Several studies have evaluated the effects of high-intensity aerobic training (HIAT) on pain severity and quality of life (QoL) among women with primary dysmenorrhea. However, to date, no studies have evaluated the effectiveness of HIAT on academic performance or absenteeism or examined the cost-effectiveness of HIAT relative to other treatments in women with primary dysmenorrhea. Furthermore, the mechanisms underlying aerobic exercise-induced analgesia in primary dysmenorrhea remain unclear. The objectives of this study are to: (1) evaluate the effects of HIAT on absenteeism and academic performance among university students, (2) identify the underlying mechanisms associated with aerobic exercise-induced analgesia in primary dysmenorrhea, and (3) determine the cost-effectiveness of HIAT compared with a wait-list control (WLC) group receiving usual care.A sequential, embedded, mixed-methods study design, including a crossover, randomised controlled trial (RCT) and semi-structured focus groups, will be conducted alongside an economic evaluation. A total of 130 women aged 18–24 years will be randomised into either HIAT (n = 65) or wait-list control (n = 65) groups. Primary outcomes will include average pain intensity, absenteeism from university, and academic performance. Primary mediators will include salivary progesterone and prostaglandin F2α levels. Outcome and meditator variables will be assessed at baseline and post-treatment, at 12 and 28 weeks. An economic analysis will be conducted from the societal and healthcare perspective of Hong Kong. Semi-structured focus groups will be conducted at 32 weeks. Of the 130 participants included in the RCT, 70 will be included in the focus groups.t-test and a two-sided, unpaired Student’s t-test will be performed to exclude carryover effects and identify within-participant differences in outcome variables between the study periods, respectively. Treatment effects will be evaluated using analysis of variance via a mixed-effects model with fixed effects for intervention, period, and sequence. In all models, random effects will include the participants nested within the sequence as a sampling cluster. The mediation effects will be assessed using the Sobel test. The EQ-5D responses will be converted into utility scores to estimate the gain or loss of quality-adjusted life-years. Seemingly unrelated regression analyses will be used to estimate the total cost differences and effect differences. Qualitative data will be analysed using the process of thematic analysis.All statistical analyses will be performed on an intention-to-treat basis, using SPSS (version 24). Preliminary analysis using an independent samples  Primary dysmenorrhea is a debilitating condition that affects nearly half of all menstruating women worldwide . A crossThe pathogenesis of primary dysmenorrhea has been linked to abnormally elevated levels of prostaglandin secretion ,6. AbnorAerobic exercise has been associated with a reduced prevalence of primary dysmenorrhea and a decrease in related symptoms in several studies –12. Our The proposed crossover randomised controlled trial (RCT) will test the following three hypotheses. (1) Engaging in HIAT for 12 weeks will lead to significant reductions in pain intensity and absenteeism and improve academic performance compared with baseline levels. (2) Pain improvements will be mediated by increased progesterone levels, resulting in decreased prostaglandin F2α (PGF2α) levels. (3) HIAT will display superior cost-effectiveness compared with usual care (WLC) for primary dysmenorrhea treatment.https://clinicaltrials.gov/ct2/show/record/NCT04665661. This trial will include: (1) a two-step screening phase for eligibility; (2) a baseline assessment (time-point [T1]); (3) 12 weeks of supervised treadmill training for the HIAT group; (4) post-treatment outcome assessments and economic evaluations at 12 weeks (T2); (5) a four-week washout period, followed by the crossover of the HIAT and WLC groups; (6) 12 weeks of supervised treadmill training for the WLC group; (7) post-treatment outcome assessments at 28 weeks (T3); and (8) semi-structured focus groups at 32 weeks. Potential participants will be recruited via advertisements (study flyer) distributed at the HK PolyU and the Education University of HK campuses and throughout the local community and by word-of-mouth. This study will be conducted at the Department of Rehabilitation Sciences, HK PolyU.A sequential, embedded, experimental mixed-methods design, including a crossover RCT and semi-structured focus groups, will be conducted alongside an economic evaluation. In this experimental model, qualitative data will be embedded within the quantitative methodology . Qualitad = 0.36) [d = 0.40), the required sample size is 54 per group . However, we plan to enrol many more than 54 participants per group  in order to ensure that we have adequate power to test all three of the study hypotheses.G-Power analysis  (3.1.9.2 = 0.36) , the sam = 0.36)   and prepare 130 sealed, opaque envelopes for allocation concealment. The sealed opaque envelopes will contain the group name (HIAT or WLC) and a personal identification number (PIN). Following the baseline assessment, participants will select an envelope containing the details of their treatment allocation. The nature of the intervention is such that neither the participants nor the therapist can be blinded to the treatment allocation. A blinded examiner will assess all study outcomes and collect saliva samples for the estimation of primary and secondary mediators. Enzyme-linked immunosorbent assays (ELISAs) will be performed in a blinded manner by research personnel not involved in the assessment of study outcomes. Data analysis will be conducted in a blinded manner, using coded groups (Group A and Group B). The group code will only be revealed after all analyses have been completed.Potential participants will undergo a two-step screening process. The first step will be conducted via telephone interview, using an eligibility checklist. Potentially eligible women will then be invited to an in-person screening session on the first day of their next menstrual period. During this session, women will complete the screening questionnaire and sf-IPAQ to determine eligibility and assess baseline physical activity levels. Written informed consent and anthropometric measurements will be acquired. Baseline assessments of the primary and secondary outcomes, information regarding the costs and absenteeism associated with each individual’s previous three menstrual cycles, and saliva samples will be collected. Participants will then be randomised into HIAT or WLC groups. Participants in the HIAT group will receive supervised treadmill training for the first 12 weeks of the trial. To minimise carryover effects, a four-week washout period will be observed for all study participants before cross-over. During the washout period, the HIAT group will be asked to discontinue exercise, and both groups will continue to manage pain as usual . After the four-week washout period, the cross-over period will begin during Week 16, and the participants who were initially randomised to the WLC group will receive the HIAT regimen from Weeks 16 to 28. Participants who were initially randomised to the HIAT group will be instructed not to engage in exercise during the remainder of the study period (i.e. Weeks 16–28). Participants will be permitted to use analgesics for menstrual pain, as needed, throughout the study and will be instructed to record the type and dose (in milligrams) of analgesics consumed in an electronic diary (e-diary). A diagnosis of primary dysmenorrhea can be confirmed by collecting detailed history ; therefoSaliva will be collected as described in a previous study . All par9, adherence was higher for supervised sessions than for unsupervised home exercise sessions. MHR will be calculated using the conventional age-predicted formula (220 − age). To achieve the target HR, the speed of the treadmill will be gradually increased, based on the participants’ HR, which will be displayed on the treadmill, and the rating of perceived exertion (RPE). Because the current proposed study does not involve graded, incremental exercise, graded exercise testing  will not be performed.The exercise intervention will last for 24 weeks (12 weeks for the HIAT group and 12 weeks for the WLC group) at PolyU. Supervised treadmill (Zebris FDM-T) training at PolyU will begin on the first day of the second menstrual period . Exercise sessions at PolyU will be instructed and supervised by a registered physiotherapist. Using this supervisory method, the participant-to-research personnel ratio will be 1:1. The rationale for providing supervised sessions is to increase adherence to study interventions because, in our previous RCTWomen will perform treadmill-based aerobic exercise for three days a week, at 70%–85% of MHR for 30 minutes and at a perceived exertion level of 14–16, based on the Borg RPE scale. This range  is consiWomen in the WLC group will be instructed to continue with their usual activities and manage their pain as normal .The anticipated adverse events include falls from the treadmill, sprained joints resulting from falls, or muscle strains. To minimise these risks, a registered physiotherapist will continuously monitor the participants while they use the treadmill. Serious adverse events are highly unlikely to occur as the intervention being studied is a low-risk intervention. Additionally, in our previous studies ,14 examiPrimary mediators will include progesterone and PGF2α levels in saliva. Salivary measures provide a reliable and non-invasive method for assessing unbound steroid hormones in humans . SalivarPain intensity during the last 24 hours of menstruation will be assessed using the 0–10 NRS , with 0 Absenteeism from the university will be recorded using an e-diary. Access to a prospective e-diary will be provided to each participant for recording university absenteeism. E-diaries have been are reported to be highly acceptable and feasible for use in research . SignifiAcademic performance will be measured using the self-reported 20-item academic performance questionnaire, which was developed to measure the impacts of menstrual symptoms on academic performance .The 0–10 visual analogue scale will be used to measure the impact of dysmenorrhea on concentration , with 0 The 10-item, disease-specific dysmenorrhea daily diary  will be used to measure the severity of menstrual bleeding, the use of analgesic (number of tablets/pills) to treat pelvic pain, and the impacts of menstrual pain on work/education, physical and social activities, and sleep . The eleIf evidence of extreme ratings is identified in the self-reported outcome measures, research personnel will contact the participant over the phone to obtain more information regarding any major changes in symptoms.excluding absenteeism costs) [We will analyse the results of HIAT versus WLC over a 12-week, within-trial period  . The cosm costs) . Cost anm costs) .https://forms.gle/gvoLHAoDqySQFr4GA) will be used to record cost expenditures and work absenteeism. All participants will be provided with access to the cost diary post-randomisation. Participants with limited access to the internet will be provided with printed hard copies of the cost and absenteeism diary, along with stamped and addressed return envelopes. Participants will be instructed to record any forms of medical intervention undertaken during the study period and will be encouraged to record the intervention in the relevant column of the cost diary. In addition, participants will be asked to retain treatment receipts. The treatment receipts will be verified against the relevant entry, and the original receipt will be returned to the participant. Participants will be instructed to complete the diary during the week of menstruation each month. The research team members will be able to access the electronic diary to obtain a summary of completed items for all study participants. A student assistant will contact participants by telephone to acquire additional details for incomplete entries or when participants fail to complete the diary.An electronic diary  will contact any participant who has missed an exercise session and attempt to re-schedule that session. Following the baseline assessment, all participants will be provided with information regarding how to access and complete the electronic diaries.To maximise retention, we (1) will obtain the contact information of participants and any friends and relatives who may know their whereabouts and obtain permission to follow up with any listed contact persons for study purposes; (2) will include appointments for data collection in the study schedule; and (3) will schedule supervised exercise at convenient times for participants.The qualitative study will provide insight into the participants’ experiences during and attitudes towards the study intervention. During the focus group discussions, attempts will be made to identify the subjective experiences of participation when engaging in HIAT, in addition to participants’ perceptions regarding any changes in menstrual pain, university attendance, or academic performance. Ethical approval for the focus groups will be obtained from the Human Subjects Ethics Committee of PolyU before participant enrolment. Written informed consent to participate in the focus groups will be obtained. A sample size of 50–60 is recommended for qualitative research ; therefoFor this component of the research, a computer-generated randomisation schedule will be generated and utilised to identify 70 participants (from the 130 participants included in the RCT), to participate in the focus groups. Invitations to participate in the focus groups will be sent to potential participants at the end of 28 weeks. Invitations will contain contact information, including a phone number, email address, and mailing address, for research personnel. Interested participants will be asked to express interest in participation by phone, email, or post. The recruitment period for the focus group will last for three weeks. A total of seven focus groups will be conducted at 32 weeks post-randomisation. Each group will consist of a random sample of 8–10 participants and will last between 30 and 45 minutes. Focus groups will be conducted in private rooms at PolyU. If the coronavirus disease 2019 pandemic continues, face-to-face seminars will be replaced with virtual meetings (via Zoom) to allow for appropriate social distancing measures. A semi-structured interview technique will be used, in which the discussion will be guided by but not limited to pre-determined (open-ended and probing) questions. At the start of the interview, participants will be assured that their anonymity will be protected through the use of participant code numbers. Two researchers, a moderator and a co-moderator, will conduct the focus groups, and a third research team member will take notes. At the conclusion of each focus group, the co-moderator will present a summary of the discussion to the group, and the participants will be provided with an opportunity to add or modify any comments. All focus groups will be audio recorded , transcribed, and anonymised.We will implement strategies, such as the digital diary, follow-up phone-call reminders, and providing routine encouragement to continue exercise, to minimise missing data. All statistical analyses will be performed on an intention-to-treat basis using SPSS (version 24). A data monitoring committee will not be required for the currently proposed trial because it does not have any of the following features: major safety concerns, unknown risks, long-term follow-up, or double-blind treatment assignment .t-test [t-test [The two phases that each participant completes during the course of a crossover trial are typically referred to as the two study periods . First, t-test . Second, [t-test ,41. Trea [t-test ,43. The 2SA2 + A2SB2, where A and B are the regression coefficients of Paths A and B, respectively, and SA and SB are the standard errors of Paths A and B, respectively [http://quantpsy.org/sobel/sobel.htm) will be used. Hypothesis 2 will be supported if the Sobel test indicates a significant (p < 0.05) mediation effect.The mediation effects of increased progesterone levels on pain will be assessed using the Sobel test . A four-ectively . The onlThe unadjusted mean costs and cost differences between HIAT and the usual care groups will be calculated for both total and disaggregated costs  . SeemingThe EQ-5D responses will be converted into utility scores to estimate the gain or loss of quality-adjusted life-years (QUALYs). The incremental cost-effectiveness ratio (ICER) will be calculated using the formula ICER = ΔC/ΔE. The advantage of this approach is that our confidence intervals will not rely on the usual normality assumption. Uncertainty surrounding the ICER values and the 95% confidence intervals around cost differences will be estimated using bias-corrected and accelerated bootstrapping, with 5,000 replications . The cosWe will determine whether the intervention is cost-effective by utilising two other thresholds to test the robustness of our conclusion: (1) GDP per capita (in terms of purchasing-power parity), which is the threshold suggested by the World Health Organisation Commission for Macroeconomics and Health for an intervention to be ‘very cost-effective’; and (2) the country-specific thresholds published by Woods et al. , based oAn independent transcription service will provide verbatim transcriptions of the focus groups. Each participant will be assigned a code number for data entry. Transcripts will be analysed using the six phases of thematic analysis, following the guidelines described by Braun and Clarke . The resThis is the first study that proposes to use a mixed-methods model approach to evaluate the effectiveness of HIAT among women with primary dysmenorrhea by exploring the effects on academic performance and absenteeism, in addition to assessing the subjective experiences of participation in the exercise intervention and evaluating the cost-effectiveness of the intervention. Each effective intervention for primary dysmenorrhea has beneficial effects that act through unique mechanisms. Understanding the mediators of these interventions may lead to targeted treatments that distil the most critical change factors into maximally effective therapies. Understanding the mechanisms underlying aerobic exercise-induced analgesia in primary dysmenorrhea pain would help design future studies that could identify mediators of pain interventions for clinical improvements, which could themselves be the target of interventions.The proposed study is the first to evaluate the effectiveness, cost-effectiveness, and physiological mechanisms underlying the beneficial effects of HIAT for primary dysmenorrhea. The other strengths of the proposed study include the robust methodology; the adequately powered design, which will be sufficient to detect statistical signficance; and the crossover design, which is less prone to confounding because each participant acts as her own control. Despite these strengths, the proposed study also has a limitation. The potential for a placebo effect is a constant concern in studies of pain , and exeS1 Checklist(DOC)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers  were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11bbright population consisted of 5.4%  of total eosinophils. After activation, this population of CD11bbright cells comprised nearly half the population . Eosinophils in COVID-19 had a similar percentage of CD11bbright cells before activation , but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11bbright fraction after activation . Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue.A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry  in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial ( Coronavirus disease 2019 (COVID-19), a rapidly emerging pandemic disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), causes an extremely variable disease ranging from asymptomatic to viral pneumonia, respiratory distress, ICU admission, and/or death  and hypeClinically severe to critical COVID-19 in the ICU has many characteristics of coagulopathy and tissue edema, particularly in the lung . This suA multitude of articles describe the importance of hyperinflammation in the pathogenesis of COVID-19 . MoThe importance of the eosinophilic compartment in the pathogenesis of COVID-19 is less clear. Early studies, particularly those coming from Wuhan, have indicated that more severe COVID-19 is associated with eosinopenia ,11. UnfoHere, we set out experiments to study the eosinophil compartment in COVID-19 patients both qualitatively and quantitatively. We will show also that our COVID-19 cohort was associated with a relative eosinopenia, but eosinopenia was also seen in some patients with other viral and bacterial infections, albeit to a lower extent. In addition, the eosinophil compartment exhibited clear signs of deregulated activation. Whether this activation is pathological or a normal response to virus infections will be discussed.n = 45) were compared to healthy controls (n = 15) and SARS-CoV2 PCR negative patients with an alternative diagnosis  or bacterial infection (n = 20)). The initial blood samples were drawn at the emergency room (ER) or within two days on the COVID-19 ward. Thereafter, samples were drawn at regular intervals during the stay at the COVID-19 ward. No blood samples were drawn at the intensive care unit. Clinical outcomes and baseline characteristics of the COVID-19 patients have been published recently [Seventy-five suspected COVID-19 patients older than 18 years of age were included who presented at the University Medical Center Utrecht (UMCU) during the Dutch epidemic between March 19th 2020 and May 17th 2020. SARS-CoV2 PCR positive patients  from each patient during standard-of-care diagnostic workup at the Emergency Department (the formation of a COVID-19-biobank (#20-175) with a reviewed issuance protocol was approved on the 21 of April 2020) or during standard blood drawing moments at the COVID-19 ward. Healthy control blood was obtained from the Mini Donor Service at the UMCU  from the medical ethical committee of the UMCU under protocol number 07-125/C. This service provides normal donors  to donate blood for any research). Healthy control subjects could not be matched for age nor gender because of the limitations for clinical experiments during the first wave of the pandemic and had to be chosen based on donor availability. The blood from healthy controls was drawn at the out-patient clinic, which is next to the emergency department. Sample handling times analysis were identical to the patients included in this study. SARS-CoV-2 was detected with the Allplex 2019-nCoV assay . Diagnostics of other viruses were performed with the ePlex RP panel Blood was drawn in one extra 4 or 9 mL VACUETTE® “Load & Go” flow cytometer  exactly as described before [Blood was analyzed in terms of total cell counts, scatter, and fluorescence by a completely automated point-of-care AQUIOS CLd before . Aspecifd before . The anad before . In shor® were imported into the online tool Cytobank . The identification of eosinophils in the blood samples was conducted by gating first on granulocytes on the basis of scatter characteristics (see bright) to enrich for eosinophils (CD16negative) and progenitors (CD16dim). Thereafter, this fraction was analyzed by the FlowSOM algorithm [high, CD16negative, and CD11bmoderate) (The .lmd data files obtained by AQUIOS CLtics see . Thereaflgorithm  (presentoderate) D,E. In tTwo eosinophil phenotypes could be discriminated by either moderate or high expression of CD11b see . The marPatients that tested PCR positive for SARS-CoV-2 at any point during admission were considered as having COVID-19. Patients that were PCR negative for COVID-19 were studied in detail for other diagnoses, explaining their clinical condition applying bacterial cultures, PCRs for other viruses, or other diagnostic tests. PCR negative patients with no other explaining diagnosis were excluded from the study.2/FiO2 ≤ 315 [Patients that were PCR positive for SARS-CoV-2 were grouped based on disease severity according WHO criteria published on 27th May 2020 . ModeraO2 ≤ 315  at any pFor this study, the IRB approved the formation of a COVID-19-biobank (#20-175) with a reviewed issuance protocol. For this procedure, informed consent was waived. The Mini Donor Service received positive approval from the medical ethical committee of the UMCU under protocol number 07-125/C. All procedures performed in this study were in accordance with the 1964 Helsinki declaration and its later amendments.bright and CD11blow cells, a Wilcoxon matched-pairs signed rank test was used. Healthy individuals and COVID-19 PCR negative patients with proven bacterial or other viral infection were used as separate control groups. Differences in receptor expression on eosinophils were tested with Kruskal–Wallis and post-hoc, and the Dunn’s test with Bonferroni correction was used for multiple comparison. GraphPad Prism version 7  was used for analysis and data visualization. Statistical significance was defined as a p-value < 0.05.Kruskal–Wallis analysis was applied for the eosinophil data when multiple groups were compared. For comparison of CD11bmoderate) and a much smaller population of CD11bbright eosinophils  determination of the phenotype of leukocytes with a minimal artificial activation due to ex vivo manipulation of the cells . In factinophils .bright population  and thipulation . Interes to fNLF . In linepulation C. The sm to fNLF .p = 0.02, bright cells in the absence or presence of the formyl peptide was similar in moderate, severe, and critical patients (see The responsiveness for fNLF was determined for eosinophils from the blood of COVID-19 patients and the different control groups such as described above. In marked contrast to healthy controls, eosinophils in the blood of COVID-19 patients were refractory to activation with fNLF see . Interesents see C,D.Next, we tested whether eosinophil counts and eosinophil responsiveness for fNLF would normalize during resolving disease. moderate and CD62Lbright, and do not shift to CD11bbright cells upon activation with fNLF (see bright population likely consists of activated cells as part of the eosinophils in the CD11bmoderate population move to the CD11bbright population after activation of whole blood with fNLF in vitro. It is, therefore, tempting to speculate that the fNLF unresponsive cells belong to a separate subset of eosinophils. Such a hypothesis is supported by the data published by Mesnil and colleagues, suggesting separate subsets of resident and inflammatory eosinophils [moderate eosinophils might belong to this resident subset as our data show that this population is characterized by a high CD62L expression as well (see The fast and automated point-of-care flow analysis allowed the definition of two separate eosinophil subsets in healthy controls: cells that are responsive to fNLF and those that are unresponsive. fNLF unresponsive cells are CD11bfNLF see . The disinophils . The reswell see C. HoweveWe next applied our methodology for eosinophil phenotyping to patients with COVID-19, as eosinophils have been implicated in the pathogenesis of the disease. This is mainly based on the finding that eosinopenia is a hallmark of the disease and the suggestion that the extent of eosinopenia is correlated with the severity of disease ,11. HoweThe eosinophil compartment in the blood of COVID-19 patients is associated with refractoriness for activation with fNLF. This impaired responsiveness is associated with low eosinophil counts. It is, therefore, tempting to speculate that the responsive cells leave the blood and migrate to tissues, leaving behind the refractory cells. This is in line with the idea that inflammatory eosinophils are highly responsive to inflammatory and infectious cues, whereas refractory eosinophils are not. This latter subset might home for the tissues that contain eosinophils in homeostasis . The conResponsiveness of eosinophils for fNLF was anticipated, as we have described fMLF-induced activation in vitro ,19. HoweResponsiveness for fNLF implies that eosinophils, historically seen as T2-effector cells involved allergic and parasitic diseases, are likely to engage in bacterial infections in humans in vivo as well. This latter hypothesis is strengthened by earlier studies showing the capability of eosinophils to kill bacteria  and viruThese data lead to the essential question whether homing of responsive eosinophils to the tissues in COVID-19 should be prevented? Obviously, this descriptive study will not lead to an answer, but several important issues should be taken into account. Firstly, eosinophils have been shown to kill viruses in vitro through the presence of RNAses present in the granules of these cells . Secondlper se, rather than specific for COVID-19. The lack of disease controls in many COVID-19 studies can lead to wrong interpretations regarding specific immune responses in this disease. In addition, the initiation of disease can still be caused by aberrations in the bradykinin metabolism [In summary, the issue whether a specific hyperinflammation in the pathogenesis of COVID-19 is the cause or rather the consequence of infection with the SARS-CoV2 is still a matter of debate. At presentation, COVID-19 patients in our cohort show only very limited signs of a specific systemic inflammation and they cannot be discriminated from patients infected with other micro-organisms or viruses. However, potential differences in co-morbidities in the patient cohorts might have led to confounding data. Moreover, eosinophil counts and eosinophil responsiveness for fNLF did not correlate with disease severity. This implies that the data in this study are associated with severe inflammatory disease tabolism  and coagtabolism . The restabolism  can lead


The majority of falls experienced by older adults occur in the home with home hazards associated with an increased risk of falling. Low-income older adults, who have more disability and live in substandard housing, need feasible interventions to help them safely age in place. The Home Hazard Removal Program (HARP) is a new home hazard removal and fall risk self-management program delivered in the home by occupational therapists to prevent falls. To evaluate the program, a randomized control trial was conducted with 310 community-dwelling older adults receiving aging services in the community. HARP had high acceptability with older adults and was feasible to deliver in the community. Adjusted for fall risk, individuals in the HARP group fell 1.4 times versus 2.2 times in the control group over 12 months. This low-cost home hazard removal program demonstrated acceptability, feasibility, and a significant reduction in falls for at-risk community-dwelling older adults.


Neonatal lupus erythematosus (NLE) is a syndrome of clinical symptoms observed in neonates born to mothers with antibodies to soluble antigens of the cell nucleus. The main factors contributing to the pathogenesis of this disease are anti-Sjögren Syndrome A (anti-SS-A) antibodies, known as anti-Ro, and anti-Sjögren Syndrome B (anti-SS-B) antibodies, known as anti-La. Recent publications have also shown the significant role of anti-ribonucleoprotein antibodies (anti-RNP). Seropositive mothers may have a diagnosed rheumatic disease or they can be asymptomatic without diagnosis at the time of childbirth. These antibodies, after crossing the placenta, may trigger a cascade of inflammatory reactions. The symptoms of NLE can be divided into reversible symptoms, which concern skin, hematological, and hepatological changes, but 2% of children develop irreversible symptoms, which include disturbances of the cardiac stimulatory and conduction system. Preconceptive care and pharmacological prophylaxis of NLE in the case of mothers from the risk group are important, as well as the monitoring of the clinical condition of the mother and fetus throughout pregnancy and the neonatal period. The aim of this manuscript is to summarize the previous literature and current state of knowledge about neonatal lupus and to discuss the role of anti-Ro in the inflammatory process.  Neonatal lupus erythematosus (NLE) is a system of clinical symptoms observed in children born to mothers with antibodies to soluble antigens of the cell nucleus. The main factors contributing to the pathogenesis of this disease are anti-Sjögren A Syndrome (anti-SS-A) antibodies, otherwise known as anti-Ro, and anti-Sjögren Syndrome B (anti-SSB) antibodies, otherwise known as anti-La. Recent publications have also shown the significant role of anti-ribonucleoprotein antibodies (anti-RNP) . Seropos‘LE’ cells (lupus erythematosus) play a major role in the pathogenesis of lupus. They were discovered by Hargraves et al., who described them as ‘mature multinuclear granulocytes that phagocytosed Feulgen-colored nuclear material’ . The proStudies on neonatal lupus date back to the 1950s. In 1954, McCuistion and Schoch  were theIt is difficult to discuss neonatal lupus without mentioning its pathogenesis because it is the source of new diagnostic and therapeutic opportunities. It is currently known that the onset of neonatal lupus is related to anti-nucleus antibodies in the mother. These antibodies are anti-nuclei antibodies, belonging to the group of autoantibodies against extractable nuclear antigens. This group of antibodies also comprises antibodies against the following antigens: Sm, RNP, SS-A/Ro, SS-B/La, Scl-70, Jo-1, Pm/Scl, Mi-1, Mi-2; however, these are anti-SSA/Ro, anti-SSB/La, and anti-RNP antibodies, which play a key role in neonatal lupus pathogenesis. Antigens for these antibodies are located intracellularly in the cytoplasm or cell nucleus. They are expressed on the cell surface only if the cell is to be apoptosis assisted.Anti-SS-A antibodies, also referred to as anti-Sjögren Syndrome A, were first described in 1970 in a patient with Sjögren’s syndrome. They are otherwise called anti-Ro, after the name of the patient (Rose) in which it was first detected. They are directed against four different antigens, each of which consists of a complex of low molecular weight RNA (micro-RNA) and protein, whose molecular weight may be 45, 52, 54, or 60 kDa. Only targeted antibodies against molecule SS-A/Ro52 and SS-A/Ro60 are used in everyday medical practice, although older studies do not distinguish between Ro52 and Ro60 antibodies, defining them together as anti-SS-A/Ro . Ro60 waThe Ro52 antigen belongs to the TRIM21 family of receptors and a family of E3 ubiquitin ligases; it takes part in protein ubiquitination and triggers pro-inflammatory action (by affecting interleukin 2 synthesis) and is also involved in cell apoptosis ,19,20,21In addition, they occur in patients with Sjögren’s syndrome and systemic scleroderma. In case of autoimmune myositis, the presence of anti-Ro52 often coincides with other autoantibodies, but it may be the only positive specific antibody ,27. AntiAnti-Ro52 epitope p200 (anti-amino acid antibodies in positions 200–239 of the Ro52 antigen) is the most likely to cause a complete heart block in the fetus and was found to be the highest risk factor for heart damage. Standberg has shown in his studies that mothers of children with a second- or third-degree atrioventricular block had significantly higher p200 antibodies levels than mothers with rheumatic disease, whose children had heart rates within the normal range. A significant difference was observed in anti-p200 levels between the groups with the first-degree block and those with the second- and third-degree block, compared to fetuses with normal conduction. The use of p200 antibodies as the second stage of analysis during pregnancy in the case of a mother with anti-Ro52 antibodies increased the positive predictive value for fetal heart block  from 0.39 (0.27–0.51) to 0.53 (0.37–0.68) . In summRo60 is a protein with a molecular weight of 60 kD. It is also called TROVE2. The Ro60 protein consists of two domains: one of them is similar in structure to the von Willebrand factor. It is also present in extracellular space and takes part in cellular adhesion. The second domain of the alpha helix structure resembles a cylinder shape and is responsible for binding nucleotide acids . The Ro6Initially, it was believed that the first antibodies in systemic lupus patients were formed by the molecular mimicry mechanism between the Epstein–Barr virus nuclear antigen-1 (EBNA1) and the Ro60 protein. However, no similarity has been found in the amino acid sequences of EBNA1 and Ro60; thus far, no explanation has been given as to why EBNA1 mimics the Ro60 protein . SubsequAnti-SS-B/La (anti-Lane antibodies) were named after a patient named Lane in whom these antibodies were found for the first time. They are directed against phosphoprotein with a molecular weight of 48 kDa, which is associated with RNA polymerase III . They arAnti-ribonucleoprotein antibodies (RNP) are directed against three antigens. For this reason, some authors specify three types of antibodies. The RNP antigen belongs to a group of small nuclear ribonucleoproteins (snRNP) containing low molecular RNA with high uridine content (U-RNA) and various core proteins with a molecular weight of 70 kDa (U1), 33 kDa (protein A), and 22 kDa (protein C). The RNA components are labelled as U1 to U6. The RNP antibody is heterogeneous and directed against various epitopes, the most important of which has a molecular weight of 70 kDa (U1). Anti-U1-snRNP antibodies are detected in high titers in 95 to 100% of patients with mixed connective tissue disease (MCTD). The presence of anti-U1-RNP antibodies in the serum is required by all MCTD diagnostic criteria. These antibodies, found in all patients and often in high titers, are not always present from the beginning of the disease. Their titers may fluctuate and persist during the disease activity, often also during clinical remissions, but their titers frequently decrease or disappear during periods of improvement. They may also be present in patients with systemic lupus erythematosus, rheumatoid arthritis, or Sjögren’s syndrome, but in these patients, their titer does not correlate with the activity of the disease . AntibodThe onset of neonatal lupus is related to the antibodies received from the mother. ENA antibodies belong to the immunoglobulin G group, within which four subclasses are differentiated: IgG1, IgG2, IgG3, and IgG4. IgG is transported by transcytosis via neonatal receptors for Fc IgG fragments present on placental trophoblast cells. The neonatal Fc receptor (FcRn) is a transbone heterodimeric glycoprotein with a major histocompatibility complex (MHC)-like structure, but unlike MHC class I, FcRn is unable to bind peptides (antigens); however, it interacts with Fc IgG and albumin. IgG and albumin are the main plasma proteins accounting for about 80% of the total plasma protein pool and have the longest half-life (about 20 days). This suggests that they can be protected against catabolism by an inverse relationship between the serum concentration of both of these proteins and their half-life. Their catabolism is directly proportional to their blood levels and increases with hyperimmunity. IgG catabolism is fully dependent on FcRn. The neonatal receptor functions throughout life, maintaining IgG and albumin levels by protecting them from intracellular degradation. This property of the FcRn receptor is used to prolong the half-life of biological drugs such as etanercept . The preMaternal sera containing anti-Ro antibodies may bind cross-reactive epitopes on calcium-regulating molecules such as ion channels type L and T , which lThere is special type of macrophages expres high levels of sialic acid-binding Ig-like lecithin 1 (SIGLEC-1) on the cell surface. Siglec-1-positive macrophages were found in the cardiac lesions in fetus with CHB . These cThe incidence rate of NLE is about 1 in 12,500 to 20,000 live births and is slightly higher in female and premature babies . HoweverIt has not yet been explained why some fetuses suffer from congenital heart block and some do not, despite the same developmental conditions. However, we started to differentiate some conditions that predispose the occurrence of neonatal lupus erythematosus. Jill Buyon has played a significant role in exploring the issue of neonatal lupus by publishing many works on this matter. Buyon was the first to notice the correlation between the specificity and number of mother’s antibodies crossing the placenta barrier and the risk of developing the congenital heart block in a fetus. The study published by her in 1993 proved an increased risk of fetal heart complications when anti-SS-A and anti-SS-B antibodies were found in mothers, especially when specific anti-Ro52 antibodies were detected. Buyon also showed the lack of correlation between the type of anti-SS-A or anti-SS-B antibodies and clinical symptoms of neonatal lupus. In the same study, Buyon showed that, in women with low antibody titers, the risk of newborn lupus was low . Jaeggi Apart from this, there are studies being conducted currently in order to find fetal factors associated with susceptibility to the development of total heart block. As we know today, neither gender nor tissue compatibility antigens (MHC) influence susceptibility, although some researchers have shown slightly higher morbidity in female fetuses . It has Ambrosi et al. conducted studies on a Swedish patient population and studied the effects of fetal sex, number of children born by the mother, and the mother’s age and birth time on the occurrence of congenital heart block. Neither the fetal sex nor the number of children born by the mother were correlated with the occurrence of heart block. Pregnancies which led to the birth of a child with heart block correlated with a higher age of the mother, the duration of the second trimester of pregnancy in winter months, childbirth in summer, and lower levels of vitamin D3 .The mothers of children with neonatal lupus erythematosus are characterized by the presence of anti-SS-A and/or anti-SS-B antibodies, but in some cases, anti-RNP antibodies are also found, even without the presence of anti-SS-A antibodies. Despite their seropositivity, the mother does not always have a diagnosed systemic connective tissue disease. Many authors have made a prospective evaluation of the clinical characteristics of patients whose children suffer from neonatal lupus. Some mothers are asymptomatic when they give birth, and their serological status is determined only after the diagnosis of abnormalities in the child. Although the women have no symptoms at the time of procreation, this does not protect them against development of the disease. Rivera et al. assessed 321 mothers listed in the Registry for Neonatal Lupus kept by American scientists. The patients were observed for 6 months to a few years. Half of the women with no visible symptoms developed the autoimmune disease. Out of 26 women who developed the disease, 14 were diagnosed with undifferentiated connective tissue disease, 7 with Sjögren’s syndrome, 4 with systemic lupus erythematosus and 1 with Sjögren’s syndrome . The proZuppa et al. evaluated 50 patients who tested positive for anti-SS-A/Ro antibodies and monitored their children for 9 months. The most frequent maternal autoimmune disease accompanying symptomatic neonatal lupus was systemic lupus erythematosus (25 patients). A total of 15 mothers had primary Sjögren’s syndrome, and 5 mothers were diagnosed with systemic lupus erythematosus and Sjögren’s syndrome; 2 patients had mixed connective tissue disease, and 3 patients were asymptomatic without diagnosis of any systemic connective tissue disease . The resIn summary, the above data indicate the need to monitor asymptomatic mothers in terms of the potential development of systemic autoimmune diseases and to ensure the in-depth monitoring of the condition of the fetus and mother with diagnosed SLE or arthritis to protect their well-being.Neonatal lupus is a rare disease and an example of passively acquired autoimmunization. We can define it as a set of clinical signs observed in the children of mothers with anti-SS-A and/or anti-SS-B antibodies . The cliSato et al. published an interesting case of a female neonate born in the 36th week of pregnancy by caesarean section because her mother went into labor. The mother was 32 at the time of delivery and did not show any symptoms of connective tissue disease. However, 3 years before, when she was 29, she was monitored for symptoms of lupus erythematosus due to erythema on her face. At that time, she did not meet the classification criteria and was not diagnosed as suffering from the condition. After birth, the child was observed to have the following conditions: skin lesions in the form of a ring-shaped erythema and subcutaneous tissue atrophy on the face, trunk, and back. In addition, fetal growth restriction (FGR) was diagnosed. Subsequent studies showed a slight increase in hepatic aminotransferases activity, thrombocytopenia, and mild heart failure with no atrioventricular block. Serologically, the neonate showed elevated moderate titers of anti-SS-A antibodies and high titers of anti-SSB. Anti-DNA and anti-RNP antibodies were absent, and complement components were not lowered. The mother’s serological test results were the same as those of the child. In order to broaden the diagnosis of inflammatory diseases of systemic connective tissue diseases, a histological examination of the minor salivary gland was performed in the mother, which showed lymphocytic infiltration. The mother was diagnosed with Sjögren’s syndrome. All symptoms in the neonate disappeared at the age of 7 months, without any health complications. The researchers carried out a histopathological examination of the placenta, which revealed a collapse of capillaries in the placenta but no visible thrombus formation; massive infiltration of inflammatory cells and apoptotic cells with hematoxylin and eosin (HE) staining was found. C4d deposition was visible in the endothelial cells of the terminal villi of placenta and umbilical cord vessels. However, no C4d deposition was detected in the maternal vessels. The findings of the researchers suggest that maternal antibody reactions in the placenta induce fetal organ damage but do not attack the placenta itself or the maternal vessels. A hypothesis was posed that the complement is activated by maternal antibodies in the fetal vessels through the classical pathway, and, later on, inactive C4d binds to the surface of the fetal endothelium. Covalently bound C4d anchors tightly in the tissue, which is useful as a trace of tissue damage through antibodies. Although it is an inactive product of the complement cascade division, it is postulated that C4d may have the function of a new marker of tissue destruction by autoimmune phenomena . DetectiIt is worth mentioning that C4d deposition in the placenta also occurs in the majority of mothers with systemic lupus erythematosus, antiphospholipid syndrome, and pregnancy-induced arterial hypertension. At the same time, it is associated with complications for the fetus, namely, preterm birth and fetal growth restriction (FGR) . It is sNeonatal skin lupus occurs in 5−16% of infants exposed to anti-Ro and/or anti-La antibodies. Exposure to anti-SS-B antibody and female sex are risk factors for developing skin lesions . DermatoCardiological complications in neonatal lupus may include transient arrhythmias; first-, second- or third-degree atrioventricular block; dilated cardiomyopathy; and fibroelastosis of the endocardium. Atrioventricular block is the most common cardiological complication of neonatal lupus; heart fibroelastosis is much less common.The population prevalence of atrioventricular block is estimated at 0.005% , i.e., 1Another cardiological complication described in the children of mothers with anti-SS-A positive is endocardial fibroelastosis, the dangerous consequence of which is dilated cardiomyopathy. Endocardial fibroelastosis (EFE), previously only diagnosed post-mortem in histopathological examinations , is now Dilated cardiomyopathy is the most common cardiomyopathy in children. Most of them suffer from idiopathic cardiomyopathy, but in some cases, endocardial and mediastinal biopsy reveals the features of fibroelastosis of the endocardium. Matitiau et al. evaluated 24 children under 2 years of age; 50% recovered without complications; 30% died; and 20% survived, but with significant left ventricular dysfunction remaining. In 16 children, heart biopsies were performed; in 45%, myocarditis was diagnosed, and in 25% of cases, endocarditis was diagnosed. In 16 children, a diagnosis of inflammation or fibroelastosis was confirmed by histopathological examination. Patients with inflammatory changes in biopsy had better prognosis than those with histological features of fibroelastosis. Unfortunately, Matitiau did not refer to the serological status of the mothers in his studies. The only known factor is that in the families of these children, there were no previously diagnosed inborn cardiomyopathies . Wisuthsarewong et al. documented that FGR was the accompanying symptom in 29.4% of cases of neonatal lupus . VasculoGathering data in registries allow the characterization of the clinical picture of mothers and children. Such registries serving scientific purposes have already been established in several countries. The first lupus registry was created in the United States in 1994 to collect data of mothers with positive anti-SS-A and/or anti-SS-B and/or anti-RNP antibodies and whose children had lupus symptoms . Fredi eCurrent recommendations include performing fetal echocardiography weekly between 16 and 26 weeks of pregnancy and every 2 weeks between 26 and 34 weeks of pregnancy in pregnant women with systemic lupus erythematosus ,94. AtriAnother aspect was investigated by Evers et al. in a study published this year. He tried to identify a strategy that would optimize the use of echocardiography since he considered weekly testing of all patients with anti-SS-A antibodies to be an exaggeration. Decision analysis of cost/utility modelling for three screening paradigms was performed: ‘standard screening’ (SS) where mothers in mid-pregnancy were subjected to weekly screening tests; ‘limited screening’ (LS) where fetal echocardiograms were avoided unless the fetus develops bradycardia, and ‘targeted screening based on maternal antibody titers’ (TS), where only high anti-Ro values justified weekly screening. While the effectiveness of fetal intervention for first- or second-degree AV block remains unclear, this analysis supports the use of antibody levels to stratify this population in order to optimize surveillance of the occurrence of a potential atrioventricular block. Standard weekly screening tests are inefficient in terms of costs and result in over-exploitation of financial resources . It appears that monitoring the fetal heart rate by the mother at home is the most effective approach. It is important to find specific markers to identify high-risk pregnancies and to ensure that women are able to monitor the fetal heart rate and have prompt access to obstetric care in the event of abnormal readings.Since atrioventricular block is usually diagnosed between 18 and 24 weeks of pregnancy, effective therapy should already be administered in the prenatal period. Mortality due to this disease varies between 15–20% . NegativIn 2008, Brucato summarized the current state of knowledge about the treatments. Non-fluorinated steroids  are recommended only if the health condition of the mother requires it and should not be administered to prevent fetal CHB in women who tested positive for anti-Ro. Fluorinated steroids (dexamethasone or betamethasone) are not metabolized by the placenta and are available to the fetus in their active form. Routine prophylactic fluorinated steroid therapy is not recommended even for women who have previously had children with CHB or a skin rash because this therapy has side effects, mainly regarding brain development. Intravenous immunoglobulin is not used prophylactically, but if administered early enough after the detection of atrioventricular block, it increases the chances of restoring the sinus rhythm. The current recommendation is that, if the mother tests positive for anti-Ro antibodies, serial echocardiograms and sonograms should be performed in accordance with the algorithm described above. The aim is to detect early fetal abnormalities, such as premature atrial contractions or moderate pericardial effusion, which may precede a total atrioventricular block and may be the aim of the preventive therapy. If there are no disease symptoms, fluorinated steroids should not be used. In the event of disturbing symptoms, betamethasone seems safer than dexamethasone .Studies conducted by Sonesson et al. on a group of 212 patients have shown that fluorinated steroids may reverse first- and second-degree atrioventricular block, but also that third-degree atrioventricular block may prove to be reversible if treatment begins shortly after its onset . These dIn 2008, Friedman et al. published the results of a large study evaluating the correlation between the length of the PR interval and the use of dexamethasone. In the literature on the subject, this study is known under the acronym PRIDE . The study group consisted of 127 mothers from 33 centers who tested positive for anti-SS-A antibodies before or up to 18 weeks of pregnancy and who were treated with prednisone at a dose below 10 mg/day or did not receive glucocorticosteroids at all . PR inteHigh-grade AV block and cardiomyopathy may occur within one week of a normal echocardiogram, without an initial first-degree block. It has been demonstrated that early intrauterine treatment of an incomplete AV block with fluorinated steroids prevents progression of cardiac block ,99,101.Since the PRIDE study, we have acquired better insight into early prenatal lupus development in neonates with cardiac conditions. It has been determined that the phenotype of the cardiac form of NLE varies from the clinically silent first-degree atrioventricular block, which does not progress, to the third-degree atrioventricular block, which develops into generalized fetal edema (hydrops) and results in death of the fetus within less than one week after the appearance of the first symptoms . It is aHydroxychloroquine (HCQ) passes through the placental barrier and is considered safe for pregnant women and fetuses. It inhibits Toll-like receptors (TLR).Hydroxychloroquine has a long half-life, and therefore it takes longer for it to achieve efficacy. Therefore, hydroxychloroquine should be implemented in the pre-conception period 2–8 weeks before the planned pregnancy.The incidence of neonatal lupus was lower in women taking hydroxychloroquine, even if the disease was asymptomatic . At presRitodrine, a β-sympathomimetic drug in Japan commonly used as a tocolytic administered to mothers, may cause acceleration in fetal heart rate patterns. Some studies were conduct in Japan and indicate the advantages of using ritodrine. Matsubara et al. indicated that maternal administration of ritodrine increased the ventricular rate and thus fameliorated the signs of fetal heart failure if this developed due to CHB ,105.Miyoshi et al. also confirmed that bradycardia was improved by beta-sympathomimetic administration, but the survival rate was not improved. The second statement was that chronic use of steroids for more than 10 weeks can lead to adverse effects in the fetus such as fetal growth restriction and oligohydramnios .In Europe, according to the latest recommendation, ritodrine is not a first choice medication due to adverse events. It can cause complications such as necrotizing enterocolitis, intraventricular hemorrhage, and acute respiratory distress syndrome in fetus. The adverse maternal cardiopulmonary effects described with beta-agonists frequently cause treatment to be interrupted, in conclusion that beta-agonists no longer be prescribed for tocolysis ,108.Before decision about the usage of ritodrine, a balance of benefits and risks should be made.The most recent recommendations from 2018 concerning systemic lupus erythematosus treatment emphasize the role of ustekinumab (IL-12 and IL-23 inhibitor) . There aCurrently approved treatment methods aimed at removing pathogenic autoantibodies in autoimmune diseases include plasmapheresis and IVIG (intravenous infusions of immunoglobulin G). They are expensive, require complicated procedures, and are burdened with the risk of adverse effects, which is why attempts are being made to develop alternative therapeutic strategies. The results of studies conducted in the USA  have revNeonatal lupus still poses many unanswered questions about its pathogenesis. Anti-Ro’s relationship with macrophage activation and IFN secretion should be further investigated. The consequences of neonatal lupus may be grave. Ill neonates should be treated in specialist tertiary medical centers. Interdisciplinary care is also advisable. Thus far, there are no clear care standards for pregnant women or mothers and children after childbirth. The published manuscripts focused on small populations in various countries and provided only limited information on different populations. Registers of neonatal lupus cases may be helpful in determining uniform international recommendations for action.The usefulness of the close monitoring of fetal heart rate in the critical weeks of pregnancy in terms of the highest exposition to the development of neonatal lupus symptoms, as well as the protective effect of hydroxychloroquine, are commonly indicated in the literature on this subject. There is a need to observe larger groups of patients in order to identify high-risk populations, improve screening protocols, and establish uniform treatment.


The present study aimed to evaluate the diagnostic ability of wide-field optical coherence tomography angiography (OCTA) density map for detection of glaucomatous damage in high myopic (HM) eyes and to further compare the diagnostic ability of OCTA with that of conventional imaging approaches including red-free photography and swept-source OCT (SS-OCT) wide-field maps. A total of 77 healthy HM eyes and 72 HM eyes with open angle glaucoma (OAG) participated in this retrospective observational study. Patients underwent a comprehensive ocular examination, including wide-field SS-OCT scan and peripapillary area and macular OCTA scans. An integrated OCTA density map thereafter was merged by vascular landmark-guided superimposition of peripapillary and macular superficial vascular density maps onto the red-free photography (resulting in the OCTA-PanoMap). Glaucoma specialists then determined the presence of glaucomatous damage in HM eyes by reading the OCTA-PanoMap and compared its sensitivity and specificity with those of conventional images. Sensitivity and specificity of OCTA-PanoMap for HM-OAG diagnosis was 94.4% and 96.1%, respectively. Compared with other imaging methods, the sensitivity of OCTA-PanoMap was significantly higher than that of red-free photography (P = 0.022) and comparable to that of wide-field SS-OCT maps. Specificity of OCTA-PanoMap was significantly higher than those of other conventional imaging methods (except for wide-field thickness map). The OCTA-PanoMap showed good diagnostic ability for discrimination of HM-OAG eyes from healthy HM eyes. As a complementary method of an alternative imaging modality, OCTA-PanoMap can be a useful tool for detection of HM-OAG.  The prevalence of myopia has been increasing dramatically, especially among East Asians5. Therefore, diagnosis and monitoring glaucoma in myopic eyes are becoming very important; however, it is challenging to diagnose this condition, especially in HM eyes6. The reasons for the difficult diagnosis of glaucoma in HM include optic nerve head tilt, increased ovality/rotation/torsion, larger peripapillary atrophy9, and the temporal shift of Bruch’s membrane opening10 and the retinal nerve fiber layer (RNFL) peaks12.Myopia has been considered as an independent risk factor for glaucoma, and moderately to highly myopic (HM) eyes have a higher risk for developing this condition than low myopic or hyperopic eyes do13; however, image analysis is not easy due to the absence of normative database for HM.Various imaging modalities are used for evaluating the glaucomatous structural damages in HM eyes. Due to structural distortion of the optic disc in these eyes (including disc tilt or large peripapillary atrophy), evaluation of the optic disc through the stereoscopic photographs is very difficult. Further, the visibility of the RNFL is poor in HM eyes on red-free photography. Optical coherence tomography (OCT) is used as a relatively objective imaging method14, its asymmetry analysis15, and asymmetrical differences across the horizontal raphe16 have shown improved diagnostic power or been adjunctive diagnostic tools for glaucoma in HM eyes. Some studies suggested approaches to improve the diagnostic power by modifying the RNFL temporalization or magnification by using the self-constructing myopic normative database in peripapillary18 or macular area19. Recently, Kim et al. reported that wide-field thickness and deviation map of swept-source OCT (SS-OCT) exhibited better accuracy and diagnostic power for detection of glaucomatous defects in myopic eyes than the conventional maps from spectral domain OCT did20.There have been many attempts to overcome these difficulties when using OCT. The macular parameters (ganglion cell complex)21. The location of focal perfusion loss in peripapillary retina showed good agreement with visual field defect22. There are many studies on the association between peripapillary choroidal microvascular dropout (MVD), which is visible in the deeper layer of OCTA, and glaucoma27. There are also studies on changes in the macular OCTA in glaucomatous eyes. Furthermore, many OCTA studies have been conducted on HM eyes, the relationship between MVD and healthy HM28 eyes and HM glaucoma29, and the diagnostic abilities of several superficial vessel density parameters30.OCT angiography (OCTA) is a relatively new imaging modality that enables us to noninvasively assess the peripapillary and macular microvasculature. Studies have shown that OCTA is useful for detecting and quantifying glaucomatous damage and its progression. Peripapillary retinal vessel density is lower in glaucomatous eyes than in healthy eyes, and the decreased vessel density in OCTA images shows an exact topographical correlation with the RNFL defect37. Nowadays, with the development of OCT for diagnosing and monitoring glaucoma, it is becoming more common to analyze the optic nerve and the macular area together (both in software and hardware) rather than each area separately. However, there were not many studies that have incorporated this concept into OCTA and furthermore, as yet, there have been no studies using wide-field OCTA Map itself.Recently, many studies have attempted to integrate the information of peripapillary and macular areas analysis together in the field of glaucomaThe purpose of the present study was to evaluate the diagnostic ability of the wide-field OCTA density map for the detection of glaucomatous damage in HM eyes and further compare the diagnostic ability of OCTA with that of conventional imaging approaches, including red-free photography and SS-OCT wide-field maps.Table Two independent glaucoma specialists (WJL and KIN) rated the presence of RNFL defects from red-free photography, wide-field thickness, and deviation map from SS-OCT and OCTA-PanoMap. The agreement obtained was moderate for red-free photography (Kappa = 0.596) and substantial for other wide-field maps (including OCTA-PanoMap) from SS-OCT (Kappa 0.741–0.771) between the two specialists.The sensitivity and specificity of the categorical variables including OCTA-PanoMap, red-free photography, wide-field RNFL thickness map, wide-field deviation map , and peripapillary RNFL, GCIPL, and GCC thicknesses using the abnormal criteria (< 5% level or < 1% level) provided by the built-in normative database for discriminating between HM healthy and HM-OAG eyes are presented in Table The AUCs in terms of quantitative measurement are presented in Fig. The present study compared diagnostic accuracy for the detection of glaucomatous defects in HM eyes between wide-field OCTA density map (OCTA-PanoMap) and conventional red-free photography or wide-field maps from SS-OCT (Topcon DRI OCT). Data demonstrated the clinical utility of OCTA-PanoMap for the diagnosis of HM-OAG.OCTA has been widely studied and used in recent years in the diagnosis of glaucoma over the peripapillary and macular areas and imaging of the superficial and deep layers.40. Using OCTA, reduced peripapillary retinal perfusion in glaucomatous eyes can be visualized as focal defects and quantified as peripapillary flow index and vessel density40. Significant reductions in the RPC density were correlated with the site of RNFL decrease and visual field loss in glaucoma41. There was also an attempt to build a normative database based on the change in the RPC density with age and to create a deviation map using this database42.OCTA demonstrated reproducible focal loss of the radial peripapillary capillary (RPC) in patients with early OAG compared with that in normal controls47.This decrease in the superficial blood vessel density in glaucoma has also been reported in the macular area. Most studies have shown decreased perfusion in the superficial vascular complex of the macular area in patients with glaucoma using OCTA and that these decreases were correlated with visual field loss37. With the development of OCT, it is becoming more common to analyze these two areas together rather than individually. Wide-field OCT scan and map from SS-OCT and the integrated map (PanoMap) of SD-OCT are representative examples of areas currently being commercialized. These may help to well visualize continuous glaucomatous structural damage topographically and well facilitate the understanding of the spatial relationship between two areas in glaucoma48. Research on applying this concept to OCTA is limited, and a few study with similar attempts are introduced below.Recently, many studies have attempted to integrate the information of peripapillary and macular areas using OCT in the field of glaucoma49. The wide-field OCTA (8 × 8 mm) scan can visualize RPC information in a large area using software montage, but it can only cover the peripapillary area50. These studies attempted to reveal wider application areas using OCTA, but all were confined to the peripapillary area and did not address glaucoma51.Wide-field montage OCTA can visualize the expansion of the RPC network. The RPC density was also correlated well with the RNFL thickness52. OCTA measurements detect changes in retinal microvasculature (in the density of both the peripapillary and macular vessels) before visual field damage is detectable in patients with OAG53. Another study also compared the vessel density in each of the two areas among the control, OAG, and angle closure glaucoma groups54. Those studies evaluated the vessel density in both the areas, but all analyses were focused on parameters and not on topographic analysis with the map.One study showed that significant microvascular damage was present in both the macular and peripapillary areas in early OAG. This study used vessel density parameters of OCTA in both the areas but evaluated only quantitative parameters, not those in HM glaucoma30. Some studies compared the vascular–function with structure–function correlations in HM glaucomatous eyes. Peripapillary vessel density, as measured using OCTA, may be useful in the evaluation of glaucomatous visual field damage in HM eyes. The performance of the vessel density was remarkable even when the RNFL measured using OCT was not reliable due to segmentation errors56.There have also been many OCTA studies related to glaucoma in myopia and HM. OCTA measurements can be useful for diagnosing glaucoma in HM eyes, especially when using calculated indices such as macular vessel density ratio (outer macular vessel density/inner macular vessel density)The present study has several differences from existing OCTA studies. First, a topographic map was used, not the parameters of OCTA. Second, two topographic maps of the optic nerve and macula were combined and analyzed. Finally, this method was applied to HM to compare the diagnostic ability of detecting glaucoma using conventional imaging methods.Because this study was only performed on HM eyes, the inter-grader agreement between glaucoma specialists in this study was not higher than that reported in other studies performed with the general population. This is because it is difficult to diagnose glaucoma itself in HM, and in the process of determining any factor that could lead to its diagnosis, the method used in this study (OCTA-PanoMap) was devised.The OCTA-PanoMap proposed in this study has two main strengths. First, the OCTA-PanoMap can visualize the structural glaucomatous damage more objectively in HM. The sensitivity of the OCTA-PanoMap was significantly superior to that of red-free photography, and the specificity of the OCTA-PanoMap was significantly superior to that of other OCT categorical parameters. As mentioned earlier, the presence of the tigroid fundus poses a significant challenge in the evaluation of glaucomatous damage in HM. Although OCT affords reproducible measurement of the RNFL thickness (swept-source technology was used in this study), the detection of RNFL abnormalities in HM eyes is complicated because of the high rate of false-positive errors , which is likely related to the lack of inclusion of individuals with HM in the normative database. Second, the OCTA-PanoMap could visualize the RNFL defect at a glance in HM, making it much easier to apply clinically than when using quantified data. Maps such as thickness and deviation maps are more intuitive data than the numbers and can help in actual practice. As the images of the two areas are combined and shown as one, the continuous decrease in the capillary plexus from the optic nerve to the macular is well visualized.58.This imaging method still has some limitations. First, until now, the scan range was not wide due to hardware limitations, and even though wide-field OCTA has been developed to capture a wide area, it took a long time to scan, and it was difficult to obtain a good quality image. Second, a tool for quantitative analysis of OCTA is still lacking, and only a qualitative analysis method was used in this study. Thus, the development of a more objective method is needed. Third, although this method can play an important role in imaging, it only plays a limited role in revealing the pathophysiology. There are debates as to whether the changes seen in the OCTA-PanoMap are primary or secondary. It is assumed that axon damage occurs first by glaucoma, and subsequently, the blood vessels in that area are reduced. Finally, topical medication might influence OCT parameters, including vessel density, and need to be taken into considerationIn conclusion, the wide-field OCTA density map (OCTA-PanoMap) showed good diagnostic ability for the discrimination of HM glaucoma from healthy HM eyes. As a complementary method of an alternative imaging modality, the OCTA-PanoMap of SS-OCT can serve as a useful tool for the detection of HM glaucoma.The study protocol was approved by the Institutional Review Board (IRB) of Hanyang University Hospital. Informed consent was waived due to its retrospective nature and it was confirmed by IRB of our institution. The study design adhered to the tenets of the Declaration of Helsinki for biomedical research.This retrospective cross-sectional study enrolled patients with healthy HM eyes and HM eyes with OAG who visited the Glaucoma Clinic of Hanyang University Hospital between August 2018 and December 2020.All participants underwent complete ophthalmologic examination, including visual acuity testing, manifest refraction assessment, slit-lamp examination, intraocular pressure (IOP) measurements using Goldmann applanation tonometry, gonioscopy, dilated fundus examination, axial length measurement , stereo disc photography, red-free photography , 24–2 perimetry using the Swedish interactive thresholding algorithm , and SS-OCT .59.A visual field was considered to be reliable if the fixation losses were < 20%, the false positive rate was < 15%, and the false-negative rate was < 15%. A normal visual field was defined as that with a mean deviation (MD) and pattern standard deviation (PSD) within 95% confidence limits, and results of the glaucoma hemifield test (GHT) within normal limits. Eyes with glaucomatous visual field defects were defined as those with a cluster of three points with probabilities of < 5% on the pattern deviation map in at least one hemifield, including at least one point with a probability of < 1%; or a cluster of two points with a probability of < 1%, and a GHT result outside 99% of the age-specific normal limits or a PSD outside 95% of the normal limits. The visual field defects were confirmed using two consecutive reliable tests29. Eyes with poor OCTA images were excluded on the basis of the following criteria53: image quality < 30, poor-clarity images, localized weak signal caused by artifacts such as floaters, residual motion artifacts visible as irregular vessel patterns or disc boundary on the en face angiogram or segmentation failure. One eye was randomly chosen for the study in cases where both the eyes met all the eligibility criteria.The inclusion criteria were as follows: best-corrected visual acuity of 20/40 or higher, HM, and an open anterior chamber angle. HM was defined as refraction less than −6.0D or Axial length (AXL) > 26 mm. The exclusion criteria were as follows: history of ophthalmic surgery , severe glaucoma with an MD worse than −20 dB, any other ocular disease that could interfere with the visual function, any media opacity that could significantly interfere with OCT acquisition, and an inability to obtain a high-quality OCT image. All of the OCT scan images had to have an image quality score ≥ 30, according to the manufacturer’s recommendationWe identified patients with OAG using several features in addition to an open angle confirmed using gonioscopy. The first feature was the presence of a characteristic optic disc with localized or diffuse neuroretinal rim thinning, increased cupping, or a cup-to-disc ratio difference > 0.2 between the eyes observed in the stereo disc photograph. The presence of an RNFL defect on red-free fundus imaging was an alternative feature. The presence of glaucomatous visual field defects was a prerequisite for glaucoma diagnosis, and patients with preperimetric glaucoma  were excluded from this study. A patient diagnosed with OAG was treated with topical medication or not according to the decision of the glaucoma specialist. Healthy eyes were defined as those without any history or evidence of intraocular surgery, intraocular pressure of ≤ 21 mmHg with no history of increased intraocular pressure, absence of glaucomatous appearance of the optic disc, and normal ophthalmologic findings. The wide-field OCT scan analysis was performed according to the right-eye orientation. A senior glaucoma specialist (MS) determined whether the participants were HM-OAG or HM healthy controls. All previous medical records, including images and visual field results, were reviewed to examine disease progression and confirm the diagnosis. Detailed objective method used to determine progression is provided in the 2) using the DRI-OCT device. The DRI-OCT is an SS-OCT device that uses a wavelength-sweeping laser with a central wavelength of 1050 nm and tuning range of approximately 100 nm. We acquired 100,000 A-scans per second with an 8-µm axial tissue resolution. A 12 × 9 mm2 scan contained 256 B-scans, each containing 512 A-scans for a total of 131,072 axial scans/volume. The wide-field RNFL thickness map and wide-field deviation map were obtained and the method has been described in detail in a previous study32.We employed a wide-field scan protocol , separately. Scans were obtained from 4.5 × 4.5 mmWith the built-in analysis software, superficial retinal layer [from the internal limiting membrane (ILM) to the inner border of the inner nuclear layer] of the macular and the density map of radial peripapillary capillary layer (from top to 70.2 µm below ILM) were used for the present study.We used a combined wide-field OCTA density map for single-image evaluation of glaucoma diagnosis. The map was created by the same-day superimposition of optic disc and macular OCTA density maps onto RNFL photography as aligned using the Photoshop software  based on vascular landmarks. Each combined map was labeled by date, and all, as collectively integrated, were defined as the OCTA-PanoMap.32.RNFL defect on red-free photography, wide-field RNFL thickness map, and wide-field deviation map has been described in detail in a previous study61. RNFL defect on the wide-field RNFL thickness map was defined as an arcuate or wedge-shaped diverging dark-blue area surrounding an abrupt color scale change that appeared less thick than neighboring areas on color-coded maps of wide RNFL thickness. The minimum defect size was larger than the diameter of a major retinal vessel63. RNFL defect on the SuperPixel map was arbitrarily defined as the presence of a wedge-shaped area of at least 20 contiguous yellow/red pixels along with RNFL thinning on the deviation map64. Since there were many yellow/red pixels on the deviation map of HM patients, the judgment was made by dividing the ones judged by the glaucoma specialist  and those judged by criteria (20 contiguous pixels). RNFL defect on the OCTA-PanoMap was defined similarly to that on wide-field RNFL thickness map, as an arcuate or wedge-shaped diverging dark area surrounding an abrupt color scale change that appeared less crowded than neighboring areas did on color-coded density maps.RNFL defect on red-free photography was defined as having a width at a 1-disc-diameter distance from the edge of the disc larger than that of a major retinal vessel, diverging in an arcuate or wedge shapeRNFL defects on above-mentioned images were evaluated independently by two glaucoma specialists  in a masked fashion. For prevention of bias, the graders who evaluated the wide-field maps were masked to the red-free photography. When the two experts disagreed, a third ophthalmologist (MS) evaluated the photographs and the disagreement was resolved after discussion.65. To assess the abilities of each categorical variable, such as wide-field RNFL, thickness map, or the criteria on the basis of the comparison of measurements with a built-in normative database to distinguish HM glaucoma from HM healthy controls, sensitivities and specificities were tested. The comparison between sensitivities or specificities were performed using the McNemar test. The areas under the receiver operating characteristic curve (AUCs) were calculated for the continuous variables and the comparison of AUC values of the parameters was assessed using the method described by DeLong et al.66. P values < 0.05 were considered statistically significant. The values are presented as the mean ± standard deviation.Statistical tests were performed using IBM SPSS Statistics 24  and MedCalc . The independent t-test was used to compare the characteristics of the continuous variables and the chi-squared test was used for the categorical variables. Inter-rater agreement between two glaucoma specialists  was evaluated using the Kappa test. Cohen suggested the Kappa result be interpreted as follows: values ≤ 0 as indicating no agreement, 0.01–0.20 as none to slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as almost perfect agreementSupplementary Information.