Opposing impacts of HLA-G haplotypes PROMO-G010104-UTR-3 and PROMO-G010101b/c-UTR-4 on risk of recurrent implantation failure.

RESEARCH QUESTION: The human leukocyte antigen (HLA) class Ib molecules HLA-F and HLA-G are implicated in pregnancy success, but how do HLA-G and HLA-F genetic polymorphisms impact recurrent implantation failure (RIF)? DESIGN: Prospective cohort study at a fertility clinic including a cohort of 84 women experiencing RIF and 35 IVF controls to assess the influence of HLA-G haplotypes and diplotypes and HLA-F single nucleotide polymorphisms (SNP) on RIF. RESULTS: Over-representation trends for HLA-F SNP genotypes rs1362126, rs2523405 and rs2523393, previously linked with a short time-to-pregnancy, were detected in female control groups compared with RIF patients with no identified pathology linked to infertility. The HLA-G promoter haplotype PROMO-G010101b/c linked with the HLA-G 3'-untranslated region (3'UTR) haplotype UTR-4, which previously has been associated with positive IVF outcome and pregnancy success, was less frequent in the RIF group. For RIF patients carrying the UTR-4 haplotype, the odds ratio (OR) was 0.27 (95% CI 0.12-0.66; P = 0.0044, Pc = 0.026). The HLA-G PROMO-G010104-UTR-3 haplotype was associated with an increased risk of RIF. For RIF patients carrying the UTR-3 haplotype, the OR was 5.86 (95% CI 1.52-26.23; P = 0.0115, Pc = 0.069). CONCLUSIONS: These results show that specific HLA-G haplotypes based on the promoter region and the 3'UTR are either associated with an increased risk of reduced fertility, including the manifestation of RIF, and lower chance of achieving pregnancy, or with a reduced risk of experiencing RIF.

A cervical screening initiative for elderly women in Denmark.

INTRODUCTION: To assess outcome of a one-time human papillomavirus (HPV)-screening in 2017 of Danish women aged 70+. MATERIAL AND METHODS: Women born 1947 or before were personally invited to have a cell-sample collected by their general practitioner. Screening- and follow-up samples were analyzed in hospital laboratories in the five Danish regions and registered centrally. Follow-up procedures varied slightly across regions. Cervical intraepithelial neoplasia 2 (CIN2) was recommended treatment threshold. Data were retrieved from the Danish Quality Database for Cervical Cancer Screening. We calculated CIN2+ and CIN3+ detection rates per 1000 screened women, and number of biopsies and conizations per detected CIN2+ case. We tabulated annual number of incident cervical cancer cases in Denmark for the years 2009-2020. RESULTS: In total, 359 763 women were invited of whom 108 585 (30% of invited) were screened; 4479 (4.1% of screened, and 4.3% of screened 70-74 years) tested HPV-positive; of whom 2419 (54% of HPV-positive) were recommended follow-up with colposcopy, biopsy and cervical sampling, and 2060 with cell-sample follow-up. In total, 2888 women had histology; of whom 1237 cone specimen and 1651 biopsy only. Out of 1000 screened women 11 (95% confidence interval [CI]: 11-12) had conization. In total, 579 women had CIN2+; 209 CIN2, 314 CIN3, and 56 cancer. Out of 1000 screened women five (95% CI: 5-6) had CIN2+. Detection rate of CIN2+ was highest in regions where conization was used as part of first-line follow-up. In 2009-2016, number of incident cervical cancers in women aged 70+ in Denmark fluctuated around 64; in 2017 it reached 83 cases; and by 2021 the number had decreased to 50. CONCLUSIONS: The prevalence of high-risk HPV of 4.3% in women aged 70-74 is in agreement with data from Australia, and the detection of five CIN+2 cases per 1000 screened women is in agreement with data for 65-69 year old women in Norway. Data are thus starting to accumulate on primary HPV-screening of elderly women. The screening resulted in a prevalence peak in incident cervical cancers, and it will therefore take some years before the cancer preventive effect of the screening can be evaluated.

Time trends in prevalence of p16 positivity and combined HPV/p16 positivity in a large cohort of Danish vulvar cancer patients.

Vulvar cancer is rare, but causes substantial morbidity in affected patients. A subset of vulvar cancers is caused by high-risk human papillomavirus (hrHPV), which primarily exerts its oncogenic effect through upregulation of tumor suppressor protein p16. Tumors positive for both hrHPV and p16 (double positive) are assumed to be HPV-driven, but only few large studies have investigated the combined prevalence of hrHPV and p16 positivity in vulvar cancer over time. In this Danish cross-sectional study, we assessed the prevalence of p16 positivity and double positivity for hrHPV and p16 in a large sample of vulvar squamous cell carcinomas (VSCCs) diagnosed during 1990 to 2017. In a nationwide register, we identified VSCCs from 13 hospitals across Denmark, and collected archival tumor tissue for hrHPV testing with INNO-LiPA and immunohistochemical p16 staining. We calculated the prevalence of hrHPV, p16 positivity and double positivity according to time, age and histological subtype and evaluated time trends through estimated annual percentage changes. We included 1278 VSCCs. Overall, 35.0% (95% confidence interval [CI]: 32.4-37.6) were positive for p16 and 31.0% (95% CI: 28.4-33.5) were positive for both hrHPV and p16. The prevalence of p16 positivity and double positivity increased over time, both in women aged ≤59 and ≥60 years. The double positive prevalence was higher in nonkeratinizing (60.7%) and warty/basaloid VSCCs (67.5%) than in keratinizing (16.1%) and verrucous VSCCs (5.0%). These results indicate that approximately one-third of vulvar cancers were caused by hrHPV infection, supporting a substantial preventive potential of the HPV vaccine.

Endometrial HLA-F expression is influenced by genotypes and correlates differently with immune cell infiltration in IVF and recurrent implantation failure patients.

STUDY QUESTION: Is human leukocyte antigen (HLA)-F protein expressed in mid-secretory endometrium, and are its expression levels influenced by HLA-F gene polymorphisms and correlated with the abundance of uterine natural killer (uNK) cells and anti-inflammatory M2 macrophages? SUMMARY ANSWER: HLA-F protein is expressed in mid-secretory endometrium, and levels are correlated with immune cell infiltration, plasma progesterone concentrations and HLA-F single-nucleotide polymorphisms (SNPs), however, women experiencing recurrent implantation failure (RIF) show differences when compared to women attending their first IVF treatment. WHAT IS KNOWN ALREADY: The immunomodulatory HLA class Ib molecules HLA-G and HLA-F are expressed on the extravillous trophoblast cells and interact with receptors on maternal immune cells. Little is known regarding HLA-F expression in endometrial stroma and HLA-F function; furthermore, HLA-F and HLA-G SNP genotypes and haplotypes have been correlated with differences in time-to-pregnancy. STUDY DESIGN, SIZE, DURATION: Primary endometrial stromal cell (ESC) cultures (n = 5) were established from endometrial biopsies from women attending IVF treatment at a fertility clinic. Basic HLA-F and HLA-G protein expression by the ESCs were investigated. A prospective controlled cohort study was performed including 85 women with a history of RIF and 36 control women beginning their first fertility treatment and with no history of RIF. In some analyses, the RIF group was divided into unknown cause, male infertility, female infertility, and both female and male infertility. Endometrial biopsies and blood samples were obtained the day equivalent to embryo transfer in a hormone-substituted cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: HLA protein expression by ESCs was characterized using flow cytometry and western blot. In the cohort study, the specific immune markers HLA-F and HLA-G, CD56 and CD16 (NK cells), CD163 (M2 macrophages), FOXP3 (regulatory T cells) and CD138 (plasma cells) were analysed by immunohistochemistry and a digital image analysis system in endometrial biopsies. Endometrial receptivity was assessed by an endometrial receptivity array test (the ERA® test). Endometrial biopsies were examined according to modified Noyes' criteria. SNPs at the HLA-F gene and HLA-G haplotypes were determined. MAIN RESULTS AND THE ROLE OF CHANCE: HLA-F protein is expressed in the endometrium at the time of implantation. Furthermore, the HLA-F protein levels were different according to the womens HLA-F SNP genotypes and diplotypes, which have previously been correlated with differences in time-to-pregnancy. Endometrial HLA-F was positively correlated with anti-inflammatory CD163+ M2 macrophage infiltration and CD56+ uNK cell abundance for the entire cohort. However, this was not the case for CD56+ in the female infertility RIF subgroup. HLA-F levels in the endometrial stroma were negatively correlated with plasma progesterone concentrations in the RIF subgroup with known female infertility. Conversely, HLA-F and progesterone were positively correlated in the RIF subgroup with infertility of the male partner and no infertility diagnosis of the woman indicating interconnections between progesterone, HLA-F and immune cell infiltration. Glandular sHLA-G expression was also positively correlated with uNK cell abundance in the RIF subgroup with no female infertility but negatively correlated in the RIF subgroup with a female infertility diagnosis. LARGE SCALE DATA: Immunohistochemistry analyses of endometrial biopsies and DNA sequencing of HLA genes. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The control group of women attending their first IVF treatment had an anticipated good prognosis but was not proven fertile. A significant age difference between the RIF group and the IVF group reflects the longer treatment period for women with a history of RIF. The standardization of hormonal endometrial preparation, which allowed consistent timing of endometrial and blood sampling, might be a strength because a more uniform hormonal background may more clearly show an influence on the immune marker profile and HLA class Ib levels in the endometrium by other factors, for example genetic polymorphisms. However, the immune marker profile might be different during a normal cycle. WIDER IMPLICATIONS OF THE FINDINGS: The findings further highlight the importance of HLA-F and HLA-G at the implantation site and in early pregnancy for pregnancy success. Diagnostic measures and modulation of the complex interactions between HLA class Ib molecules, maternal immune cells and hormonal factors may have potential to improve fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Region Zealand Health Sciences Research Foundation and the Zealand University Hospital through the ReproHealth Research Consortium ZUH. The authors declared there are no conflicts of interest.

Changes in HPV prevalence in Danish women with vulvar cancer during 28 years - A nationwide study of >1300 cancer cases.

OBJECTIVE: A substantial proportion of vulvar cancers are caused by high-risk human papillomavirus (hrHPV), but hrHPV prevalence in vulvar cancer has mainly been investigated in smaller studies which did not evaluate time trends. Our aim was to assess hrHPV prevalence in >1300 Danish vulvar cancers diagnosed during 1990-2017, including changes in hrHPV prevalence over time. METHODS: In a nationwide pathology register, we identified women diagnosed with vulvar cancer at thirteen hospitals from all Danish regions. Archival tumor tissue was collected from local repositories and, upon pathology review, sent to a central laboratory for HPV testing using INNO-LiPA. We calculated hrHPV prevalence according to time, age and histology, and evaluated the overall and age-specific estimated annual percentage change (EAPC). RESULTS: We included 1308 vulvar cancer cases, with a median age of 72 years at diagnosis. The overall hrHPV prevalence was 52.0% (95% CI: 49.3-54.7). HPV types 16/18 were found in 39.6% of cases, whereas nine-valent HPV (9vHPV) vaccine types 16, 18, 31, 33, 45, 52, and 58 were found in 50.8%. The hrHPV prevalence showed an increasing trend over time, with an EAPC of 0.35% (95% CI: 0.00-0.71). The hrHPV prevalence was higher in younger women throughout the study period, and increasing trends over time were seen in both older (age ≥ 60) and younger (age < 60) women. The hrHPV prevalence was higher in non-keratinizing (71.0%) and warty/basaloid (78.0%) carcinomas than in keratinizing (39.4%) and verrucous (36.4%) carcinomas. CONCLUSIONS: Our results indicate that the 9vHPV vaccine could potentially prevent a substantial proportion of vulvar cancers in Denmark.

Conventional microscopy versus digital image analysis for histopathologic evaluation of immune cells in the endometrium.

In the search for a reliable biomarker able to diagnose immunological causes of infertility, uterine immune cells have been widely investigated. As a result, heterogeneous methods and cutoff values of what constitutes an aberrant number of immune cells have been reported, and a standardized method for quantification is needed. The objective of this study was to compare methods for quantification of immune cells visualized with immunohistochemistry in the endometrium of women in fertility treatment. Evaluation of the density of CD56+, CD16+ and CD163+ cells by conventional microscopy on a semiquantitative scale (low, medium and high) was compared to a continuous count using digital image analysis (DIA) reported as percentage positive cells out of the total number of stromal cells and number of positive cells per mm2, respectively. We previously reported the CD56/CD16 ratio as a possible prognostic marker, and therefore the ratios of CD56/CD16 were compared using two different methods for selecting fields for counting with DIA: one method using principles of systematic random sampling, where glands were excluded, and one method analyzing large parts of the tissue including glands. A significant association between conventional microscopy and DIA was found when the semiquantitative scale was compared to medians of positive cells in CD56, CD16 and CD163, respectively, p < 0.001. A systematic significant difference in the ratios of CD56/CD16 was found when comparing the two methods for field selection, p < 0.001. To determine the possible use of these methods, more knowledge of the correlation to clinical outcome is warranted.

Histological outcomes in HPV-screened elderly women in Denmark.

INTRODUCTION: Danish women exit cervical cancer screening at age 65 years, but 23% of cervical cancer cases occur beyond this age. In addition, due to gradual implementation of cervical cancer screening, older women are underscreened by today´s standards. A one-time screening with HPV test was therefore offered to Danish women born before 1948. METHODS: Register based study reporting histology diagnoses and conizations in women found HPV positive in the one-time screening. Number and proportion of women with severe or non-severe histology results were calculated for screened and HPV-positive women by age group or region of residence. Number of women with biopsy and/or conization per case of cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2+) or CIN3+ were also calculated by age groups and region. RESULTS: 4,479 (4.1% of screened women) had positive HPV test. 94% of these had one or more additional tests. 2,785 (62%) of HPV-positive women had histology results, and conization was performed in 1,076 (24% of HPV-positive and 1% of all screened women). HPV positivity and CIN3+ detection varied little between regions, but the proportions of HPV positive women undergoing histology varied between regions from 40% to 86% and the proportion with conization from 13% to 36%. Correspondingly, the number of histologies and conizations per CIN3+ detected varied from 5.9 to 11.2 and 1.8 to 4.7, respectively. In total, 514 CIN2+ (0.47% of screened women, 11% of HPV-positive) and 337 CIN3+ (0.31% of screened women, 7.5% of HPV-positive) were diagnosed, including 37 cervical cancer cases. DISCUSSION: HPV screening of insufficiently screened birth cohorts can potentially prevent morbidity and mortality from cervical cancer but longer follow-up is needed to see if cancer incidence declines in the screened women in the coming years. Management strategies differed among regions which influenced the proportions undergoing biopsy/conization.

Management of low-grade cervical cytology in young women. Cohort study from Denmark.

OBJECTIVE: Cytology findings of atypical squamous cells of unknown significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) are common among women under 30, but evidence on best management strategy is insufficient. We therefore investigated how different management strategies used in Denmark influenced biopsy rates and detection of cervical intraepithelial neoplasia (CIN). METHODS: Register-based cohort study including Danish women aged less than 30 years and born 1980-95, with ASCUS/LSIL as their first abnormal cervical cytology in 2008-16. Rates and relative risks (RR) of biopsy and detection of CIN3+, CIN2 and < CIN2 during two years follow-up were compared between women referred directly to colposcopy after ASCUS/LSIL or undergoing additional testing, including mRNA or DNA test for high risk HPV or repeat cytology. RESULTS: 19,946 women with ASCUS and 19,825 with LSIL were included in the study of whom 92% had adequate information about follow-up. Among women referred directly to biopsy, CIN3+ was detected among 21%, CIN2 in 17%, while 62% had < CIN2. Repeating cytology after 6 months reduced the biopsy rate to 44% of which 53% had < CIN2. Biopsy rates with HPV test were 67% for DNA test, 77% with 14-type mRNA test and 58% with 5-type mRNA test. The detection of CIN3+ was somewhat higher, between 13% and 14% for the three HPV tests vs. 11% with repeat cytology. However, the detection of < CIN2 (not indicating treatment) also increased with RR 2.11 (95% CI 2.01-2.21) for 14-type mRNA test, 1.35 (95% CI 1.29-1.41) for 5-type mRNA test, and 1.86 (95% CI 1.76-1.97) with HPV DNA test. CONCLUSIONS: The choice of management strategy influences both the detection rate for severe lesions (CIN3+) and the proportion of women followed up for potentially insignificant findings.

Are different markers of endometrial receptivity telling us different things about endometrial function?

PROBLEM: To what extent do endocrine, immunological, gene expression and histological markers of endometrial receptivity correlate? METHOD OF STUDY: Between November 2017 and September 2019, 121 women referred to a University Hospitals Fertility Clinic consented to inclusion in this cohort study. The women underwent timed endometrial biopsy followed by blood samples in a hormone-substituted cycle. Of these, 37 women had just started IVF treatment, and the remaining 84 had experienced recurrent implantation failure following IVF/ICSI. The hormone-substituted cycle consisted of initiation with oral oestradiol followed by addition of vaginal progesterone treatment for five full days. Endometrial biopsies were subject to histological examination, immune cell markers by immunohistochemistry (CD56+ , CD16+ , CD163+ , FoxP3) and gene expression microarray analyses with the endometrial receptivity array (ERA® ) test (Igenomix). Plasma progesterone and oestradiol were measured on the day of biopsy. RESULTS: CD56+ uterine natural killer (uNK) cell counts correlate with transcriptional markers of endometrial receptivity assessed by the ERA test. Endometrial maturation, receptivity and immunological markers were not correlated with mid-luteal blood plasma progesterone level. Mid-luteal serum oestradiol level correlated with markers of endometrial maturation and receptivity. The tests were carried out during a standard hormone substitution cycle, and the findings may not apply in the natural cycle. CONCLUSION: CD56+ uNK cell counts and endometrial receptivity assessed by the ERA test appear to be linked. Mid-luteal progesterone levels were not correlated to the tested markers of endometrial receptivity. In contrast, mid-luteal oestradiol level was inversely related to markers of endometrial receptivity and maturation.

Assessing endometrial receptivity after recurrent implantation failure: a prospective controlled cohort study.

RESEARCH QUESTION: What is the prevalence of disrupted markers of endometrial function among women experiencing recurrent implantation failure (RIF), and does the prevalence differ from a control cohort? DESIGN: Prospective controlled cohort study. In total, 86 women with a history of RIF and 37 women starting their first fertility treatment were recruited for this study. Endometrial and blood profiling were carried out in a hormone-substituted cycle using oestradiol and progesterone. Endometrial biopsies were analysed by histology, immune cell profiling, and the endometrial receptivity array (ERA®) test (Igenomix, Valencia, Spain). The vaginal microbiome was analysed using a NGS-based technology (ArtPRED, Amsterdam, the Netherlands). Blood tests included oestradiol, progesterone, prolactin, thyroid-stimulating hormone, vitamin D and anti-phospholipid antibody levels. RESULTS: Patients who had experienced RIF produced a range of test abnormalities. Compared with controls, women with RIF had a higher prevalence of chronic endometritis (24% versus 6%), a lower vitamin D level and a borderline lower progesterone level. Women who had experienced RIF had a more favourable vaginal microbiome compared with controls. Although the RIF cohort was older than the controls (mean age 33.8 years versus 30.2 years), no differences between the groups were observed in immune cell profiling and the ERA test. CONCLUSION: These data demonstrate that a single test or treatment for the endometrial factor in RIF is unlikely to be clinically effective. Diagnosing the endometrium in women with RIF permits targeted rather than blind interventions. Relative vitamin D deficiency, lower mid-luteal progesterone and chronic endometritis are ready targets for treatment. Understanding the role and treatment of an unfavourable vaginal microbiome in RIF needs further investigation.

High-grade cervical lesions after vaccination against human papillomavirus: A Danish cohort study.

INTRODUCTION: It is pertinent to evaluate the impact of vaccination against human papillomavirus (HPV) in real life. The aim of the study was to evaluate the real-life impact of HPV vaccination in the first birth cohort of Danish women offered free HPV vaccination as girls and invited to screening at the age of 23 years. MATERIAL AND METHODS: Women born in 1993 were offered free HPV vaccination at the age of 15 years but women born in 1983 have never been offered free HPV vaccination. We followed these two birth cohorts for 10 years from the age of 15 to after their first invitation to screening, and compared the risk of high-grade cervical intraepithelial neoplasia (CIN). Data were obtained from Danish national health registers. RESULTS: Vaccination coverage was 91% in the 1993 birth cohort and <0.1% in the 1983 cohort. Screening coverage was close to 80% in both cohorts. CIN2+ was detected in 4% of the 15 748 screened women born in 1983 and in 3% of the 19 951 screened women born in 1993. The risk of high-grade CIN was reduced by about 30% in the 1993 cohort compared with the 1983 cohort; for CIN2+ relative risk 0.74 (95% CI 0.66-0.82) and for CIN3+ relative risk 0.68 (95% CI 0.58-0.79). CONCLUSIONS: This study investigated the real-life impact of quadrivalent HPV vaccination by comparing a cohort of women offered HPV vaccination with a cohort of women not offered HPV vaccination. The observed decrease in the detection of high-grade cervical lesions following HPV vaccination is in line with results from the randomized trials and has important implications for future cervical screening of HPV vaccinated cohorts.

Prevalence of high-risk human papillomavirus after HPV-vaccination in Denmark.

Vaccination against human papillomavirus (HPV) has been introduced as a public health initiative in many countries, including Denmark since October 2008. It is important to monitor postimplementation effectiveness of HPV-vaccination at the population-level. We studied HPV-prevalence after first invitation to screening at age 23 years in women offered the quadrivalent HPV-vaccine at the age of 14 years. Randomly selected screening samples from women born in 1994 in four out of five Danish regions were subjected to analysis for HPV in addition to routine cytology. Cobas4800 was used in all participating pathology departments. Data from a Danish prevaccination cross-sectional study using Hybrid Capture 2, and a Danish split-sample study using Cobas4800 were used for comparison. In the period from February 2017 to April 2019, 6233 screening samples from women born in 1994 were selected for HPV-analysis; 27 samples had no HPV-test and 3 samples had no HPV-diagnosis, leaving 6203 samples with an HPV-diagnosis. Prevalence of any high-risk (HR) HPV was 35%; only 0.9% were positive for vaccine HPV types 16/18 while the remaining 34% were positive for other HR HPV. When comparing with prevaccination prevalence data, HPV-16/18 decreased by 95%; RR = 0.05 (95% CI 0.04-0.06), while other HR HPV remained fairly constant; RR = 0.88 (95% CI 0.82-0.94) and RR = 0.95 (95% CI 0.88-1.03), respectively. One-third of women vaccinated as girls with the quadrivalent HPV-vaccine were HR HPV-positive at time of first invitation to screening. Vaccine HPV-types 16 and 18 were almost eliminated, while the prevalence of nonvaccine HR HPV-types remained constant.

FOETAL for NCD-FOetal Exposure and Epidemiological Transitions: the role of Anaemia in early Life for Non-Communicable Diseases in later life: a prospective preconception study in rural Tanzania.

PURPOSE: Low-income and middle-income countries such as Tanzania experience a high prevalence of non-communicable diseases (NCDs), including anaemia. Studying if and how anaemia affects growth, placenta development, epigenetic patterns and newborns' risk of NCDs may provide approaches to prevent NCDs. PARTICIPANTS: The FOETALforNCD (FOetal Exposure and Epidemiological Transitions: the role of Anaemia in early Life for Non-Communicable Diseases in later life) Study is a population-based preconception, pregnancy and birth cohort study (n=1415, n=538, n=427, respectively), conducted in a rural region of North-East Tanzania. All participants were recruited prior to conception or early in pregnancy and followed throughout pregnancy as well as at birth. Data collection included: maternal blood, screening for NCDs and malaria, ultrasound in each trimester, neonatal anthropometry at birth and at 1 month of age, cord blood, placental and cord biopsies for stereology and epigenetic analyses. FINDINGS TO DATE: At preconception, the average age, body mass index and blood pressure of the women were 28 years, 23 kg/m2 and 117/75 mm Hg, respectively. In total, 458 (36.7%) women had anaemia (haemoglobin Hb <12 g/dL) and 34 (3.6%) women were HIV-positive at preconception. During pregnancy 359 (66.7%) women had anaemia of which 85 (15.8%) women had moderate-to-severe anaemia (Hb ≤9 g/dL) and 33 (6.1%) women had severe anaemia (Hb ≤8 g/dL). In total, 185 (34.4%) women were diagnosed with malaria during pregnancy. FUTURE PLANS: The project will provide new knowledge on how health, even before conception, might modify the risk of developing NCDs and how to promote better health during pregnancy. The present project ended data collection 1 month after giving birth, but follow-up is continuing through regular monitoring of growth and development and health events according to the National Road Map Strategic Plan in Tanzania. This data will link fetal adverse event to childhood development, and depending on further grant allocation, through a life course follow-up.

HPV-prevalence in elderly women in Denmark.

AIM: In countries like Denmark, cervical cancer incidence is at present relatively high in elderly women, while routine screening stops at age 65 years. On this background, all women aged 69 and above were invited to human papillomavirus (HPV)-screening in Denmark in 2017. METHODS: Women were identified from the Central Population Register and personally invited by digital or ordinary mail to have a screening sample taken by their general practitioner. In four regions, samples were tested for high risk (HPV) with the cobas 4800® HPV-assay, and in the last region with the BD Onclarity® HPV-assay. Participation rate, prevalence of high risk HPV, and proportion of positive samples with HPV16, HPV18, and other high risk HPV-types were tabulated by 5-year age-groups. RESULTS: 455,612 women were invited, and 30.2% (95 confidence interval (CI) 30.0-30.3) participated. Average age of participants was 74.6 years. Overall, 4.3% (95% CI 4.1-4.4) of participants were HPV-positive, of whom 24% had HPV 16/18. HPV-prevalence decreased slightly from 4.5% in women aged 69-73 years to 3.1% in women aged 84-88 years, but was 5.2% in the very small group of participants aged 89+ years. CONCLUSION: Invitation to HPV-screening was well received by elderly women. The HPV-prevalence decreased slightly with increasing age. No rebound of HPV-prevalence after menopause was found when our data were combined with previously published Danish data from younger women. The presently relatively high cervical cancer incidence in elderly women was not reflected in the HPV-prevalence.

Impact of HPV vaccination on outcome of cervical cytology screening in Denmark-A register-based cohort study.

4vHPV vaccination has been tested in randomized controlled trials under almost ideal conditions, and studies of real-life use have compared outcome between vaccinated and unvaccinated women from the same birth cohort and mostly before screening age. Here we present the first-to our knowledge-evaluation of the impact of the 4vHPV vaccination in real life without selection bias in the reported data. The study has been carried out by comparing the results after first cervical screening between an HPV-vaccinated and an unvaccinated birth cohort, consisting of women born in Denmark in 1993 and 1983, respectively. Cytology data covering an 8-year period, from the age of 15 (age of HPV-vaccination) to age 23 (age of invitation to first cervical screening), were retrieved from the Danish National Pathology Register. Abnormal cytology, defined as atypical squamous cell of undetermined significance and worse (ASCUS+) was detected in 9.4% of women born in 1993 as compared with 9.0% of women born in 1983; RR = 1.04 (95% CI 0.96-1.12), p = .29. Detection of high-grade squamous intraepithelial lesion (HSIL) was statistically significantly lower in the 1993 than in the 1983 cohort, RR = 0.6 (95% CI 0.5-0.7), p < .0001, while the opposite pattern was seen for ASCUS RR = 1.4 (95% CI 1.2-1.6), p < .0001. The decrease in HSIL means that more women can be spared referral for colposcopy and biopsy. The increase of ASCUS could be explained by transition from conventional to liquid-based cytology, but this observation requires further monitoring.

Danish method study on cervical screening in women offered HPV vaccination as girls (Trial23): a study protocol.

INTRODUCTION: The first birth cohorts of women offered human papillomavirus (HPV) vaccination as girls are now entering cervical screening. However, there is no international consensus on how to screen HPV vaccinated women. These women are better protected against cervical cancer and could therefore be offered less intensive screening. Primary HPV testing is more sensitive than cytology, allowing for a longer screening interval. The aim of Trial23 is to investigate if primary HPV testing with cytology triage of HPV positive samples is a reasonable screening scheme for women offered HPV vaccination as girls. METHODS: Trial23 is a method study embedded in the existing cervical screening programme in four out of five Danish regions. Without affecting the screening programme, women born in 1994 are randomised to present screening with liquid-based cytology every third year (present programme arm) or present screening plus an HPV test (HPV arm). The study started 1 February 2017 and will run over three screening rounds corresponding to 7-8 years. ANALYSES: The primary endpoint is cervical intraepithelial neoplasia grade 3 or above. The trial is undertaken as a non-inferiority study including intention-to-treat and per-protocol analyses. The potential effect of primary HPV screening with a 6-year interval will be calculated from the observed data. ETHICS AND DISSEMINATION: The study protocol has been submitted to the ethical committee and deemed a method study. All women are screened according to routine guidelines. The study will contribute new evidence on the future screening of HPV vaccinated birth cohorts of women. All results will be published in open-access journal. TRIAL REGISTRATION: NCT03049553; Pre-results.

Cervical screening in Denmark - a success followed by stagnation.

INTRODUCTION: Despite an intensive screening activity, the incidence of cervical cancer in Denmark has remained stable for the last 15 years, while regional differences have increased. To search for explanations, we investigated possible weaknesses in the screening program. MATERIAL AND METHODS: Data on the screen-targeted women were retrieved from Statistics Denmark. Data on screening activity were retrieved from the annual reports from 2009 to 2015 on quality of cervical screening. Coverage was calculated as proportion of screen-targeted women with at least one cytology sample within recommended time intervals. Insufficient follow-up was calculated as proportion of abnormal and unsatisfactory samples not followed up within recommended time intervals. Diagnostic distribution was calculated for samples with a satisfactory cytology diagnosis. RESULTS: Coverage remained stable at 75%-76% during the study period. Annually, approximately 100,000 women are screened before they are eligible for invitation, and 600,000 invitations and reminders are issued resulting in screening of 200,000 women. In 2009, 21% of abnormal and unsatisfactory samples were not followed up within the recommended time interval; a proportion that had decreased to 15% in 2015. Overall, 11% of satisfactory samples with a cytology diagnosis were abnormal, but with surprising variation from 6% to 15% across regions. DISCUSSION: The success of a screening program depends first of all on coverage and timely follow-up of abnormal findings. Our analysis indicated that the currently high incidence of cervical cancer in Denmark may partly be due to low screening coverage. Also worrisome is a high proportion of non-timely follow-up of abnormal findings. Innovative ways to improve coverage and follow-up are urgently needed.

Endometrial immune markers are potential predictors of normal fertility and pregnancy after in vitro fertilization.

PROBLEM: Elucidating immune mechanisms in the endometrium, which lead to the success of implantation and pregnancy, is important in reproductive medicine. Studies of immune cell abundance have shown conflicting results, and the expression and importance of HLA class Ib proteins in pre-implantation endometrium have not yet been investigated. METHOD OF STUDY: The study population consisted of four subgroups: a hydrosalpinx, a salpingectomy, an unexplained infertility, and a fertile control group. Endometrial samples were collected during the implantation window. Immune markers (CD56+ and CD16+ cells, FoxP3+ Tregs, HLA-G, HLA-F) were quantified in the samples. The outcome of the subsequent IVF treatment was recorded. RESULTS: Increased CD56+ uNK cells and high HLA-G expression served as predictor for successful pregnancy outcome. HLA-F expression was positively correlated with uNK cells, being indirectly predictive for achieving pregnancy. CONCLUSION: Endometrial uNK cell abundance in the pre-implantation endometrium seems to be important for normal fertility and pregnancy success, and they may be used as clinical markers to predict implantation success in IVF.

Consultation on urological specimens from referred cancer patients using real-time digital microscopy: Optimizing the workflow.

INTRODUCTION: Centralization of cancer treatment entails a reassessment of the diagnostic tissue specimens. Packaging and shipment of glass slides from the local to the central pathology unit means that the standard procedure is time-consuming and that it is difficult to comply with governmental requirements. The aim was to evaluate whether real-time digital microscopy for urological cancer specimens during the primary diagnostic process can replace subsequent physical slide referral and reassessment without compromising diagnostic safety. METHODS: From May to October 2014, tissue specimens from 130 patients with urological cancer received at Næstved Hospital's Pathology Department, and expected to be referred for further treatment at cancer unit of a university hospital, were diagnosed using standard light microscopy. In the event of diagnostic uncertainty, the VisionTek digital microscope (Sakura Finetek) was employed. The Pathology Department at Næstved Hospital was equipped with a digital microscope and three consultant pathologists were stationed at Rigshospitalet with workstations optimized for digital microscopy. Representative slides for each case were selected for consultation and live digital consultation took place over the telephone using remote access software. Time of start and finish for each case was logged. For the physically referred cases, time from arrival to sign-out was logged in the national pathology information system, and time spent on microscopy and reporting was noted manually. Diagnosis, number of involved biopsies, grade, and stage were compared between digital microscopy and conventional microscopy. RESULTS: Complete data were available for all 130 cases. Standard procedure with referral of urological cancer specimens took a mean of 8 min 56 s for microscopy, reporting and sign-out per case. For live digital consultations, a mean of 18 min 37 s was spent on each consultation with 4 min 43 s for each case, depending on the number of digital slides included. Only in two cases could a consensus regarding the diagnosis not be reached during live consultation; this did not, it should be noted, affect patient treatment. Complete agreement between conventional and digital histopathology diagnosis was reached in all the 53 patients referred to central pathology units. The participating pathologists were in general comfortable using live digital microscopy, but they emphasized that a fast internet connection was essential for a smooth consultation. DISCUSSION AND CONCLUSION: An almost perfect agreement between live digital and conventional microscopy was observed in this study. Live digital consultation allowed cases to be referred from local hospitals to central cancer units without the standard delay caused by shipment. Only a few preselected specimen slides for each patient were presented in live consultation, which reduced the time spent on diagnosis compared to using the conventional method. Implementation of real-time digital microscopy would result in quicker turnaround and patient referral time, and with careful selection of relevant specimen slides for consultation, diagnostic safety would not be compromised.

Pathologic evaluation of normal and perfused term placental tissue.

This study reports for the 1st time the incidence and interobserver variation of morphologic findings in a series of 34 term placentas from pregnancies with normal outcome used for perfusion studies. Histologic evaluation of placental tissue is challenging, especially when it comes to defining "normal tissue" versus "pathologic lesions." A scoring system for registration of abnormal morphologic findings was developed. Light microscopic examination was performed independently by 2 pathologists, and interobserver variation was analyzed. Findings in normal and perfused tissue were compared and selected findings were tested against success parameters from the perfusions. Finally, the criteria for frequent lesions with fair to poor interobserver variation in the nonperfused tissue were revised and reanalyzed. In the perfused tissue, the perfusion artefact "trophoblastic vacuolization," which is believed to represent dilated transtrophoblastic channels, was reproducible and significantly correlated to the perfusion marker "fetal leakage." In longer perfusions, microscopy of the perfused cotyledon revealed bacteria in the fetal vessels. This finding led to an adjustment in the perfusion protocol with addition of antibiotics to the medium. In the "normal" tissue, certain lesions were very frequent and showed only fair or poor interobserver agreement. Revised minimum criteria for these lesions were defined and found reproducible. This study has emphasized the value of pathologic examination as a supplement in placental perfusion models. Examination of the perfused cotyledon for trophoblastic vacuolization is recommended as an additional quality marker in perfusion models. The study also underlines the need for exact definitions of abnormality in frequent placental lesions.