—a setting in which orders are given and obeyed. A male veteran in his mid 20s described the structure as a key reason that some people return to the military after separation, “they want that structured life again because… you get used to it if you do it long enough. And then you get out, then all of a sudden you've got to take care of yourself.” This description also illustrates how structure is an important component of military family caretaking. Similarly, the career Reservist who gave soldiers concrete and simple tasks in stressful situations was providing structure as a way to take care of his soldiers.normal is alien captures veterans’ experience of alienation upon return to civilian life. Veterans frequently talked about civilian life as “normal” while it was clear that many aspects of civilian life no longer felt normal to them upon return from military service. This reflected the mismatch between expectations that coming home would be a welcome return to “normal”, and the reality that what used to feel “normal” felt alien due to the changes in the veteran and changes at home. This overarching theme is directly connected to military as family, which contains the subsidiary themes of caretaker and structure, because the departure from the structure and caretaking within the military environment was a key part of what generated a feeling of alienation from “normal” civilian life. Normal is alien includes four subsidiary themes: a) disconnection, b) unsupportive institutions c) lack of civilian structure and d) loss of purpose.The overarching theme of Upon return to civilian life the vast majority veterans (19 of 24) felt disconnection from people at home, including family and friends, who had not shared the experience of military service. Veterans felt that those who had not served in the war could not truly understand them or their experiences during service. As one respondent lamented, “I can tell stories all night long and [my family] probably won’t really grasp what’s going on.” Most families were making major efforts to provide support to veterans, but due to this feeling of disconnection, veterans faced difficulties in accessing the support offered. Veterans also described feelings of disconnection when friends, acquaintances and strangers made “unwarranted assumptions” about people in the military and military service, asked insensitive questions about veterans’ experiences in service , or tried to connect veterans experiences to their own in a way that was not seen as respectful.Veterans expected their service to be honored, but many (15 of 24) felt they did not receive deserved support from the military, the Department of Veterans Affairs (VA), and other institutions in the transition back to civilian life. This generated and exacerbated feelings of alienation. Some veterans reported that the military did not provide needed resources for the transition to civilian life. As one respondent explained, “A lot of the [Officers] don't send people to [the class to facilitate transition to civilian life]. And the class is available. It's not like they don't offer it.”Other respondents reported that their mental health problems were not appropriately diagnosed or handled. As a veteran who had recently retired from a long military career explained, “All too often, they refer to these injuries as the invisible injuries of warfare… They're not invisible to loved ones… So who are they invisible to?… They're invisible to leaders who don't want to recognize the mental and emotional and spiritual sides of warriors.” Others reported that the medical care provided by the VA was inadequate.Beyond the VA, some veterans reported inflexibility of community institutions. For example, one respondent explained the frustrations of being unable to translate military emergency medical technician (EMT) training and experience into a civilian EMT job, “‘You can drive an ambulance… [but] you can’t treat anybody.’ But I’ve done all these things. Look at my training certificate. ‘Oh we don’t honor that certificate.’ That’s really hard to deal with.” Another veteran described being unable to take advantage of a veteran loan program because the banks he approached did not want to deal with the restrictions and costs.…there are certain expectations that you can rely on while you're over there, such as… [when] things need to be done, they're done, [because] your life depends upon it. Whereas here, there's a lot more leeway. And initially when I came back, I just couldn't deal with that gray area that's neither black nor white, and people talking back [and] making excuses… So I was pretty quick to get mad or frustrated.A quarter of respondents (6 of 24) expressed that the difference between the highly structured military environment and the less structured civilian environment created challenges in organizing their lives, and frustration in dealing with people at home. For example, one respondent said “… everything [in the military] is structured…. And then you get out and all of a sudden you've got to take care of yourself.” Another veteran talked about this issue as one of the biggest challenges, “…just switching from that military style of chain of command and clear goal into all of a sudden having to make your own decisions. I think a lot of guys have trouble with that.” This problem also manifested in frustration with family members, friends and classmates (for veterans who attended college) when they were not punctual and did not respect authority. One respondent noted:It’s really hard to put in words but I just miss the environment. I miss the common goals… the way people put aside their own personal [agendas]… I used to run a lot and [the] feeling is just like that…‘yeah man, let’s go do it!’ I’ve never really gotten the same thing on the civilian side, even though I try and pour my heart into things.A substantial proportion of respondents (10 of 24) noted that civilian life lacked meaning and purpose, and that they no longer felt they were contributing to an important communal effort. As a female veteran in her mid 50s explained:When I did start working at the surplus store… I had a very mixed feelings about it… I saw myself doing security [or] first aid, something that validated my experiences over there. But then after searching for a job for two months, I was grateful to have found one. So, it was such a mixed feeling and I guess a wake-up call, you know, that it just didn't matter that I had been to Iraq.The experience of a lack of meaning was intensified when veterans could not find jobs they felt were important or drew upon their skills. As a young male veteran reflected:searching for a new normal captures the approaches, resources and perspectives that helped support veterans’ successful transition to civilian life. For many veterans, family members were an important foundation that offered practical help such as a place to live, and tried to understand and support them. However, veterans often found it hard to engage that support due to feelings of alienation from individuals who had not shared the experience of military service. Three subsidiary themes emerged about connections or perspectives that eased the challenges of transition and supported veterans to find their way to a fulfilling civilian life: a) support from a navigator, b) embracing an ambassador role, and c) ease with time.The overarching theme of Veterans who had help from a veteran peer or a veteran specific support system to navigate the transition to civilian life had substantial advantages. For 13 of 24 this was an important source of support. Veteran peers provided invaluable help with practical issues such as how to access veteran benefits, and how to manage more general civilian tasks. As one veteran explained, “What’s been helpful, really, is just friends [who] have gone out before me. They’ve been really helpful in telling me… what needs to be done after the military. They had nobody to really guide them, they had to find out the hard way.” A key feature was that these veterans had successfully navigated the transition out of the military; peers who were themselves in the throes of transition challenges were not consistently perceived as helpful or supportive. Another veteran found that an uncle who was also a veteran was the only one who was able to continue to support him as he struggled with a drug addiction, “He would call, and he would always let me know that he'd be there, and whenever I was ready to come back to earth, he would be there. But my mom and everybody, they just didn't--no one understood what I was going through.”Veterans coordinators at colleges and universities also provided important support and practical help, “There was [a veterans representative at the University]… She was always asking me how I was doing in my classes, and then she pointed me in the right direction [to get services from] the VA.” While most of the comments about navigators focused on help provided with practical tasks, it was clear that emotional support was also conferred as part of the help provided by navigators.In contrast, veterans who had been mistreated or betrayed while in the military, such as those who experienced sexual assault, often did not feel comfortable with those who served in the military. Thus they could not access veteran peers as a source of support and had to contend with psychological effects of the mistreatment, as well as alienation from people at home. Women veterans were less able to connect to other veterans, although they did find veterans coordinators at colleges and universities to provide helpful support. A few veterans recovering from substance use problems found that spending time with veteran peers was not supportive of recovery because alcohol use was common, and thus they also faced challenges in accessing peer support.Some veterans (7 of 24) found that embracing the role of being an ambassador to the military experience was a way to connect to people at home and to feel like they were doing something important. One veteran explained how he tried to help civilians understand his experience in the service, “they think ‘Saving Private Ryan’. And that’s not true… so every time I talk to a civilian I have to explain. It’s like giving a lesson.” In addition to explaining the military experience to civilians, some veterans were asked to describe their experiences at public events, “Memorial Day they asked me to wear my uniform and say a couple of things. So I did, and it was fun… I got a lot of handshakes. It was cool.” This approach helped mitigate feelings of alienation.Some veterans (8 of 24) discussed the challenges of transition as inevitable but easing over time. The initial months of transition were often characterized by considerable alienation, more frequent substance use, and conflicts with others. As one veteran described, “I know I always offend people. And then I just say ‘Oh, I’m sorry. It’s only been 90 days. It’s the crazy period.’ And then I always calm down afterward.” Simply knowing that the challenges of transition would ease with time helped veterans be patient with the experience. Overall, veterans who discussed this topic indicated that about a year was required to readjust to civilian life. Veterans who described struggles lasting well beyond a year were generally dealing with serious exacerbating issues such as psychopathology, substance use problems, or experiences of sexual assault in the military.Overall, a substantial proportion of veterans experienced the military as a family that took care of them and provided structure. Upon return to civilian life, many veterans experienced alienation and lack of support from institutions—including from the military and veterans’ agencies themselves—and faced a substantial lack of structure and loss of purpose. In the face of these challenges, the most common resource for a successful transition back to civilian life was a veteran who had successfully transitioned, and provided support and advice as a peer navigator. A peer who has successfully adapted to civilian life may uniquely understand another veteran’s experiences and be able to provide timely help navigating benefits  and other practicalities. A minority of respondents—those who reported the military system had turned against them, women veterans, and veterans recovering from substance abuse problems—were less able to access peer support.Consistent with homecoming theory, many veterans reported experiences during military service that they viewed as unique, including combat and environmental challenges . These sThe experience of alienation described by many participants is consistent with homecoming theory, which posits that environmental differences and personal change lead to a mismatch between expectations and reality when the veteran returns; this experience has also been documented in research on US veterans of previous wars –7. RecenProblems with unsupportive institutions have also emerged in work with college student veterans, who described “feeling abandoned” when they did not receive helpful transition training from the military, and when they faced difficulties accessing VA services . Lack ofThe struggles veterans experience with a lack of structure and purpose upon return to civilian life are key emerging issues in research in Afghanistan and Iraq veterans. Differences between military and civilian culture have increased over the past century, and issues of structure and purpose are exacerbated in an environment in which it is difficult to find employment . ProblemWhile the challenges faced by returning veterans are in many ways unique, homecoming theory postulates certain commonalities of homecoming transitions . ConsistHomecoming theory posits that reestablishing connections is critical to a successful transition . AlthougOther work with veterans has recommended fostering relationships with military peers as a way to support reintegration, but the focus has been primarily on emotional support rather than navigational and practical support . InterveSome veterans were able to take on the role of an ambassador as a way to connect with civilians, and it is possible that embracing an ambassador role might be helpful for more veterans. Reaching out in this way to bridge the divide required a degree of stability and confidence, but also might help build a sense of confidence that could assist veterans in making a healthy transition. Overall, the findings indicate that veterans are helping one another, and helping civilians to understand them. It would be worth exploring how civilians could do more to effectively bridge the gap, and to reduce the perception that individuals who have not been in service cannot understand veterans’ experiences. Although the experiences at war may be difficult to understand, most civilians have experienced the feeling of alienation and could empathize with that aspect of the experience. In addition, there are many civilians who have made transitions between cultural contexts during their lives who would understand issues of separation, change and disconnection. The notable success of some representatives at colleges in helping veterans indicates that with the right training, support and connection is possible. There are high quality materials and trainings on what the community can do to support veterans, such as those developed by Swords to Plowshares , and it To our knowledge, this is the first study of a sample drawn from a general population of veterans to examine the transition experience in depth; a recent study examined the transition experience of seven veterans in college . A majorOverall, it is critically important to better support veterans in transition given the long-term risks to those who do not transition successfully ,15,48. OS1 Interview Guide(PDF)Click here for additional data file.


Introduction. LeFort colpocleisis (LFC) is a safe and effective obliterative surgical option for older women with advanced pelvic organ prolapse who no longer desire coital activity. A major disadvantage is the limited ability to evaluate for post-LFC gynecologic malignancies. Methods. We present the first case of endometrioid ovarian cancer diagnosed after LFC and review all reported gynecologic malignancies post-LFC in the English medical literature. Results. This is the second reported ovarian cancer post-LFC and the first of the endometrioid subtype. A total of nine other gynecologic malignancies post-LFC have been reported in the English medical literature. Conclusions. Gynecologic malignancies post-LFC are rare. We propose a simple 3-step strategy in evaluating post-LFC malignancies.  Symptomatic pelvic organ prolapse affects millions of women. Approximately 200,000 surgeries for POP are performed in the United States annually  and repr2. She reported a family history of breast cancer in her maternal aunt but denied any other personal or family history of any other malignancies. Her medications included daily low-dose acetylsalicylic acid and a calcium supplement. She denied being on any hormonal replacement therapy. Recent colonoscopy, mammogram, and Papanicolaou test were normal. She denied smoking, alcohol use, and use of illicit drugs. Urogynecologic evaluation using the Pelvic Organ Prolapse Quantification (POP-Q) system revealed stage 4 uterovaginal prolapse. The patient also had postmenopausal atrophic vaginitis. There were no palpable masses. In favor of a definitive and long-lasting treatment for her POP, she was counseled about vaginal reconstructive and obliterative surgery options. Uterine and ovarian conservation were requested by the patient following normal clinical examination. She underwent LeFort colpocleisis, levator plication, perineorrhaphy, and cystourethroscopy. She had a quick recovery and was doing well at her 8-week postoperative visit with no recurrent prolapse. She presented 6 months later with painless vaginal spotting with no other associated symptoms. Atrophic vulvovaginitis was suspected as a cause of this spotting and the patient was treated with topical vaginal conjugated equine estrogen. Her bleeding persisted despite a month of therapy. At this point, further evaluation included abdominal and rectal exams that were normal. Transvaginal ultrasonography was not feasible with an obliterated vagina. An abdominal ultrasound was limited by body habitus but showed a questionable 4 × 4 cm pelvic fluid collection with no particular pattern on Doppler mode and was thought to resemble a hematoma. Endometrial thickness was 5 mm. In light of this undiagnosed vaginal bleeding, the suspected pelvic fluid collection and thickened endometrium the patient was counseled about the differential diagnosis of vaginal bleeding including atrophic vaginitis and other gynecologic sources, namely, cervical and endometrial pathology and she was informed of the need to further evaluate her bleeding. We offered the patient the option of colpocleisis “reversal” by taking the repair down and reaccessing the cervix for a proper endometrial evaluation but she had refused given the excellent prolapse correction. Furthermore, our experience in accessing the cervix and endometrium through the lateral vaginal canals is minimal and thus the decision to perform a total abdominal hysterectomy (TAH) to evaluate the endometrial lining and the questionable fluid collection was taken. During the TAH, an 8 cm × 5 cm × 5 cm left ovarian irregular mass was identified. Initial visual and tactile evaluation of the pelvic and abdominal structures was normal except for cecal and posterior vaginal nodules. Intraoperative gynecologic oncology consultation was obtained. A frozen section of the left ovary revealed adenocarcinoma. Staging then followed. Pelvic washings were obtained upon initial abdominal entry. Uterus, fallopian tubes, right ovary, and omentum were resected. Peritoneal biopsies were also obtained. All these specimens were negative for metastatic disease. Resected cecal and posterior vaginal nodules were positive for metastatic adenocarcinoma. She was diagnosed with stage IIIB grade 1 endometrioid ovarian cancer.A previously healthy nulligravid and menopausal 76-year-old female presented with worsening pelvic organ prolapse (POP) symptoms. Her medical and surgical histories were significant for left shoulder and right knee surgeries. She denied any history of abnormal Papanicolaou tests and sexually transmitted illnesses. Besides obesity, she denied having any medical problems. Her body mass index was 35 kg/mThe patient underwent adjuvant chemotherapy with paclitaxel and carboplatin thereafter. She tolerated the treatments well and was doing well at her 3-month postchemotherapy follow up. Her CA-125 dropped from 25 u/mL to 6 u/mL at the time of initial diagnosis and by her last chemotherapy session, respectively. Both limited vaginal exam and digital rectal exam were normal. Chest X-ray and abdominal/pelvic computed tomography scan were unremarkable. She remains in remission 2 years after chemotherapy and regularly follows up with the gynecologic oncologist.Although the development of gynecologic malignancies post-LFC is rare, it is more than a theoretical risk . We reviColpocleisis is a safe and effective obliterative surgical option for older women with advanced pelvic organ prolapse (POP) who no longer desire coital activity . CompareDue to the obliterative nature of LFC, postoperative gynecologic evaluation is limited. This reflects the importance of preoperative assessment and counseling. Patients should be made aware of the potential delay and limitation in diagnosing gynecologic pathology post-LFC. The precolpocleisis exam should include a careful speculum exam with visual inspection of the cervix and vagina followed by palpation of the entire vagina  as well The postcolpocleisis gynecologic exam includes an evaluation of the external genitalia in addition to a limited vaginal and complete rectal exam. There are currently no practice guidelines for the evaluation of gynecologic malignancies post-LFC. In light of the limited literature available, we compile a simple 3-step approach in evaluating post-LFC malignancies: (1) routine gynecologic exams; 2) cytology/biopsy; and (3) an imaging study. This 3-step approach should be used in any suspected gynecologic malignancy post-LFC, and not with suspected endometrial pathology only. As in prior reported cases, sending a sample of the drainage for cytology may be diagnostic particularly with vaginal and cervical lesions  cytology. ImagingLeFort colpocleisis is a safe and effective obliterative surgical option for older women with advanced pelvic organ prolapse who no longer desire coital activity. Significant tissue pathology is not anticipated; however, many patients with prolapse are elderly and are at a risk of developing a gynecologic malignancy . Commonl


The early detection of intraepithelial lesions of the cervix, through the periodic examination of cervical cells, has been fundamental for the prevention of invasive cervical cancer and its related mortality. In this report, we summarise the cervical cancer screening activities carried out in Catalonia, Spain, within the National Health System during 2008–2011. The study population covers over two million women resident in the area. The evaluation includes 758,690 cervical cytologies performed on a total of 595,868 women. The three-year coverage of cervical cytology among women aged between 25 and 65 years was 40.8%. About 50% of first screened women with negative results had not returned to the second screening round. The introduction of high-risk human papillomavirus DNA (HPV) detection, as a primary screening cotest with cytology among women over age 40 with a poor screening history, significantly improved the detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+), being far superior to cytology alone. Cotesting did not improve the detection of CIN2+. The use of the HPV test for the triage of atypical squamous cell undetermined significance (ASC-US) improved the selection of women at high risk of CIN2+.Sampling (both cytology and HPV test) was largely performed by midwives (66.7%), followed by obstetricians (23.8%) and nurses (7%). Over half of the centres (54.8%) had full use of online medical records. During the study period, educational activities for professionals and for women were carried out periodically.The organisation of screening as a population activity in which women are actively called to the screening visit and the introduction of HPV testing as a primary screening tool are strongly recommended to ensure the maximum population impact in the reduction of the cervical cancer burden. The early detection of intraepithelial lesions of the cervix through the periodic examination of cervical cells has been fundamental for the prevention of invasive cervical cancer and its related mortality . The impIn 2006, the autonomic region of Catalonia established specific guidelines for the prevention of cervical cancer within the public health sector , with a The aim of this report was to provide a summary evaluation of the activities performed during the period 2006–2012 to facilitate better planning for new updated guidelines. For this, we estimated the coverage of screening, the interval between cytologies within a first screening round and the rescue of underscreened women. We also evaluated the value of HPV testing as a cotest with cervical cytology in the underscreened women and in the follow-up of women with a diagnosis of atypical squamous cell of undetermined significance (ASC-US) –12. FinaThe information was derived from the following sources:1. Information system of primary care services (SISAP)The SISAP reports the medical care activities within the primary health care system of 75% of the residents in Catalonia. Through this system, we were able to evaluate the coverage, the interval between cytologies, the number of HPV tests requested and the results of the different tests by age categories for centres requesting the exams, health regions, and for the overall population of Catalonia. We included information on women over age 15 years. The evaluation of the interval between cytologies was measured as the follow-up of women that were screened for the first time in 2008 and had a normal cytology test as specified elsewhere ..10].We evaluated the triage of ASC-US using HPV test for the prediction of CIN2+ in 611 women with a mean age of 34.5 years (range 15–79 years) [Finally, for the purpose of monitoring the interlaboratory reproducibility of performance of HPV detection technique, a total of 946 samples were re-tested in a second laboratory during the period 2008–2011. A total of 44 (4.6%) discrepancies were found, and the overall correlation coefficient between the two measurements was 0.95 (p < 0.05) for the continuous evaluation of RLU values, while the kappa value for positive/negative was 0.91, demonstrating an almost excellent interlaboratory agreement for all 12 participating laboratories [Among all ASSIR centres, 83.3% declared to follow the 2006 protocol. ASSIR centres reported that cervical cancer screening activities involved multidisciplinary professionals. In 52% of the ASSIR centres, midwives were responsible for the identification of women susceptible to cervical cancer screening. In 23.8% of the centres, the administrative staff helped in detecting underscreened women through the routine registration of cytologies. Sampling (both cytology and HPV test) was largely performed by the midwives (66.7%), followed by obstetricians (23.8%) and nurses (7%). The communication of Pap results was carried out by obstetricians and midwives in similar percentages , while communicating the results of HPV tests was performed more often by obstetricians (59.5%) compared to midwives (35.7%).About 5% of the centres reported warning systems to identify women who were not routinely screened. Some centres referred that coordination with primary health care teams facilitated the recruitment of women with inadequate screening, while highlighting the facilitating role of social entities working with socially disadvantaged groups. Almost all centres (97.6%) reported specific activities  to contact women with an abnormal cytology or a positive HPV test.Most centres (54.8%) used the online medical records that were compatible with the systems of other centres, allowing an automatic download. However, still less than 40% of the centres had this technology available. Specifically, the results from 40.5% of cytologies, 28.6% of HPV testing, 40.5% of colposcopy, and 35.7% of biopsies were registered manually. In 7% of the centres, cytological results were communicated via email.In most centres (97.6%), indicators related to screening for cervical cancer were part of the NHS yearly quality evaluation of the centres. Also, 73.8% of centres trained their professionals on preventive activities including cervical cancer prevention, although educational programmes were reported on an irregular basis.http://www20.gencat.cat/portal/site/cancer) [During the period 2006–2012, there were two information campaigns aimed at the general population, and many scientific conferences were held for professionals at the primary health level at the start of the protocol implementation. A free online course was offered to professionals involved in screening, in order to reinforce the contents of the protocol and its applicability in clinical practice. The course also addresses basic knowledge about the HPV vaccine. A total of more than 1,500 professionals in Catalonia were enrolled. Further, the detailed protocol was available on the website of Canal Cancer of the Health Department (/cancer) .Leaflets were distributed to all centres to increase awareness in the general population in the Catalan and Spanish languages. Furthermore, dissemination activities in the regular press and in the local radio channel were occasionally conducted.This report summarises the cervical screening coverage within the NHS in Catalonia, Spain. Data are based on electronic medical records of over two million women including cervical cytologies and/or HPV tests perfomed per women and in some instances including also pathology records . The selAfter evaluation of over 750,000 cytologies performed in the study period, the coverage per women in a three-year period was estimated to be 40% among those aged 25–65 years. In an ancillary study, when non-attenders were asked about the reasons for not attending the NHS centres, 30% of women reported visiting a private gynaecologist and largely on an annual basis . Thus, tThrough the follow-up of screened women, we were able to identify that about half of the women that were screen-negative at first round had not returned to be screened at the recommended three-year interval. In the 2006 protocol, no active follow-up was included in the majority of centres, thus when the recommended interval is long (3 years) women may either forget the time of the next appointment or go to a private practice within a shorter interval. We do not know the underlying cause of such a failure to return but special action is needed to retain attenders. Further analysis at a longer interval is currently being done to further evaluate the spontaneous return to screening visits.Overall, there was a very satisfactory evaluation of the introduction of the HPV test for its high validity in CIN2+ detection, and easiness to be implemented in laboratories of different complexity , 11. MosFurther, the results of the proficiency testing assessment among laboratories for the HPV test showed that the HC2 assay has a high interlaboratory concordance .Given these satisfactory results, HPV testing seems a clear candidate to substitute by large the use of cervical cytology as the primary screening tool. However, it is relevant to consider that the extension of HPV testing in the general population as primary screening should be done in a controlled manner for proper implementation. Strict monitoring of age, intervals between the test and referral rates should be established together with regular quality controls . It is rAnother key element in the evaluation of this protocol was the availability of the information systems at the primary health level . The facWhen exploring organisational aspects, It was found that teams were multidisciplinary with an important contribution of midwives in the identification of women susceptible to screening, sampling and reporting of cytology results if negative.The introduction of the protocol has been complemented by training activities in which a free online course was included for professionals on cervical cancer prevention with a good record of acceptance.Based on the results of this evaluation , the urg


Oncoviruses such as HPV, KSHV, and EBV have been reported in patients with HIV infection and AIDS. How oncovirus-associated cancers rise in AIDS patients has not been fully established. The purpose of our study was to identify the viral agents in vulvar cancer and to assess their contribution to pathogenesis.We retrospectively identified a total of 13 vulva tissue samples from HIV-1 positive and 9 vulvar samples from HIV-1 negative patients from the Botswana National Health Laboratory in Gaborone, Botswana, a Southern African country with a high incidence of HIV. We utilized PCR and IHC to identify HPV, EBV, KSHV, and JC virus in FFPE preserved tissue samples.+/GP6+ primer set we detected several HPV types in tissue samples. EBV was detected in all of the positive cases (100%) and in most of the negative cases (89%). KSHV was detected in 39% of the HIV-1 positive samples and in 11% of the negative samples, and no JC virus was detected in any of the samples.Using the GP5Using IHC we demonstrated that LANA was expressed in 61% of the positive samples and in 44% of the negative samples. The ubiquitous EBV was more consistently expressed in negative cases (100%) than in positive cases (69%). Interestingly, the HPV-16 E6 transcript was detected in 56% of the negative samples compared to 31% of the positive samples. However, the cell cycle protein P21 used as a surrogate marker for HPV was detected in 77% of the positive samples and in 44% of the negative samples, while VEGF signals were similar in both positive (92%) and negative samples (89%).Our study, suggests that in Botswana, vulvar squamous cell carcinoma (VSCC) is associated with oncogenic viruses present in the niche but the contribution and progression may be regulated by HPV and other immunosuppressive infections that include HIV-1. Chlamydia trachomatis in vulvar intraepithelial neoplasia Kwasniewska et al. reported that 73% were HPV-16 positive, 14.63% were HSV-2 positive, and 14.63 Chlamydia trachomatis positive [There are two types of vulvar cancer. The first type that develops from intraepithelial neoplasia caused by human papillomavirus (HPV) infection, shows multifocality, and is increasing in prevalence among young women and especially those with multiple co-infections including HIV . The secpositive . Hampl eIn sub-Saharan Africa there has been a paucity of vulvar carcinoma studies and in the referenced studies the rates of vulvar carcinomas are low and recently more associated with HIV infection -9. ImporThe aim of this study was to identify the oncogenic viruses in vulvar carcinoma tissues in HIV positive and negative vulvar cancer patients from Botswana and to assess the differential expression of cell cycle proteins and growth factors that may be associated with transformation of cells. Further, from strong evidence associating these proteins with G1 phase arrest dysregulation and angiogenesis in some cancers we wanted to confirm their expression in VSCC -15.+/GP6+ 94°C for 5 minutes, 94°C for 30 seconds, 48°C for 30 seconds, and 72°C for 30 seconds, for 40 cycles; elongation at 72°C for 5 minutes; and then incubation at 4°C. For EBV-94°C for 5 minutes, 94°C for 30 seconds, 47°C for 30 seconds, and 72°C for 30 seconds, for 40 cycles; elongation at 72°C for 5 minutes; and then incubation at 4°C. For KSHV- 94°C for 5 minutes, 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 30 seconds, for 40 cycles; elongation at 72°C for 5 minutes; and then incubation at 4°C. The PCR products were run on 2.5% agarose gel at 100 V for 1 hour.We retrospectively identified a total of 13 vulva tissue samples from HIV-1 positive and 9 vulvar samples from HIV-1 negative patients from the Botswana National Laboratory in Gaborone, Botswana  . We usedThe data for this study was collected in Excel and imported into a statistical software program. Analysis encompassed determination of proportions  was used for analysis.+/GP6+ primers we detected HPV in 67% of the negative cases and 54% of the positive cases  ; 18 ; 45 ; 33 ; and 31 were detected in the tissues of patients   was identified in positive cases more than in the negative controls by PCR  Table  and by IWe assayed for the cell cycle regulators p21 Figure .John Cunningham (JC) virus , which hCD4 has been used as a marker of immunocompetence in HIV infected patients, with higher CD4 counts indicating better immune response in these patients to opportunistic infections and other pathogenic infections. We assessed CD4 counts in relation to Cyclin D1 in the VSCC cases and controls. We observed that in the HIV positive VSCC cases the counts were 437.9 compared to 409.8 for the controls. For p21 signals the CD4 counts were 410 for the HIV positive cases and 457 for the HIV negative controls. For VEGF positive samples CD4 counts were 430.1 for the HIV positive cases and 477 for the HIV negative controls.The CD4 range for HIV positive individuals who were not currently taking HAART average was 392 compared to HIV positive cases taking HAART with an average of 457.5.There was a significant difference between cases who were currently on HAART and those not taking HAART . When comparison was made between HAART therapy and human papilloma virus detection by PCR HPV-18 , HPV-33 , HPV-31 , HPV-6 , as well as oncogenic herpesviruses EBV , and KSHV  were significant while HPV-16 , HPV-45 , and HPV-11  were borderline significant . This strongly suggests that in vulvar cancer, HIV may be contributing to the initiation of the cancer in younger females through pathways that have not been fully delineated , whereas there were significant differences for p21 . For the growth factor VEGF  also showed a significant difference for women who are on HAART compared to controls (Table Studies indicate that a majority of keratinizing squamous cell carcinomas usually found in older women are likely to be non-HPV related ,25. Yet,INK4A resulting in hyperproliferation that can be detected by antibodies against Ki-67 nuclear antigen present in all cell cycle stages of proliferating cells except for the G0 phase that was expressed to a lower level than HPV-16 E7 in our samples (Figure The viral genes of HPV-16, E6 and E7 have been shown to interfere with 2 pathways associated with cell cycle deregulations. E6 has been shown to interact with p53, causing p53 dysfunction and eventually disrupting cell cycle arrest . HPV-16 Perturbation of cell cycle proteins was also considered a factor in VSCC, thus we assayed for Cyclin D1 which is in the p53 pathway, p21 which is downstream target of p53, and VEGF whose contribution to vascularity is considered essential for tumor progression -15,31,32Though our sample size is small we do suspect that similar immunological surveillance dysfunction and dysregulation of specific pathways associated with viral oncogenesis may be a factor in vulvar carcinoma pathogenesis. Additional levels of investigation are needed in HIV-1 associated vulvar carcinoma to delineate the dysregulation these viruses will initiate that leads to oncogenesis.The findings of different oncogenic viruses and expression of cancer associated markers in tissues of women with and without HIV in Botswana with VSCC warrants further investigation with a larger sample size that may lead to identification of molecular moieties for targeted therapeutic and vaccine development. This Botswana cohort was unique in exposure to different environmental and infectious agents that may have additional epigenetic effects on the outcome of VSCC .The authors declare that they have no competing interests.KOS designed the study, conducted experiments, interpreted data and drafted the manuscript and supervised the study, HCJ interpreted data and drafted the manuscript, MK identified VSCC tissues, CK identified VSCC tissues, and ESR designed the study, interpreted data, edited the manuscript, and supervised the study. All authors have read and approved the final manuscript.


Human papillomavirus (HPV) serology is a main factor for designing vaccination programs and surveillance strategies; nevertheless, there are few reports of HPV seroprevalence in the general population, especially in Latin America. This study aimed to describe high-risk HPV serological prevalence, persistence, and association with concurrent cervical infection, in Chilean women.1021 women from the general population, aged 15–85 years, were studied in 2001 of whom 600 were reexamined in 2006. The assessments at both time points included cervical HPV DNA testing, HPV antibody testing, cervical cytology and a sociodemographic/behavioral questionnaire. HPV DNA and antibodies against L1 protein of types 16, 18, 31, 33, 35, 45, 52, and 58 were assessed by reverse line blot and multiplex serology, respectively.Seropositivity was high at both baseline (43.2%) and follow-up (50.2%) and increased with age (p < 0.001); corresponding DNA prevalences were 6.7% and 8.7%. DNA and seroprevalence were associated at baseline (p = 0.01 for any HPV). Early age at first sexual intercourse and having had two or more sexual partners were independently associated with seropositivity. Most (82.0%) initially seropositive women remained seropositive at follow-up; 21.6% of initially seronegative women seroconverted, reaching 17.5% among women older than 60 years of age. ASCUS or worse cytology was correlated with HPV DNA positivity but not with HPV seropositivity.HPV seroprevalence studies are a useful tool for learning about the dynamics of HPV infection in a community. This study contributes to understanding the natural history of HPV infection and provides a baseline assessment before the incorporation of HPV vaccination into a national program. Chile is a Latin American country where screening programs for cervical cancer have been effective in decreasing the burden of this malignancy . NeverthHuman papillomavirus (HPV) is the most common sexually transmitted infection in the world and persistent infection with high-risk HPV (HR-HPV) types is a necessary cause of cervical cancer . A populWhile HPV DNA informs about current cervical infection, incident or persistent, it is not a marker for cumulative exposure to the virus . CumulatAs part of a population-based study of the natural history of HPV infection in women from Santiago, Chile ,16, we eThe present analysis was performed on a random sample of 1393 Chilean women from the general population of a low socio-economic area of Santiago, Chile. A detailed description of the study design, enrollment, data collection procedures and results of HPV DNA infection has been published previously . BrieflyAll participants gave written informed consent. The study protocol was approved by the ethical committees of the Ministry of Health’s South-Eastern Health Service and of the International Agency for Research on Cancer.Collection of cervical samples for HPV DNA testing has been described previously ,16. BrieThe method used for HPV DNA testing has been described in detail elsewhere ,16. BrieSerum samples were analyzed at the German Cancer Research Center (DKFZ), Heidelberg, Germany. The presence of IgG antibodies against the major capsid protein L1 of HR-HPV types 16, 18, 31, 33, 35, 45, 52 and 58 was assessed using multiplex serology based on glutathione S-transferase fusion proteins, which has been broadly used in epidemiological studies . The incSeropositivity cutoffs for each specific HPV type have been previously defined ,21. BrieFor the following analyses, “any HPV” refers to seropositivity or DNA positivity for at least one of the following HR-HPV types: 16, 18, 31, 33, 35, 45, 52, and 58.Any, type-specific and age-specific  HR-HPV seroprevalence (at baseline and follow-up), seropersistence, seroclearance and seroconversion (at follow-up) were assessed. Seropersistence was defined as the number of women who were seropositive at baseline and follow-up among all women who were seropositive at baseline. Seroclearance was the number of women who were seropositive at baseline and seronegative at follow-up among all those seropositive at baseline. Seroconversion was the number of women seronegative at baseline and seropositive at follow-up among those seronegative at baseline.Baseline seropositivity was compared with concurrent cervical DNA positivity to assess concordance, which can only be examined at the type-specific level, as women are often infected with multiple types of HPV and the concordance status can be different for each type. To overcome such limitation, analyses were performed at the genotype level using generalized estimating equation (GEE) models that account for the lack of independence between multiple observations from the same woman ,23.Univariable and multivariable GEE models were performed to identify predictors of HR-HPV seroprevalence at baseline. Risk estimates were adjusted by potential risk factors for HPV seropositivity: age at baseline, marital status, education level, number of children, age at first sexual intercourse, lifetime number of sexual partners, high-risk sexual partner , history of sexually transmitted disease, hormonal contraception, smoking, condom use and cervical HR-HPV DNA infection. Variables with p-value < = 0.2 in the univariate analysis  were then included in multivariate models. Tests for linear trend of ORs were performed by giving an increasing score for each level of the categorized variable and fitting them into the model as continuous variables. Predictors of HR-HPV seropersistence were evaluated by using GEE models, as described above for HPV seroprevalence; no variables had p-value < = 0.2 in the univariate analyses, so estimates presented were adjusted by women’s age at baseline.All tests were two-sided and the results with p < 0.05 were interpreted as statistically significant. Statistical analyses were performed using STATA version 11.The median age of the 1021 women studied at baseline was 43 years (range: 15-86) and for the 600 women at follow-up it was 48 years (range: 19-87). Prevalence of ASCUS+ cytology was 3.7% (37/994) at baseline and 2.8% (16/573) at follow-up. ASCUS+ cytology was five times more prevalent in DNA-positive women (12.8%) than in DNA-negative women (2.6%). On the other hand, ASCUS+ cytology was similarly prevalent in seropositive (3.6%) and seronegative women (3.1%) (data not shown).Cervical HR-HPV DNA positivity for at least one of the eight studied HR-HPV types was 6.7% at baseline and 8.7% at follow-up. At baseline, the most prevalent types were HPVs 16 (2.5%), 58 (1.3%), 31 (1.1%), 45 (0.7%) and 52 (0.7%), representing 83.3% of DNA-positive women; at follow-up, most prevalent were HPVs 16 (3.0%), 18 (2.5%), 45 (1.7%), 31 (1.0%) and 52 (1.0%), representing 92.3% of DNA-positive women (data not shown).At baseline, almost half of the participants were seropositive for at least one of the eight studied HR-HPV types, and seropositivity increased by 16% at follow-up  women were negative for both HPV DNA and antibodies, while 33 (3.3%) women were DNA and antibody positive. Seropositivity was 50.0% among DNA-positive women and 57.2% among DNA-negative women  presented seroclearance, 12.0% (21.6% of seronegative at baseline) seroconverted, and 36.5% (82.0% of seropositive at baseline) presented seropersistence. HPV 16 seropersistence and seroconversion were 80.5% (95% CI: 72.8-82.3) and 7.4% (95% CI: 4.8-9.2), respectively (data not shown). Our dataset was under-powered to detect significant differences in seropersistence and seroconversion by age, though seropersistence was high (74-89%) in all age groups, except for ages 15-20 years , and seroconversion was between 18% and 27%, also excluding the youngest age group  of the eight most common HR-HPV types; 41% of women already had antibodies at age 15-19 years. This supports the recommendations that HPV vaccination should begin at younger ages. Interestingly, any HPV and type-specific seroprevalence increased with age even at older ages, consistent with data from cross-sectional studies in Taiwan , China , MongoliLife-time number of sexual partners, marital status, HSV-2 infection, oral contraception, smoking and viral load have been postulated in several studies as determinants of HPV seropositivity ,27,32,33Data on the natural history of HPV antibody response is limited; few studies report longitudinal measurements of both DNA and antibodies. In our study, the majority of the seropositive women at baseline were also seropositive five years later (82% for any HPV). Previous cohort studies have shown that most women who seroconverted remained seropositive until the end of follow-up (follow-up periods from 18 months to 5 years) -36. ObseLimitations of the present study include the referred lack of intermediate assessments and the relatively low sample size that prevented us from performing more informative type-specific analyses. Strengths of the study include the population-based study design and prospective analysis, and the use of a high-throughput serologic test that allows the simultaneous identification of antibodies against eight HPV types.This study provides for the first time estimates of seroprevalence, seropersistence and seroconversion of eight HR-HPV types in Chile. These findings are useful to better understand the epidemiology of the immune response to HPV and also to inform cervical cancer prevention strategies in middle developing countries in Latin-America and elsewhere. The high HPV seroprevalence, already elevated below age 20 years, as well as the increase after age 50 years support the initiation of vaccination programs at young ages and the established screening practice at older ages in Chile. This data constitutes a baseline assessment before the incorporation of HPV technologies in cervical cancer prevention.HPV: Human papillomavirus; HR: High-risk; MFI: Median fluorescence intensity; GEE: Generalized estimating equation; OR: Odds ratio; CI: Confidence interval; ASCUS+: Atypical squamous cells of undetermined significance or worse cytology.The authors declare that there is no competing interests.CF was responsible for the study conception, design and implementation, and analysis and interpretation of data. KP coordinated the field work. PS was responsible for the DNA testing and collaborated with data analysis. MP was responsible for the serological testing and collaborated with data analysis. AD performed the statistical analyses. FC, SF, and VV were responsible for data analysis and interpretation. CF, FC, VV and AD were responsible for the manuscript preparation. All authors revised the manuscript critically and approved the final version for publication.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/361/prepubBaseline characteristics of women who participated only at baseline and those who participated at both baseline and follow-up. Santiago, Chile, 2001 and 2006.Click here for fileBaseline characteristics of women by serological status. Santiago, Chile, 2001.Click here for filea high-risk human papillomavirus among women in Santiago, Chile, 2001-2006.Determinants for seropersistence of anyClick here for file