In South Africa (SA), approximately 16 000 stillbirths occur annually. Most are classified as unexplained and occur in district hospitals. Many of these deaths may be caused by undetected foetal growth restriction. Continuous wave Doppler ultrasound of the umbilical artery (CWDU-UmA) is a simple method for assessing placental function. This screening method may detect the foetus at risk of dying and growth-restricted foetuses, allowing for appropriate management.A cohort study was conducted across South Africa. Pregnant women attending primary health care clinics at 28–34 weeks gestation were screened using CWDU-UmA. Women not screened at those antenatal clinics served as control group 1. Control group 2 consisted of the subset of control group 1 with women detected with antenatal complications excluded. Women with foetuses identified with an abnormal CWDU-UmA test were referred and managed according to a standardised protocol. A comparison between the study and control groups was performed.The study group consisted of 6536 pregnancies, and there were 66 stillbirths (stillbirth rate [SBR]: 10.1/1000 births). In control group 1, there were 193 stillbirths in 10 832 women (SBR: 17.8/1000 births), and in control group 2, 152 stillbirths in 9811 women (SBR: 15.5/1000 births) .Screening a low-risk pregnant population identified the low-risk mother with a high-risk foetus, and acting on the information as described was associated with a significant reduction (35% – 43%) in stillbirths. This demonstrates a step-change reduction in stillbirths and warrants screening in SA. Few clinicians, except for the midwife and doctor directly involved, see the stillbirth, and the woman (mostly) does not discuss the event with others outside of the immediate family. Stillbirth leads to great anguish for the health care providers and the family.2 Approximately 2.0 million stillbirths occur worldwide each year.2 Approximately 55% of stillbirths occur in sub-Saharan Africa.3 South Africa (SA) is classified as an upper middle-income country and is ranked 50th of 54 similar countries, and within Africa, SA is ranked 6th of the seven upper middle-income countries.2Stillbirths are a global public health challenge and the majority occur in low- and middle-income countries (LMICs).4 Perinatal deaths include both stillbirths  and a neonatal death (defined as a death of a neonate in the first 28 days of life). The most common category of perinatal deaths is unexplained stillbirth, which accounts for approximately a quarter of perinatal deaths  and for 36% of stillbirths .5 The majority of the mothers and foetuses were regarded as healthy at the time of the foetal demise and had not been referred for more specialised care.5 Foetal growth restriction (FGR) is a major factor associated with stillbirth.9District hospitals and community health centres reported 2.5 stillbirths for every neonatal death, and 43% of the approximately 16 000 stillbirths per year  occur in SA.11 Foetal growth restriction increases the risk of adverse outcomes by eightfold and is associated with perinatal morbidity and mortality and adulthood diseases.13 Three-quarters of growth-restricted infants are not recognised before delivery,13 and in low-risk pregnancies with a lower threshold of suspicion, the detection rate is even lower (15% – 20%).13Small for gestational age (SGA) foetuses  not detected during the antepartum period had a fourfold increased risk of adverse foetal complications compared to SGA foetuses detected before delivery.14 Symphysis-fundus measurements have a poor ability to detect growth-restricted babies, and routine foetal movements counting has also been shown to be ineffective.15 Routine imaging ultrasound16 in LMICs, surprisingly, was also shown to have no effect on perinatal or maternal death or on antenatal attendance.16The tools required to detect FGR in LMICs and especially at the level of primary care, where most women with low-risk pregnancies attend, perform extremely poorly in detecting FGR, potentially explaining the high unexplained stillbirth rate. Palpation and symphysis-fundal height measurement are commonly used in LMICs to detect FGR, despite the limited evidence to support this as an effective method to detect growth restriction and show improved maternal or neonatal outcomes.17 Reduced blood flow because of increased resistance downstream in the placenta leads to ineffective transfer of nutrients and oxygen and correlates very well with placental insufficiency, which is one of the major causes of FGR.18 This reduced blood flow is detected by the Doppler ultrasound and measured as the resistance index : the higher the RI, the more compromised the blood flow.19 A rise in RI is associated with FGR, and once absent end-diastolic flow (AEDF) is detected, there is end-stage placental disease; this is associated with adverse perinatal outcomes.18 When the placenta is damaged to the extent that no blood flow can be detected during diastole of the foetal heart, the placenta is close to failure and the foetus is at high risk of intra-uterine demise. This phenomenon is called AEDF.19 Clinical care informed by the result of the Doppler ultrasound of the umbilical artery has been shown to reduce perinatal deaths by more than one-third in high-risk pregnancies.20 Another Cochrane systematic review found there was insufficient evidence to support its use in low-risk pregnancies.21 However, this recommendation was based on study populations from high-income countries. Screening for AEDF, or a clearly defined abnormally high RI, could potentially reduce stillbirths if the prevalence of AEDF and abnormal RI was high enough in a pregnant population to warrant screening. These circumstances might be found in LMICs with high stillbirth numbers.Better detection of FGR babies is desperately needed in SA and other LMICs, especially at the primary level of care. Doppler ultrasound of the umbilical artery measures the arterial blood velocity through the placenta.TM device is a low-cost mobile CWDU device developed by the Council for Scientific and Industrial Research (CSIR) and SAMRC in South Africa.22 It measures the RI in the umbilical artery and plots it against the estimated gestational age to identify the foetus at risk for FGR.23 The accuracy of UmbiflowTM in measuring the RI in the foetal umbilical artery continuous wave Doppler ultrasound of the umbilical artery (CWDU-UmA) has been proven to be comparable to the commercial standard unit ‘gold standard’.23 The device has significant advantages in that it can be used by primary health care providers and non-specialist healthcare providers, requires only a week of training, is portable and is less expensive and less technical than imaging ultrasound; the data are recorded on the device, which allows for quality control at a later date, and it can run on battery power.24 These factors make it a good choice for a screening tool if it is effective in detecting FGR. A previous study performed in Mamelodi township using UmbiflowTM found that the use of UmbiflowTM reduced the stillbirth rate in women classified as having low-risk pregnancies by 43%.24The UmbiflowTM in primary healthcare clinics throughout SA, together with a standard referral protocol for foetuses with abnormal RI, would result in a reduction in the stillbirth rate.Building on the previous study, we investigated whether screening a low-risk pregnant population using UmbiflowTM device) were screened. The population of low-risk women were defined as women attending non-specialist primary antenatal care clinics who have been classified as ‘low risk’ at the time of recruitment and screening according to local clinical guidelines. This is based on the South African basic antenatal care plus guideline grounded on the World Health Organization (WHO) guideline.26A cohort of low-risk pregnant women attending primary health care antenatal care eligible for screening using the CWDU-UmA  and those not screened (control group) was performed. Control group 1 represents all women aged 18 years or more who attended the antenatal clinics, had a singleton pregnancy, delivered a neonate of 1000 g or more from the clinic and did not receive CWDU-UmA screening; control group 2 represents the same population, but excludes those who subsequently developed antenatal complications . This was to ensure that the screened population was as low-risk a population as possible.Women from a specific geographic area attending the primary health care clinic were considered for screening. The inclusion criteria were as follows: all candidates must be women with a singleton pregnancy, aged 18 years or more and classified as low risk between 28 and 34 weeks gestation ; all candidates were also required to provide written consent.27The study sites included nine diverse catchment areas linked to regional or tertiary hospitals. This included rural, peri-urban and urban sites across eight provinces in SA. The sites had a healthcare worker screening eligible women on specific days of the week, and there was an established communication and referral route to refer women with abnormal RI.19 Screened women with RI findings below the 75th centile for their GA were considered as having a normal RI result, and they continued their routine antenatal care at their local primary health care clinics.27 The Mamelodi study found that approximately 10% of a low-risk population with abnormal RI was identified by using the 75th centile cut-off.24 Those with RI findings above the 75th centile for their GA were considered to have abnormal RI and were referred to a high-risk clinic at their local referral hospitals for further review and management.27The CWDU-UmA screening was classified as either normal RI or abnormal RI, depending on the Doppler value in relation to GA and RI. This was automatically plotted on a graphic representation using the 75th centile as a cut-off.TM result. The stillbirth rates in the screened population (study group) were compared with those of control groups 1 and 2.Women with abnormal RIs were followed up weekly or fortnightly at the high-risk clinic and received a Doppler ultrasound at each visit and a foetal growth scan every two weeks. All were managed according to standard protocol. 28Data collection of the screened women and delivery outcomes started in September 2017 and stopped in February 2020, allowing time for the pregnant women to deliver by the end of February 2020. The CWDU-UmA screening outcome was recorded. Maternal clinical information was collected at the time of enrolment in the study. The outcome of all women screened (study group) or not screened (control groups) was recorded. Outcome data were obtained from the electronic birth register at the various delivery sites. Small for gestational age was defined as a birth weight for gestational age < 10th centile, according to the WHO foetal growth chart.z-tests for cases where only certain categories were compared. The WHO multinational foetal growth charts were used for categorising birth weight according to centile and corrected for GA and neonatal sex at delivery.28 All tests were performed at a 5% level of significance. A p-value of < 0.05 was considered as statistically significant. The relative risk was calculated using the incidence proportions of total stillbirth rate and perinatal mortality rate between the screened and not screened groups. All statistical analyses were done with R Core Team.27The maternal demographics are reported as frequencies, means and standard deviations. Categorical characteristics were investigated using chi-square tests to express differences between the screened and not screened groups and the two-proportion All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All participants provided written informed consent. Participation was voluntary and participants were informed about their rights to withdraw from the study at any stage. Ethical approval was obtained from the University of Pretoria’s Faculty of Health Sciences (clearance no. 473/2014).During the study period, there were 20 330 women who attended antenatal care at the designated primary health care clinics and delivered at the clinic or hospital. After excluding women below the age of 18 years, deliveries below 28 weeks’ gestation (or women who gave birth to newborns weighing less than 1000 g if GA was unknown) and women with no maternal and neonatal clinical notes available and multiple pregnancies, 17 368 pregnancies were analysed.The screened study group consisted of 7088 women who had CWDU-UmA screening, of which 6536 (92.2%) women had pregnancy outcomes that were analysed. Control group 1 had 10 832 women, and control group 2 had 9811 women. 27The incidence of abnormal RI in the screened group was 13.0%, and 1.2% of the screened population had AEDF. Details are documented elsewhere.p = 0.155), but there were more caesarean section deliveries in the study group than in control group 2 . A p-value of < 0.05 was considered as statistically significant.There were 66 stillbirths in the screened group , 193 in the control group 1 and 152 in control group 2. The stillbirth rate was significantly lower in the CWDU-UmA screened study group compared with both control groups .Unfortunately, we have been unable to reliably trace all the neonatal outcomes of the women that attended the same primary health care clinics as the study group. Our electronic birth register records all stillbirths, but it does not record all the neonatal deaths. The Perinatal Problem Identification Programme records all the neonatal deaths for the maternity units, but it is not granular enough to identify the clinics that the women attended. The overall neonatal deaths for the nine catchment areas were 658 neonates from 69 301 live births for women aged 18 years or older, who attended antenatal care, had a singleton pregnancy and delivered an alive baby of 1000 g or more (excluding the neonates of the study group). This gives a neonatal death rate of 9.5/1000 live births. The major neonatal causes included intrapartum asphyxia (29.9%), spontaneous preterm labour (29.3%), foetal anomalies (10.5%), HDP (10.3%), antepartum haemorrhage (4.9%) and congenital infections (3.8%).p = 0.0001) and increased caesarean section rate . This finding is similar to the Mamelodi study which showed a reduction in stillbirths, an increase in preterm deliveries without increasing neonatal mortality.24Screening a low-risk pregnant population with CWDU-UmA at primary care clinics and referral of foetuses with abnormal RIs to the next level of care resulted in a significant (43%) reduction in the stillbirth rate ; even with the unscreened population who developed antenatal complications excluded, there was a significant (35%) reduction in the stillbirth rate . There was no increase in the neonatal death rate that was discernible. This reduction in the stillbirth rate was achieved with only a slight increase in resources, namely increased use of the neonatal nursery  in the healthy woman. Current methods of detecting FGR are ineffective.16 We now know that CWDU-UmA is a feasible tool to detect FGR, and its use in screening at primary health care clinics is associated with a step-change reduction in stillbirths, thus potentially solving this problem.The largest category of stillbirths delivering at district hospitals and community health centres is unexplained stillbirth, and the majority of the mothers are clinically healthy, but the majority of these stillbirths had undetected FGR or were SGA.29 and as such they were more likely to have attended on the days of the UmbiflowTM screening. However, there were more low-birthweight (LBW) babies in the control groups (13.3% and 12.3% vs 11.2% in the study group), but the study group had more SGA babies (24.2% vs 20.6% and 20.2%). This imbalance between the study group and the control groups might have been avoided if we had screened every day of the week and if a randomised trial had been conducted.There was no difference in age or HIV status between the study group and control groups, but the screened group had more primigravid women. Primigravid women are more likely to attend antenatal care more frequently,Other limitations of this study include that CWDU-UmA screening was only performed in women aged 18 years or more, and women were screened on specific days of the week; those not attending on those days did not get a CWDU-UmA screening. We chose not to randomise women so that a larger sample of women could be recruited in a shorter period of time for budgetary reasons. There was some loss to follow-up, but unknown outcomes were only 7.8%, and this is particularly good for low-income and middle-income settings where women are considerably more mobile and relocate frequently. Another limitation of the study was its inability to trace the neonatal outcomes of all the women that attended the same clinics as the screened study group. We did not have a neonatal follow-up system that was granular enough to record neonatal deaths at clinic level, and the contact details in the birth register were insufficient to trace all the neonates from the specific clinics. However, the neonatal outcomes of all the neonates whose mothers were screened were available, as we had complete contact details of the women. This data indicates that there was not a shift to an increased neonatal death rate in the screened group, despite significantly reducing the stillbirths.A strength of the study is in having two control groups; control group 2 excluded women who developed antenatal complications. We did not remove women from the study group who developed complications, and if anything, they were a slightly higher-risk group than control group 2. This demonstrated that the screened population compared with as low-risk a population as possible with similar conditions and still had a 35%  reduction in stillbirths. This reinforces the value of screening the umbilical artery for an abnormal RI in low-risk populations. Another strength of the study was the use of nine different geographical sites and the large number of women involved.30Of the 66 stillbirths in the study group, 30 were still unexplained. Only one screening test was performed around the 30-week visit. It is possible that a subsequent screening at a later time (e.g. 36 weeks) in those women whose foetuses had a normal RI might detect more foetuses at risk. Further research is needed to answer this question. Alternatively, these foetuses might have died because of undiagnosed congenital infection, as described by Madhi et al.This study demonstrates that screening for FGR using CWDU-UmA was feasible at primary health care clinics and highly effective in preventing stillbirths. As mentioned previously, this screening system is ideal for a primary health care setting. It is recommended that CWDU-UmA should be used to screen pregnant populations for abnormal RIs of the umbilical arteries. Research should concentrate on the timing of screening and methods to implement the CWDU-UmA screening in primary health care clinics in districts throughout SA. Special attention should be paid to the resources that would be required at the referral hospital, namely increased maternity and neonatal high care and intensive care resources.Continuous wave Doppler ultrasound of the umbilical artery offers the solution to provide a simple and accurate method of detecting FGR in pregnant women and preventing stillbirths. The high prevalence of AEDF found in SA warrants population screening.


The transbilayer distribution of cholesterol (CHL) in complex asymmetric lipid membranes remains controversial, with contrasting investigations suggesting that there is more CHL either in the exoplasmic, outer leaflet (OL) or the cytoplasmic, inner leaflet (IL) depending on cell type or model, membrane composition, and method of investigation. Here, we launch systematic coarse-grained molecular dynamics simulations to investigate the impact of the sphingomyelin (SM) acyl chain length upon CHL distribution in asymmetric lipid membrane mixtures which account for the variation of the most abundant headgroups and acyl chain unsaturation in the two membrane leaflets. We find that there is always more CHL in the OL, but longer chain SM depletes more CHL from the IL than short chain SM in simple membrane mixtures containing SM and 16 : 0, 18 : 1 phospholipids. The difference between longer and shorter chain SM is neutralised in a more complex asymmetric membrane, where there are more saturated tails in the outer leaflet. We propose that interdigitation of long-chain SM into the opposing IL pushes cytoplasmic CHL towards the OL, but higher chain saturation of the outer leaflet compensates for the effect of SM chain length. Long acyl chain sphingomyelin and saturated phospholipid tails in the outer membrane leaflet deplete cholesterol from the inner leaflet in mammalian membranes. However, the PM of eukaryotic cells is highly asymmetric with respect to the lipid composition of the two leaflets (trans-bilayer asymmetry). The asymmetry drives important cellular functions and pathways. Most of the sphingomyelin (SM) and phosphatidycholine (PC) lipids are within the outer, exoplasmic leaflet (OL) while the inner, cytoplasmic leaflet (IL) mostly consists of the phosphatidylethanolamines (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) lipids.7,8 Trans-bilayer asymmetry is not an equilibrium configuration of a membrane . The cell uses active processes carry out by flippase proteins to maintain trans-bilayer asymmetry because there is a large free energy barrier associated with the translocation of a charged lipid from one bilayer leaflet to the other across the membrane hydrophobic core. The transbilayer distribution of the major phospholipid components of the PM can now be carefully determined using highly sensitive lipidomics experiments.9 However, data pertaining to the distribution of CHL across the two bilayer leaflets remains contradictory owing to the low free energy barrier of CHL to flip flop across the two leaflets. Some results show that most of the CHL molecules are concentrated in the IL while others suggest that the majority exist within the OL, reviewed in ref. 12 The distribution of CHL across the bilayer leaflets is likely to be determined by a passive thermodynamic equilibrium distribution that equalizes the CHL chemical potential in the two leaflets. An active process that maintains CHL asymmetry (if it exists) is unlikely considering the rapid flip–flop of the CHL, and remains to be discovered. Based on the above arguments, a theoretical model suggested that most CHL molecules accumulate in the IL due to the negative curvature of the PE lipids present in this leaflet.10,11 Another mechanism that can also drive CHL to the IL is the presence of the PS and PI lipids, because a coarse grained (CG) simulation study demonstrated that CHL has a higher affinity to the charged leaflets.13 However, the same simulation study suggested that CHL prefers saturated lipids, which are more abundant in the OL. Another factor that can drive CHL to the OL is the presence of SM lipids that exclusively exist in this leaflet. Theoretical models predict that 80% of CHL resides in the OL owing to the presence of SM, but contributions from the bending energy reduce this abundance to 63%.14 The majority of SM in mammalian cells in the outer leaflet have a long C24 acyl chain,9 which can interdigitate into the opposing leaflet.15,16 However, there are also more than 4% short-chain SM in the outer leaflet. In the inner leaflet, the small fraction (less than 0.1%) of SM are all exclusively short-chain lipids.9 Mutations which replace C24 SM by C18 SM have physiological consequences related to obesity and insulin resistance.17,18 We previously showed that a delicate balance between SM–CHL hydrogen bonding and interactions with the solvent result in weaker interactions with C24 SM with CHL, compared to shorter chain C18 SM lipids.16 At the same time, free energy calculations showed that CHL preferred the OL in the presence of C18 SM and the IL in the presence of C24 SM.16 However, no PE or PS lipids were present in the lower leaflet in these all-atom simulations. In addition, all-atom simulations suffer from the constraint that CHL cannot spontaneously flip flop on the simulated timescales (100 s of nanoseconds).19CHL is an important and the most abundant  molecule in the plasma membrane (PM) that surrounds all eukaryotic cells. It has several vital roles in the PM −1,20–23 depending on the presence of unsaturated lipids (more unsaturated lipids increase flip–flop rates), cholesterol content (more cholesterol lowers flip–flop rates) and overall bilayer composition . Simulations of asymmetric membranes have to be set up with care to ensure either that the tension in each leaflet is zero, or equivalently, the area of each leaflet is identical.24 When investigating CHL asymmetry, if the overall area of each leaflet is not calculated carefully, CHL will simply accumulate into the leaflet containing too few lipids. We therefore employ a careful methodology which minimizes this area difference (described in detail in the Methods section). We find that long-chain SM lipids interdigitate into the IL, and push more CHL into the OL to achieve mechanical equilibrium and balance the free volume in the two leaflets.Overall, one can argue that SM and saturated lipids pull more CHL into the OL, while PE and charged lipids attract more lipids to the IL. The competition between these determinants decides the equilibrium distribution of the CHL in the PM. Here, we exploit the possibility of spontaneous CHL flip–flop in molecular dynamics (MD) simulations employing the CG MARTINI force field for lipids, to investigate the effect of SM chain length on the distribution of CHL in the two bilayer leaflets with compositions reminiscent of biological membranes, but simplified to eliminate minor lipid species. The flip–flop rate of CHL in simulations of CG lipid bilayers can range from 1–20 μs22.19 simplified to eliminate minor lipid species and all proteins. Two compositions were chosen. First, a simple asymmetric membrane was modelled with predominantly PS and PE in the IL, and PC and SM predominantly in the OL. The lipid acyl chains were set to 16 : 0–18 : 1 for PS, PE and PC, while the SM was either all 18 : 1–18 : 0 (DPSM) or 18 : 1–24 : 0 (DSSM). The overall CHL fraction was 0.29, similar to prior simulations of plasma membrane models.25 The final system compositions are shown in We chose our bilayer compositions based on the lipidome of human erythrocytes,2.2Unlike symmetric bilayers, the construction of asymmetric MD models of bilayers is challenging because the total area of the two leaflets must be as close as possible to each other to avoid artifacts arising from imperfect lipid packing. For example, if one leaflet has too few lipids, cholesterol will accumulate in this leaflet to fill up the free volume and establish mechanical equilibrium. To avoid such artifacts, we devised the following workflow to eliminate area mismatch as much as possible.26 From these APLs, the number of lipids required to model a bilayer of a target total area was obtained.To calculate optimal area per lipid (APL) of each component, we simulated symmetric bilayers, where each monolayer had the desired composition of a single leaflet of the target asymmetric bilayer. For example, for the simple systems, we ran simulations of symmetric POPC–SM–CHL bilayers of composition of the OL to obtain the most likely area per lipids of POPC, SM and CHL, and a symmetric POPC–POPE–POPS–CHL bilayer of composition of the IL, to obtain the most likely APL of POPC, POPE and POPS in the IL. The simulation box size was set to 14 × 14 × 10 nm for these simulations, and APLs were obtained from Voronoi triangulation using apl VORO.We then constructed an asymmetric bilayer using the number of lipids calculated in the previous step. Such an asymmetric bilayer should have minimal area mismatch between the two leaflets, presuming that the CHL content is identical in the two leaflets. The preceding assumption is certainly not correct if CHL distribution is asymmetric, and we account for this in the following manner.Achol in the two leaflets, because CHL occupies additional area in the leaflet with more CHL.After a 10 μs production run, a trans-bilayer CHL asymmetry is created by overall flip–flop of CHL molecules to equalize the chemical potential of CHL in the two leaflets. However, such an asymmetry agains creates an area mismatch of ΔAchol, and repeat another 10 μs production iteration. The overall CHL concentration is kept constant across all iterations. After several such iterations, it is expected that the CHL distribution will stabilise across the two leaflets.To eliminate area mismatch again, we fill the CHL-depleted leaflet with additional lipids which occupy an area corresponding to Δi.e. CHL was always richer in the OL, as indicated by the first set of simulations (see Results section). Note that the number of phospholipids for each leaflet remained the same. For each setup, we simulated 5 replicas with different initial velocity distributions. Only the systems with simple lipid compositions were simulated in this way. This set of simulation is referred to as the REPLICA simulations hereafter.A second approach to minimising the area mismatch is to choose different initial ratio of CHL in the two leaflets in the starting configuration of the simulations. We launched simulations where the ratio between the number of CHL in the OL to the total number of CHL in the IL varied from 0.5 to 0.62, 27Overall, we have ran close to 770 μs of simulations in MARTINI time, corresponding to a real sampling time of close to 3.1 ms.2.327–29 is used to model the lipid bilayers (required files were obtained from the MARTINI web site).30 In MARTINI, 4 heavy atoms are typically represented by a single particle. For example, CHL is modelled by 8 and POPC by 12 beads. A CG force field is necessary to permit CHL flip flop to measure trans-bilayer CHL asymmetry. All lipid bilayers are modeled using the insane.py script obtained from the MARTINI web site,30 in combination with home-made Matlab scripts. The bilayers are solvated with non-polarizable water beads along with antifreeze beads.27 Na+ and Cl− ions are present to ensure a salt concentration of 150 mM. All simulations are performed using GROMACS 2019.x.31,32 The system was equilibrated using the Berendsen coupling33 for both temperature and pressure. Production runs are carried out in the NPT ensemble for 2 μs using a time step of 20 fs. The temperature is controlled using the Nosé–Hoover thermostat,34,35 with a reference value of 310 K, and the pressure is coupled semi-isotropically using the Parrinello–Rahman barostat,36 with a reference value of 1 bar. The van der Waal's forces are shifted to zero from 0.9 nm to 1.2 nm. Electrostatic interactions are calculated using the reaction-field method as recommended.37The coarse-grained (CG) MARTINI force fieldz-coordinate  of the hydrophilic ROH bead of CHL. Note that CHL can occupy the bilayer center in MARTINI simulations, but for the sake of simplicity, all CHL molecules are assigned either to one leaflet or the other based on the position of the ROH bead. No CHL molecules are classified as being in the center of the bilayer. Lipid tail order parameters are calculated from a modified version of scripts available on the MARTINI web pages.30 All other quantities are measured using GROMACS tools. All reported properties are calculated for the last μs of simulation.The CHL distribution is measured from the 33.1versus the IL converges after 9 and 12 iterations of 10 μs each for the simple DSSM and DPSM systems respectively. More CHL partitions into the OL in the presence of longer chain SM (C24), than when C18 SM is present instead  and the DSSM (C24) simple membrane simulations.Transbilayer asymmetry in acyl chain saturation is identical in all our simple systems. The presence of SM in the OL, and the length of the SM chain are the other two key driving forces which determine the difference in the CHL distribution ratio. In the remainder of this article, we will analyse the simple systems further in an attempt to converge on a hypothesis of why long-chain SM drives more CHL into the OL, in apparent contrast to our earlier findings using all-atom simulations in conjunction with a host of analytical techniques.For analysing bilayer properties, we chose the final iterations of the DPSM and DSSM simple system simulations. For time-averaged properties, the last 1 μs of the simulations were analysed.3.2The thickness of the bilayers is calculated from the average distances between the phosphate beads in the two leaflets. The thickness of the longer chain DSSM containing bilayers is expectedly higher, by about 0.5 nm . There iThe lipid tail order parameters demonstrate expected trends . The SM CHL and SM form stable nanodomains in both the DPSM and DSSM systems, as apparent from 2D-density plots  and Fig. Althoug13 are present in equal amounts in both the DPSM and DSSM systems. Therefore, we conclude that the interdigitation of lipid tails in the IL makes the IL denser, and reduces the free volume available for CHL. CHL is the only molecule which can spontaneously flip–flop in the simulations on the μs timescale, and therefore CHL accumulates in the OL when the C24 SM tail interdigitates into the IL. The hypothesis is in conflict with our previous findings, which were based on free energy calculations from all-atom simulations.16 The lipid composition in the said systems did not contain PS or PE lipids and therefore dissimilar from the ones we have used in the current CG simulations. More CHL in the OL (54%) was also observed in CG simulations of a bilayer with composition mimicking a plasma membrane.25 There are several key differences in the said simulations from our current work. Firstly, we have focussed on the differences between the effects of long-chain and short-chain SMs, while the systems in25 more resemble a realistic plasma membrane containing a mixture of short-chain and long-chain SM. Secondly, our systems are less likely to suffer from an initial area mismatch owing to reasons discussed earlier. Finally, our systems are much simpler in composition, with all lipids with an abundance of less than 5% being ignored.The C24 DSSM acyl chain protrudes into the opposing IL, while the C18 DPSM does not . Since hThe protein content in a real PM is nearly 30%, and CHL affinity to peripheral of specific regions of TM proteins can influence the overall distribution of CHL. Although inclusion of proteins could not be performed in the current set of simulations, our scheme of carefully equilibrating the systems to keep the areas of each monolayer constant can be used in the future to analyse complex membrane systems also containing proteins.4Overall, our data indicate that long-chain SM in the OL attracts more CHL to the OL, than short-chain SM does. There are, however, many other compositional determinants which will ultimately determine the CHL distribution ratio in both experiments and simulations. Since lipid composition has such an important role to play, the distribution of CHL will be sensitive to the cell and organelle type, as has been proposed in the past. Our simulations indicate that short-chain SM, when present in cells in diseased conditions, result in less CHL accumulation in the OL. A different CHL distribution can in principle affect nanodomain formation in complex membrane environments, and also influence signaling processes associated with these domains.There are no conflicts to declare.


New diagnostic devices for schistosomiasis should be designed to function best within the local endemic health care context and support stakeholders at various levels of the health care system in performing the tasks to help control and eventually eliminate schistosomiasis. Q-methodology served as a rigorous tool to identify the needs and product-service design requirements of stakeholders throughout the health care system.Stakeholders at various levels of the health care system shared that new diagnostic devices for schistosomiasis should: be available at the point of need and require minimal infrastructure to increase diagnostic capacity and surveillance, particularly in rural or distant settings; be less expensive to make diagnostic testing more affordable; and be able to identify infection status before treatment to support the identification and prioritization of patients or areas in need of treatment.Medical device designers and technology companies should ensure the development of new diagnostic devices for health care facilities with minimal infrastructure; these devices should be affordable and deployable, as well as able to identify infection status before treatment.Health care systems and community stakeholders in Nigeria should continuously participate and collaborate with the design industry in the human-centered design process of new diagnostic devices to ensure that the viewpoints of stakeholders at various levels of the health care system are explored and considered in the design process. Schistosoma haematobium that affects approximately 30 million people annually in Nigeria. Treatment and eradication of this infection require effective diagnostics. However, current diagnostic tests have critical shortcomings and consequently are of limited value to stakeholders throughout the health care system who are involved in targeting the diagnosis and subsequent control of schistosomiasis. New diagnostic devices that fit the local health care infrastructure and support the different stakeholder diagnostic strategies remain a critical need. This study focuses on understanding, by means of Q-methodology, the context of use and application of a new diagnostic device that is needed to effectively diagnose urinary schistosomiasis in Oyo State, Nigeria. Q-methodology is a technique that investigates subjectivity by exploring how stakeholders rank-order opinion statements about a phenomenon. In this study, 40 statements were administered to evaluate stakeholder perspectives on the context of use and application of potential new diagnostic devices and how these perspectives or viewpoints are shared with other stakeholders. Potential new diagnostic devices will need to be deployable to remote or distant communities, be affordable, identify and confirm infection status before treatment in patients whose diagnosis of urinary schistosomiasis is based on self-reporting, and equip health care facilities with diagnostic devices optimized for the local setting while requiring local minimal infrastructural settings. Similarly, the context of use and application of a potential new diagnostic device for urinary schistosomiasis is primarily associated with the tasks stakeholders throughout the health care system perform or procedures employed. These findings will guide the development of new diagnostic devices for schistosomiasis that match the contextual landscape and diagnostic strategies in Oyo.Urinary schistosomiasis is a waterborne parasitic infection caused by  Schistosoma haematobium. This disease is prevalent in Nigeria, especially in rural areas, and affects approximately 30 million people annually.–,,S. haematobium are predominately school based and report a disease prevalence of 17%–21% in urban areas and 32.7% in rural areas.–Urinary schistosomiasis is a water-borne parasitic infection caused by ,,–,Efforts to control and eliminate schistosomiasis involve diagnosing individuals and gathering prevalence data that can be used for strategy development, program planning, and monitoring.Onasanya et al. identified different stakeholders within 4 levels of the health care system in Oyo that are involved in diagnosing, controlling, and eliminating schistosomiasis:,,,Even though case management and control and elimination efforts are widely used simultaneously, the different stakeholders follow various procedures in implementing them. For example, at the health care level  collection of statements ; (2) selection, development, and validation of statements deducted from the concourse ; (3) participant selection (P-set), (4) selecting sorting distribution, (5) conducting the Q-sorting, (6) analysis, and (7) interpretation .50 The sSupplement 1) were gathered from literature (n=93) and stakeholder interviews (n=35) to build the concourse. Statements from literature were sourced from journal articles, conference proceedings, and NGO and government reports that contained insights on diagnosing urinary schistosomiasis in Nigeria and sub-Saharan Africa.S. haematobium, approaches, and strategies. Statements pertaining to diagnosis, treatment, and patient-related diagnostic needs were identified by interviewing stakeholders from the 4 levels of the health care system . A Q-set of 40 opinion statements provided good coverage of the study, was sufficient to elicit existing viewpoints, and fell within the sample range  that is generally accepted in Q-methodology.,,,,A Q-set is a selection of statements deducted from a concourse (step 1).Supplement 2 Table 2.) Every statement was clear and made its own original contribution to the Q-set, without overlaps.Supplement 3) was used to compute a statistical analysis of Cronbach’s alpha reliability coefficient using Windows SPSS 26. The resulting Cronbach’s alpha reliability coefficient was 0.99, which is acceptable and higher than the Nunnally norm of 0.70Supplement 4). Similarly, the expert ratings were used to compute an I-CVI value for each of the 40 statements (Supplement 3): 32 statements scored an I-CVI value of 1.00, 8 statements had an I-CVI value of 0.88, and 1 statement had an I-CVI score of less than 0.80. Experts suggested minor changes in statement wording to improve clarity and readability, especially for the Q-statements with I-CVI values of less than 0.80.Supplement 2 Table 1).In measuring the content reliability and validity, the experts rated each of the Q-statements for readability, clarity of statement, and heterogeneity (breadth and depth)A total of 29 participants were purposively selected from 3 of the 4 health care system levels in Oyo . We seleData collection involved participants’ rank-ordering statements on a fixed distribution. Compared with a free distribution, a fixed sorting distribution created an opportunity to standardize the process of ranking the statements.–,Q-sort involved rank-ordering the 40 statements on the Flatten-Gaussian 13-point scale sorting distribution grid. A total of 29 Q-sorts were conducted within this research. Q-sorts were conducted using Easy-HtmlQ 2.0; a web-based platform for online Q-administration.The study was conducted with ethical approval from the Research Ethics Committees at the University of Ibadan-Nigeria (REF UI/EC/21/0100) and Delft University of Technology, the Netherlands. Participants were assured that participating in the study was voluntary and that information collected was anonymized and treated with confidentiality. All participants signed and provided informed consent before participating in the study and were allowed to withdraw from the study at any time. No participants expressed any hesitation to participate in the study.,,Analysis was carried out using KADE  version 1.2.1 program.,Supplements 5 and 6). A factor array table is a configuration of a Q-sort showing the viewpoint of a particular factor, grouping of statements, and their specific ranking value.,Factor loadings sorted from highest to lowest provided a solution towards working our way down from the strongest factor load accounting for the maximum amount of study variance and produced clear and consistent factor interpretation and clustering.P≤.05) were considered distinguishing statements.,The factor array scores and crib sheets were interpreted to produce factor themes. Factor arrays provided the best possible estimates of relevant and holistic viewpoints and crib sheets were used to enforce holism by forcing engagement with every item in a factor array.,,Supplement 5). A concise and consistent interpretation and explanation of factors or viewpoints were best achieved with a 4-factor solution (Supplement 6).To understand the stakeholder perspectives on the context of use and application of new diagnostic devices for urinary schistosomiasis in Oyo, Nigeria, a by-person factor analysis of 29 Q-sorts was performed. A factor array score of 4 distinct factors emerged . These 4The interpretation of the factors revealed that the context of use and the application of new diagnostic devices for urinary schistosomiasis are both strongly influenced by 4 distinct factor themes. That is, new diagnostic devices will need to (1) be deployable to remote or distant communities, (2) be affordable, (3) identify and confirm infection status before treatment in patients with a diagnosis of urinary schistosomiasis based on self-reporting and be optimized for the local setting, and (4) fit within local minimal infrastructural settings .Factor 1 (F1) had an EV of 4.77 and explained 16% of the total variance. A total of 10 respondents loaded significantly in this factor, and all in the positive pole. All 10 respondents were decision makers and performed supervisory, financing, and technical roles targeting the diagnosis of urinary schistosomiasis in Oyo. Four of the 10 respondents in F1 represented the policy and economic level of the health care system, including 2 individuals in the donor financing sector, 1 schistosomiasis-control coordinator within the NGO sector, and 1 state university researcher. F1 also involved a DSNO and a medical officer of health at the state level . They represented the organizational level of the health care system that focuses on the pragmatic parts of schistosomiasis control, such as gathering information about urinary schistosomiasis that is then used for program planning. Similarly, F1 included 2 laboratory technologists, 1 medical doctor, and 1 CHEW at a state-run PHC facility. These participants represented the health care level that is interested in devices that improve the speed and accuracy of diagnosing urinary schistosomiasis.P≤.05) (P≤.05). This will increase the ability to reach remote communities to detect S. haematobium cases and map areas with a large spread of the infection  (P≤.05). Deployable diagnostic devices will need to be sensitive and able to quantify eggs to estimate the intensity of infection  (P≤.05). Estimating the population exposed to urinary schistosomiasis should not be limited to school-age children but should also include adults . The ability to identify populations exposed to this infection could be enhanced by leveraging the social power of community leaders . However, F1 respondents raised concerns about including nonclinical professionals in diagnostic processes .F1 respondents emphasized the need for new diagnostic devices for urinary schistosomiasis that are deployable to remote communities and health care facilities  (P≤.05) . New diaFactor 2 (F2) had an EV of 1.88 and accounted for 6% of the total variance. Five participants loaded significantly in the positive pole: 2 PHC laboratory technicians, 1 CHEW, 1 researcher, and 1 DSNO. All 5 participants operated in local government areas (LGAs) in Oyo. The PHC laboratory technician and CHO were interested in new devices that improve the speed and accuracy of diagnostics. They were proactive in the community and demonstrated high social relationships with the community members. The researcher represented the policy and economy level and provided technical expertise targeting the control and elimination of schistosomiasis at LGAs. The DSNO was interested in gathering and using prevalence data for planning.P≤.05) , approximately US$2, to be considered affordable or be covered by the government  (P≤.05) . AffordaAffordable or free diagnostic tests will help increase diagnostic coverage in the communities and improve the ability to identify infected areas in need of treatment.P≤.01). Having data on infection severity will enable stakeholders to plan various tasks targeting the diagnosis and subsequent control of urinary schistosomiasis . These new diagnostic devices should be deployed in communities and PHC facilities that are most lacking in general laboratory equipment .Respondents in F2 further emphasized developing novel diagnostic devices that are affordable and deployable, have high sensitivity and specificity, and present  data on the severity of urinary schistosomiasis  (P≤.01). (Factor 3 (F3) has an EV of 1.65 and accounts for 6% of the total variance. Four participants loaded significantly in this bipolar factor. Two in the positive pole (F3+) and 2 in the negative pole (F3−). The F3+ respondents were both CHOs interested in a new diagnostic device increasing the speed and accuracy in identifying urinary schistosomiasis cases in the LGAs. Respondents in F3− included a laboratory technician at a private laboratory and a medical doctor at a tertiary health facility that offers specialized health care in the form of community-based outreach. F3+ respondents confirmed that passive case identification based on people’s self-reporting is an affordable strategy to identify urinary schistosomiasis  (P≤.01). . HoweverCase identification based on people’s self-reporting is affordable but should be supported with a diagnostic device that can identify and/or confirm infection status.P≤.01). F3− respondents emphasized the need to detect and obtain data on the severity of infections in multiple biological samples  to increase the capacity to identify infected groups and areas. Similarly, such identification will guide the development of strategies to manage and control schistosomiasis within such groups and areas. Community leaders who have high social power can support strategies to manage and control schistosomiasis in infected areas . However, F3− respondents raised concerns about training community members with no formal clinical expertise to detect and diagnose schistosomiasis   (P≤.01). F3− respondents also emphasized equipping PHC facilities that currently have limited or no diagnostic devices to detect S. haematobium   has an EV of 1.35 and accounts for 5% of the total variance. Five participants loaded significantly in the positive pole. That is, NTD officers and medical doctors providing supervisory activities targeting urinary schistosomiasis in multiple LGAs. A CHEW and a CHO stationed at a community PHC facility were also included. F4 respondents were interested in new diagnostic devices that can improve the speed and accuracy of detecting F4 respondents emphasized the need for simple point-of-care (POC) diagnostic devices or tests that require minimal infrastructure   where paF4 respondents emphasized the need for simple POC diagnostic devices or tests that require minimal infrastructure where patients often travel long distances to health care facilities.To validate the results of this study, qualitative discussions were carried out with 5 experts about the 4 factors that emerged on the context of use and application of new diagnostic devices in Oyo, Nigeria. The experts included a laboratory technician, a researcher, a CHO, a medical doctor, and an NTD officer. Each expert was representative of loaders in each of the 4-factor themes . The expThis study explored stakeholder perspectives on the context of use and application of potential new diagnostic devices for urinary schistosomiasis in Oyo. The diagnosis and subsequent control of this infection currently rely on expensive diagnostic tools that are laborious to use in the local endemic context, depend on well-trained personnel often lacking in rural communities, and are not field deployable. These factors limit the capacity for stakeholders within the health system in Oyo  to perfoIn investigating this issue, our findings revealed that new diagnostic devices that fit within the context of use will need to (1) be deployable to remote or distant communities, (2) be affordable, (3) identify and confirm infection status before treatment in patients with a diagnosis based on self-reporting, and (4) be optimized for the local setting and fit within local minimal infrastructural settings. Similarly, the study revealed that the context of use and application of potential new diagnostic devices is largely associated with the stakeholder tasks or diagnostic strategies employed. These findings will contribute to driving progress in developing new diagnostic devices for urinary schistosomiasis in Oyo and globally in several ways.The study findings will contribute to driving progress in developing new diagnostic devices for urinary schistosomiasis in Oyo and globally in several ways.First, new diagnostic devices that are deployable will increase the capacity to diagnose and provide surveillance, especially in rural regions.,–Second, affordable diagnostic tests or devices that are supported by policies that reduce the overall cost of diagnosis will make diagnostic testing more affordable in Nigeria, where urinary schistosomiasis diagnostic tests costing between US$1 and US$2 (approximately ₦400 to ₦1000) are either lacking or not commercially available.–Third, new diagnostic devices that can identify infection status before treatment will support the identification and prioritization of patients or areas in need of treatment. However, MDA programs have not led to morbidity reduction since estimates that prioritize the number of people eligible for treatment from the number of people treated has often been problematic.New diagnostic devices that can equip health facilities while requiring minimal infrastructure will increase diagnostic coverage especially in rural regions. In addition, equipping health facilities in rural regions will reduce the burden on patients who often travel long distances to access treatment and are unable to return for test results.Q-methodology served as a rigorous tool in this study because it is scientifically valid and reproducible in the domain of human-centered design.Although a small size of diverse respondents is not a limitation within this study, as large sample sizes are not required for a Q-methodology study, the findings are not generalizable beyond the small participant pool . Further,,,This study provides 2 key implications related to the development of potential new diagnostic devices and the stakeholders who perform tasks targeting urinary schistosomiasis control and elimination. First, medical device designers and technology companies should ensure the development of new diagnostic devices that can equip health care facilities with minimal infrastructure, be affordable, be deployable, and identify infection status before treatment. Second, the health care system and community stakeholders in Nigeria  should c


Distal symmetric polyneuropathy (DSPN) is a common microvascular complication of both type 1 and 2 diabetes with substantial morbidity burden and reduced quality of life. Its association with mortality is equivocal.To describe the association between DSPN and all-cause mortality in people with diabetes and further stratify by the type of diabetes based on a meta-analysis of published observational studies.We searched Medline from inception to May 2021.Original data were collected from case-control and cohort studies that reported on diabetes and DSPN status at baseline and all-cause mortality during follow-up.was completed by diabetes specialists with clinical experience in neuropathy assessment.Data was synthesized using random-effects meta-analysis. The difference between type 1 and 2 diabetes was investigated using meta-regression.2 = 91.7%) compared to those without DSPN that was partly explained by baseline risk factors . The association was stronger in type 1 compared to type 2 diabetes . Findings were robust in sensitivity analyses without significant publication bias.A total of 31 cohorts  were included. Diabetes patients with DSPN had an almost twofold mortality , INot all papers reported multiple adjusted estimates. The definition of DSPN was heterogeneous.DSPN is associated with an almost twofold risk of death. If this association is causal, targeted therapy for DSPN could improve life expectancy of diabetic patients. The prevalence of diabetes mellitus is increasing rapidly worldwide: it is expected to reach 578 million in 2030 . Meta-anDiabetic microvascular complications could be partially responsible for this unexplained vascular and mortality burden. Indeed, there is strong evidence from a meta-analysis of observational studies that decreased estimated glomerular filtration rate and urinary albumin to creatinine ratio are both associated with an up to 2 times increased mortality . FurtherGiven the equivocal data on the association of DSPN with all-cause mortality, we aimed to conduct a systematic review and meta-analysis on the impact of DSPN on all-cause mortality. Our hypotheses were that  DSPN is The search strategy was designed by a diabetes specialist with experience in diabetes epidemiology (AGT) with input from all other investigators in accordance with the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guideline . MEDLINESeveral approaches  were accepted for the diagnosis of DSPN Table 1 Reviewers worked independently and checked all abstracts and selected full-text manuscripts for eligibility. Preselected manuscripts were evaluated by 2 independent reviewers for eligibility. The total number of included and excluded articles was documented by the reviewers, including reasons for exclusion or non-eligibility. Any disagreements were discussed with the study designer (AGT). Whenever reviewers disagreed and no consensus was found, the article was included into the full-text phase. Disagreements at full-text screening were resolved by consensus of all authors.For all studies, we extracted information on study design, number and characteristics of participants , prevalence of DSPN, method of DSPN assessment, duration of follow-up, all-cause mortality, matching, and confounding factors. Additionally, in the case of multiple publications, we included the most up-to-date or comprehensive information.From the given publications, we sought data on the association between DSPN and all-cause mortality with their respective 95% confidence intervals (CI) based on unadjusted  and fully adjusted models. For the unadjusted models, we collected raw numbers, odds ratios (OR), hazard ratios (HR), or incidence rate ratios (IRR) that were either unadjusted or adjusted for only age and sex. If any other variable  was taken into account, the estimate was considered to be fully adjusted. If more than one model was reported, the one with the most co-variables was selected. At this stage, we excluded unadjusted estimates from those studies, where the population was referred specifically for the examination of sensory or autonomic neuropathy, given the high risk of collider bias in this setting. If a given paper reported adjusted estimates, those were used in the analysis.The outcome measure of this meta-analysis was pooled all-cause mortality among individuals with DSPN compared to those without DSPN. We pooled estimates with their 95% confidence intervals (CIs) using random effect meta-analysis, as this methodology works well even when heterogeneity between studies is substantial . We also2-statistic. This provides an estimate of the percentage of variability across studies due to heterogeneity rather than chance: I2 < 40% may represent low heterogeneity; 30-60% may represent moderate heterogeneity; 50-90% represent substantial heterogeneity; and 75-100% represent considerable heterogeneity  were also carried out by restricting the analysis to  studies All statistical tests were two sided and used a significance level of p<0.05. We used STATA 15.1  for all statistical analyses.The flow chart of the search strategy is presented in Detailed characteristics of the included cohorts are summarized in Nine of the cohorts included population-based samples, while most were performed in tertiary care . Six cohorts used data from randomized trials or their follow-up cohorts Table 1.Diagnosis of DSPN varied widely between the different cohorts. Five cohorts used medical records as the source of data, leading to potential bias related to the non-standardized ways of examination. 3 cohorts used non-standardized symptom-checks only for diagnosis, 6 cohorts standardized questionnaires, 8 physical examination, and 7 studies used different equipments that provide quantitative measures of neuronal function. Only 5 studies used a combination of the above methods to provide the diagnosis of DSPN. When quantitative or semi-quantitative methods were considered as more reliable tools for the diagnosis of DSPN, 18 studies fulfilled this requirement Table 1.Altogether over 150 thousand diabetes patients  were included in the main analysis of whom 12.3% died during the median 7.5 (range 1.9-15.0) years of follow-up. The median proportion of participants with DSPN was 27.4% (range 8.8%-73.6%).The median age of participants was 60 years (range 20.8-68.0 years), diabetes duration ranged 0 to 25 years (median 11.1 years) in the different cohorts Table 1.While all studies provided some estimates of mortality, unadjusted (including those only adjusted for age and sex) estimates were available for 29 cohorts, while multiple adjusted estimates were reported in 17 cohorts Table 1.According to the NOS classification, 18 studies had good, 1 study had fair, and 9 poor quality  . While the point estimates in the individual studies were over one for all but one studies, the heterogeneity between studies was large (Ip<0.001) Figure 22 value  was somewhat lower in these studies compared to the overall value, heterogeneity remained large  times higher in the presence of DSPN compared to those without DSPN. While the Ied large Figure 22 value , partly related to the study by Foryoung et al. that reported a decreased risk of mortality in patients with DSPN , although DSPN still was a strong predictor of all-cause mortality. There was large heterogeneity in the estimates between studies reflected by the high Iith DSPN Figure 22 0%, p=0.48), suggesting that these estimates may have come from a common background population (probably mostly type 2 diabetes patients) . There was a low heterogeneity between the individual risk estimates (Iatients) Figure 2When we formally tested for a difference in the estimates between type 1 and type 2 diabetes by meta-regression, we found that the risk associated with DSPN was more than 2 times higher in type 1 compared to type 2 diabetes .We also investigated the potential for publication bias in our estimates. Given the large difference in risks between diabetes types, we drew funnel plots separately for each type of diabetes. These funnel plots confirmed the large difference between type 1 and type 2  groups but were incompatible with the presence of strong publication bias. The Egger tests were all non-significant  further confirming the visual interpretation  estimates arguing against the role of some influential studies in the findings. Our pre-planned sensitivity analyses restricted included cohorts by level of covariate adjustment, type of study setting, diagnostic test used to define DSPN, and overall quality of studies.First, we run separate analyses including 29 studies that reported unadjusted, and 17 studies that reported adjusted estimates. The overall risk associated with DSPN was smaller according to the adjusted estimates (pooled HR 2.0 vs. 1.6) suggesting that the included covariates explained some of the increased mortality in DSPN. In contrast, the difference between type 1 and type 2 diabetes remained statistically significant showing stronger associations between DSPN and mortality in type 1 compared to type 2 diabetes Table 2.When we restricted the analysis to population-based studies, no cohort performed on type 1 diabetes patients remained. The overall risk estimates however confirmed the association between DSPN and mortality with an almost 2 times higher risk in patients with DSPN Table 2.When we restricted the analysis to cohorts that used quantitative or semi-quantitative methods to define DSPN, the overall association between DSPN and mortality remained and the point estimate was similar to those of the other analyses. Furthermore, heterogeneity was somewhat smaller for both types of diabetes (<75%), suggesting that the use of different methods could increase heterogeneity in the mortality estimates. Although the absolute difference in hazard ratios between type 1 and type 2 diabetes was smaller in this analysis, the estimates remained statistically significantly different further supporting our hypothesis that a stronger association exists between DSPN and mortality in type 1 diabetes Table 2.When we restricted our analysis to cohorts that deemed to be of good quality according to the NOS scale, we found point estimates similar to those from the main analysis, confirming the association between DSPN and mortality. Although the difference between type 1 and type 2 diabetes pointed to the same direction as in the main analysis, the difference between hazard ratios was statistically not different probably due to the limited number of studies included in this analysis Table 2.Finally, in a meta-regression adjusted for diabetes duration, we found a similarly increased mortality in type 1 diabetes compared to type 2 diabetes as in the main analysis, although due to the wide confidence intervals, it lost its statistical significance.Based on a meta-analysis of 31 cohorts comprising over 150 thousand diabetic patients, we found an almost doubling  of all-cause mortality among diabetes patients with distal symmetric polyneuropathy compared to those without DSPN. The association was somewhat attenuated but still statistically and clinically significant  in those studies that take into account multiple risk factors of vascular disease and all-cause mortality. The observed association was more than two times stronger in type 1 compared to type 2 diabetes , most likely reflecting the much smaller background mortality of younger patients without DSPN. The observed findings were robust in several sensitivity analyses that aimed to remove bias related to different study designs, methods for DSPN definitions, and overall study quality. Furthermore, we found no sign of publication bias, or any individual study with a major influence on the estimates.Our overall finding of a statistically significantly increased mortality among people with diabetes is not very surprising given the fact that more than two third (22/31) of the included cohorts found a significantly increased risk of mortality and a further 8 studies had point estimates above one. It should be noted however, that only 4 cohorts aimed at specifically investigating the association between DSPN and all-cause mortality , 23, 37.Most of the other cohorts that reported adjusted estimates for the DSPN — all-cause mortality association, aimed to investigate either microvascular complications (or just risk factors) as predictors or cardiovascular outcomes according to their primary hypothesis. Some of the cohorts reported only unadjusted estimates that is related to the fact that these cohorts were aiming at investigating completely different questions, although reported on crude frequencies of DSPN and mortality.The fact that the association between CAN and all-cause mortality was found to be substantially attenuated and even became non-significant after adjustment for known hypertension or nephropathy in the Pittsburgh Epidemiology of Diabetes Complications (EDC) Study  highlighGiven the above reasoning, we preplanned to analyze cohorts separately that reported estimates on the DSPN-mortality association using multiple adjustments for risk factors in addition to age and sex. The role of these confounders is obvious, given that in addition to glycemic control, other cardiovascular risk factors  and micro- and macrovascular complications of diabetes are strong predictors of DSPN , 46, 47.Altogether 55% (17/31) cohorts reported adjusted estimates that confirmed our hypothesis that the association would be attenuated compared to findings of the main analysis. It should be noted however that the association still remained strong with a 60% increased point estimate  and a confidence interval that also suggests an important effect size.Although 7 studies included both type 1 and type 2 diabetes patients, only 2 of these , 37 repoWe suspect that this observation is mostly related to the huge age difference between type 1 and type 2 diabetes patients. Younger diabetes patients without DSPN are likely to be free of diabetes complications (given the strong grouping of these complications) and have a good general health, while those with DSPN are likely to have other complications, hence a hugely increased mortality. In contrast older patients with type 2 diabetes have several vascular risk factors  and an elevated mortality already independent of DSPN status leading to a smaller relative mortality increase. However it is important to note here that given the much lower absolute risk in younger people, the absolute risks still remain much higher in type 2 compared to type 1 diabetes , 48. It It should be noted that the relative mortality difference between type 1 and type 2 diabetes became non-significant in some of the sensitivity analyses. Given that the point estimates even in those models remained well above unity (1.6-2.0), we suspect that this is most likely a power issue.In contrast to our hypothesis, study design had no strong effect on the association of DSPN with all-cause mortality supported by the similar overall estimates in our main analysis and in a sensitivity analysis restricted to population-based studies. Furthermore, we found no influential study that would significantly alter the findings. We excluded one study at the selection phase based on the possibility of referral bias . This stThe meta-analyses that evaluated the effect of diabetes related CAN with all-cause mortality suggested that the method used for the assessment of neuropathy has a strong effect on the association with larger estimates if more than one modality was taken into account , 7. BaseThe definition of DSPN in the different studies varied widely. Some of the cohorts used administrative data for the definition of DSPN that is prone to (for example) indication bias, we defined these measures as high risk of bias. Another, unstandardized way for the definition is the use of simple questions on the symptoms of DSPN. Given that their wording is different between studies, these could introduce variability when meta-analyzed. Simple physical examination of absent reflexes by themselves are weak instruments for the diagnosis of DSPN and they are becoming less frequently used in research partly due to their subjectivity.A potential way to improve the symptom-based definition of DSPN is the use of standardized questionnaires. Several studies used the validated Michigan Neuropathy Screening Instrument (MNSI) for the screening of DSPN . It shouWe selected studies that used different instruments for the definition of DSPN as the most reliable tests. While the used tests are heterogeneous, given their quantitative nature and the use of equipment, they are in general less subjective. Furthermore, a skilled assistant could complete the investigation, making them suitable for the testing of large populations. These methods are also advocated in clinical guidelines for the screening of DSPN. However, it should be noted that these tests measure different modalities of sensation  , 53.A potential way to improve the definition of DSPN would be the use of a combination of a standardized questionnaire and equipment-based methods. However the number of studies that used both of these are so limited that we were unable to perform a meta-analysis with this definition Table 1.Our finding that the association was stronger in studies that used semiquantitative methods for the definition of DSPN suggests that the risk associated with DSPN is probably underestimated in our main analysis. Furthermore, this sensitivity analysis is also compatible with a higher relative mortality in type 1 vs type 2 diabetes with DSPN.We used the Newcastle-Ottawa scale to measure the overall quality of studies that includes all the above detailed aspects of study design.(NOS) The findings of the analysis restricted to good quality studies confirmed our findings in the main analysis with mostly overlapping confidence intervals , 41.The mechanism responsible for the association between DSPN and mortality is not well investigated. In general, DSPN is thought to be a determinant of reduced quality of life through disturbed sleep, physical functioning, recreation, and diminished physical and emotional well-being . HoweverFurthermore, diabetic pain per se (through similar mechanisms as DSPN itself) could be associated with an even further increased risk of mortality, as suggested by the observation of Lapin et al. on increased mortality only in those patients with painful DSPN but not in those with DSPN without pain compared to DSPN free controls . This hyAlternatively, it is also possible that DSPN is a marker of other diseases that increase mortality. Indeed, microvascular diabetes complications  show remarkable clustering . Thus, iOur meta-analysis was based on a predefined protocol standardizing the inclusion of studies and also the testing of our hypotheses. Overall, our current analysis is based on a sample with over 150 thousand participants with sufficiently high rates of mortality and DSPN to provide stable estimates. The main outcome  is a hard outcome that is unlikely to be imprecisely reported. The observed effect size is clinically relevant and statistically significant. The similar results in our main and sensitivity analyses further confirm the observed strong associations. Furthermore, our tests for influential studies and publication bias argues against major effects of individual studies or selective publication.As the quality of any meta-analysis is mostly determined by the quality of the included studies, the main limitations of our report are mostly related to the quality of the included studies. First, a large proportion of the included studies include patients from tertiary care centers, from high-income countries with Caucasian origin. All of these factors limit the external validity of our findings. Furthermore, although some of the studies reported on multiple adjusted associations between DSPN and all-cause mortality, the role of unmeasured confounding cannot be excluded. The included studies had limited data on important mortality risk factors, such as different medications, laboratory parameters, proportion of participants with risk factors at target, or other comorbidities with increased mortality. Furthermore, the differences in the definition of DSPN could have biased our results. Given that we included only cohort studies, bias related to lost to follow-up cannot be excluded. While all-cause mortality is an easily obtainable outcome, it would be of interest to see the associations of DSPN with different causes of death.Our meta-analysis strongly suggests that distal symmetric neuropathy in diabetes patients is associated with a substantially increased all-cause mortality in both type 1 and type 2 diabetes. Even if this finding is not causal, this observation should have an effect on clinical practice: the known modifiable risk factors of mortality should be treated more stringently in the presence of DSPN similarly to people with type 2 diabetes and chronic kidney disease . If thisThe original contributions presented in the study are included in the article/AT and MS contributed to the study conception and design. Analysis and interpretation were handled by all authors. Drafting of the article was handled by OV, MS, BD, VH, and AT. Critical revision for intellectual content was handled by all authors. All authors contributed to the article and approved the submitted version.


Medication adherence to CDK4/6 inhibitors such as palbociclib, prescribed as a cyclic oral anticancer therapy in women diagnosed with advanced breast cancer, may be suboptimal. We evaluated adherence to palbociclib and its impact on pharmacokinetic and pharmacodynamic (PK-PD) profiles. Patients included in the OpTAT study used an electronic monitor to register each drug intake event and were randomized into the intervention  or control group . Patients in the intervention group (n = 19) had a higher and more stable adherence than control patients (n = 19). The intervention had a larger effect on patients older than 65 and in patients with longer treatment and disease experience. The PK-PD analysis showed that catching up on a missed dose at the end of the cycle increases the risk of severe neutropenia in the next cycle. The interprofessional healthcare team should closely monitor patients’ cycle management to improve prescriptions and decrease toxicity.The cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) palbociclib is administered orally and cyclically, causing medication adherence challenges. We evaluated components of adherence to palbociclib, its relationship with pharmacokinetics (PK), and drug-induced neutropenia. Patients with metastatic breast cancer (MBC) receiving palbociclib, delivered in electronic monitors (EM), were randomized 1:1 to an intervention and a control group. The intervention was a 12-month interprofessional medication adherence program (IMAP) along with monthly motivational interviews by a pharmacist. Implementation adherence was compared between groups using generalized estimating equation models, in which covariates were included. Model-based palbociclib PK and neutrophil profiles were simulated under real-life implementation scenarios: (1) optimal, (2) 2 doses omitted and caught up at cycle end. At 6 months, implementation was slightly higher and more stable in the intervention (n = 19) than in the control (n = 19) group, 99.2% and 97.3% , respectively. The impact of the intervention was larger in patients diagnosed with MBC for >2 years , patients who received >4 cycles before inclusion  and patients >65 . Simulations showed that 25% of patients had neutropenia grade ≥3 during the next cycle in scenario 1 versus 30% in scenario 2. Education and monitoring of patient CDK4/6i cycle management and adherence along with therapeutic drug monitoring can help clinicians improve prescription and decrease toxicity. With 2.3 million new cases and 685,000 deaths in 2020, breast cancer (BC) is the most frequently diagnosed cancer worldwide . In SwitIt is estimated that 30–60% of BC patients are nonadherent to endocrine therapies, which has a direct impact on their survival ,12. ResiIn this context, we initiated the randomized controlled trial “Optimizing Targeted Anticancer Therapies” (OpTAT) at the Center for Primary Care and Public Health Unisanté and the Lausanne University Hospital (CHUV), Lausanne, Switzerland , to evalThe first objective of this study was to evaluate and compare adherence to palbociclib, a cyclic CDK4/6i, with a special focus on implementation and 12-month persistence, in patients included in the intervention group versus those in the control group. The second objective was to simulate palbociclib PK and neutrophil profiles to describe the impact of adherence on drug concentrations and neutropenia.The main outcome is medication implementation, described as a binary variable measured for each patient each day over time (1 = the observed number of electronic monitor (EM) openings is exactly as expected; 0 = the number of observed EM openings diverges from the expected number of EM openings). Medication implementation is defined, at each day t, as the proportion of patients correctly taking the medication (proportion of outcome = 1) among patients still under observation that day.We hypothesized that patients included in the intervention group would implement and persist better on palbociclib and that suboptimal implementation would influence palbociclib PK  and the risk of neutropenia.The OpTAT study was approved by the local ethics committee  in 2015 (ID 65/15) and was conducted in accordance with the Declaration of Helsinki principles. We reported findings following the ESPACOMP Medication Adherence Reporting Guideline (EMERGE)  and the The protocol of the OpTAT study has been published elsewhere . BrieflyPatients were eligible if they were adults with an OAT prescription for solid cancer. Patients were excluded from the adherence study if their treatment was not self-managed  or if they were diagnosed with cognitive disorders. Patients could either participate in the medication adherence study, the PK-PD study or both by signing a specific informed consent form. In total, 130 patients were included in the medication adherence part of the OpTAT study. In this paper, we analysed a subgroup of MBC patients (n = 38) treated with palbociclib.Patient randomization was stratified by cancer type and time since OAT initiation . Patients were randomized after a baseline period of EM use of at least 21 days. The date of the randomization was determined based on the “per-protocol” statement, i.e., the date of the first intervention delivered. Indeed, because of the coronavirus disease COVID-19 pandemic, the first intervention did not always coincide with randomization and could happen some weeks later. Patients who left the study at any moment before randomization were considered control patients .Patients randomized to the intervention group attended the routine Interprofessional Medication Adherence Program (IMAP). It is a theory-based intervention  developPatients randomized in the control group used the EM but did not have any feedback on their medication intake. Control patients visited the pharmacy only for EM refill. EM data were blinded to patients, clinicians, including the pharmacy team and researchers. Pharmacy technicians only counted the number of pills returned to the pharmacy at each intervisit monitored period so that the concordance of treatment implementation between pill count and EM adherence feedback could be verified at the end of the study.At each visit, patients in both groups were asked to state any EM deviation use during the last monitored period without (in control patients) and before (in intervention patients) seeing the temporal graph representing medication intake  . Deviati®, Cerner) at enrolment and were registered in the secure data depository RedCap  [Patient data were extracted from patients’ electronic medical records (Soarianversity) . This inThe number of observed EM daily openings was reconciled rigorously and manually with pill counts and patient reports about EM deviation use. Both the ON and OFF phases were monitored: one EM opening is expected during the ON phase, and no EM opening is expected during the OFF phase. The end date of the monitoring is defined as the date when the EM is returned or the last day of the last 15-day period with an implementation of >50% if EM has not been used in the last period, as previously performed in adherence research .Palbociclib cycle dates were determined based on three different information levels: (1) the electronic medical record reports and the prescription sheet written by the oncologist, (2) the regimen labelled on the EM pill bottle at each pharmacy dispensation and (3) the patient’s report. In the case of discrepancies between these three sources, the cycle dates were established based on the congruency of two sources. Otherwise, the patient’s report was considered the most updated information, as the cycle dates in the electronic medical record are not always updated according to the last blood cell count results.Main reasons for prescribing treatment transient interruptions or cycle start deferrals  were classified as follows: (1) toxicity or adverse effects, (2) alternate treatment  and (3) intercurent infection . Transient interruptions may also be caused by the healthcare system  or upon patients’ requests .Implementation was considered optimal (= 1) if the EM daily observed opening was equal to the expected EM opening and was considered suboptimal (= 0) otherwise. Each reason for a suboptimal implementation was coded with a specific code, which allowed us to describe reasons for nonimplementation . Codes were also attributed to each reason for the cycle start deferral , transient palbociclib interruptions and discrepancies of cycle dates notification in the electronic medical record.p < 0.05.Demographic and clinical variables are presented with proportions in both groups; quantitative variables are presented as medians and interquartile ranges (IQR). Clinical, administrative or patients’ personal events that altered the palbociclib cycle dates are described with proportions. Chi-squared or Fisher exact tests were performed to compare the proportions of events that occurred during the palbociclib cycle between groups. A statistically significant test result is considered if The underlying overall implementation was estimated by applying a generalized estimating equation (GEE) model on the daily 0/1 data with an autoregressive correlation structure and a linear and quadratic time effect.Since all patients started baseline in the control group and some of them moved to the intervention group upon randomization after a varying number of days, the group variable was introduced into the model as two time-dependent variables, representing at each follow-up time t (1) the time spent in the control group and (2) the time spent in the intervention group (linear and quadratic terms). This allowed a different time pattern to be estimated for a patient always staying in the control group or a patient switching to the intervention at a given time point . The result is summarized by the difference in implementation  between these two hypothetical patients at 6 months, when the intervention is fully effective, and enough patients are still participating.Patient age, time since MBC diagnosis and number of palbociclib cycles received at inclusion were entered one by one as covariates into the model. The covariates were dichotomized using the median of the variable as the threshold . The results are summarized by the difference in implementation (95% CI) between the intervention and control groups at 6 months for each covariate category.The effect of the treatment phase (ON or OFF) on implementation is estimated in a separate model, where the phase is represented by two time-dependent variables: time spent in ON and OFF phases, both considered in linear and quadratic forms.All statistical analyses were performed with the R statistical package .Treatment discontinuation was defined based on previously published criteria , i.e., wThe population PK-PD model previously developed with data from the OpTAT study  was usedThe population median prediction and the 95% prediction intervals (95% PI) for palbociclib plasma concentrations and absolute neutrophil count as a function of time were derived. For visual interpretation of the impact of real-life implementation scenarios on palbociclib PK, we superimposed to the simulated concentration-time profiles the real palbociclib plasma concentrations measured in one patient receiving a standard palbociclib dosage regimen who generally had an optimal palbociclib implementation (scenario A) but who missed a dose at Day 18 once (scenario B).The demographic and clinical variables of the 38 patients are presented in In total, 155 cycles were monitored and analysed in the 19 intervention patients and 184 cycles in the 18 control patients.In the control group, patients older than 65 years, patients diagnosed with MBC for more than 2 years and patients who received more than 4 cycles of palbociclib before inclusion had a lower implementation, whereas in the intervention group, these covariates did not impact implementation. Thus, the impact of the intervention on implementation was larger in patients diagnosed with MBC for more than 2 years in the intervention and control groups, 99.5% and 95.9%  b, in patThe analysis of ON and OFF phases of all participants—regardless of the randomized group–estimated that implementation decreased over time during ON phases but remained stable in OFF phases during the first 6 months . Equatiop = 0.046), and more cycles were impacted by a missed dose in control versus intervention patients  (p = 1.000). In the case of a missed dose, some patients did not behave consistently; they caught up the missed dose in some cycles but did not in others . Some OFF phases were shortened to 6 instead of 7 days in the intervention and control groups, respectively, because a dose was caught up  (p = 1.000), and none of these dose reductions were caused by a reduced length of the OFF phase due to the catch-up of missed doses.Forgetting a dose was not uncommon in our population. Significantly more control patients versus intervention missed at least one dose during the monitoring  . In pati= 0.658) . Few patAs a reminder, the simulated scenarios of palbociclib implementation were (A) optimal implementation, (B1) one dose is omitted at day 18, which is not caught up at cycle end, 7 days of treatment break are initiated from day 22, (B2) one dose is omitted at day 18, which is caught up at cycle end at day 22, 6 days of treatment break are initiated from day 23 (C) the 2 first doses are omitted at day 1 and 2 and are caught up at cycle end at day 22 and 23, 5 days of treatment break are initiated from day 24.Model-based PK-PD simulations showed that missing one (scenarios B1 and B2) or two doses (scenario C) occasionally resulted in a decrease in the palbociclib plasma concentration-time profile compared to optimal implementation (scenario A) immediately after the missed dose(s), as expected. As shown in the real patient palbociclib plasma concentration represented by the blue dot in p = 0.447) (p = 0.049). Half of the interruptions occurred because of an infection. Other reasons reported were surgery, side effects and synchronization with the fulvestrant cycle or synchronization with previous palbociclib cycles. Among the cycles interrupted, the number of cycles that were resumed after the interruption was 4/10 (40%) in the intervention group and 2/4 (50%) in the control group (p = 1.000), whereas the other cycles were stopped for at least 7 days before starting another cycle.At least one transient interruption of palbociclib during the ON phase was experienced in 6/19 (32%) patients in the intervention group and 3/18 (17%) in the control group (= 0.447) , with mop = 0.447) experienced at least one cycle start deferral due to clinical reasons , administrative reasons  or patient’s personal reasons   .These frequent transient palbociclib interruptions and deferrals of cycle start are probably the cause of discrepancies in the cycle dates notification in patients’ electronic medical record and prescription sheets .In the OpTAT study, patients taking OAT for solid cancer were randomized to the Interprofessional Medication Adherence Program (IMAP) versus controls. In this paper, we present the impact of this intervention in the subgroup of patients treated with palbociclib for MBC. The IMAP, offered to patients in the intervention group, allowed maintaining a higher and more stable palbociclib implementation compared to control patients. The intervention had a larger effect in patients older than 65 years, those diagnosed with MBC for more than 2 years and patients who received more than 4 cycles of palbociclib before inclusion. The intervention did not impact persistence to palbociclib. Model-based PK-PD simulations showed that catching up missed doses at the end of the ON phase, followed by a shortening of the OFF phase, leads to a higher risk of severe neutropenia during the following cycle. Our results show that cycles start deferral occurred due to neutropenia, but also administrative reasons or patients’ personal requests. The interprofessional health care team should closely monitor patients’ cycle management to update cycle dates in the prescription sheets and electronic medical record.Overall, patient implementation was high >95%) at 6 months in both randomized groups, which indicates efficient treatment self-management. Facilitators for adherence may have been the frequent medical appointments , sometimes coupled with other consultations from the interprofessional health care team  to help the patient cope with treatment and disease self-management. Such added consultations to the medical appointments are not offered systematically but according to the patient’s request and needs. Supporting medication adherence is not the core of these interventions and is not embedded in any theoretical framework. Interestingly, our population is not polymedicated  , which m% at 6 moEven if patients with BC describe OAT as convenient to use compared to intravenous chemotherapy , the numIn our study, various behaviours were observed in the same patients after a missed dose, which highlights that consensual education provided by HCPs to self-manage a missed dose might not be sufficient. If oncologists are not aware of patient missed doses and end-of-cycle catching-up behaviours, a low neutrophil blood count can be misinterpreted, leading (i) to unnecessary dose reductions, (ii) treatment interruptions, (iii) next cycle start deferral, and so on to desynchronization with concomitant cyclic treatments, e.g., fulvestrant injections. Interprofessional partnerships are to be strengthened and better defined to synergistically improve patient adherence to palbociclib.In this study, implementation was monitored in OFF phases , even though the patient’s effort to reach an optimal implementation during the ON phase  is higher than in the OFF phase. Indeed, we showed that implementation during the ON phases decreased with increasing cycles, whereas it was stable in the OFF phases. Notably, extended OFF phases  must be considered differently . Further scientific methodologies to characterize the implementation of treatment with cyclic regimens must be investigated . Data cleaning is of particular importance when numerous prescribed treatment interruptions are monitored to avoid misinterpreting EM nonopenings. As automated and robust methodologies to clean EM databases are needed, we are currently developing a script in the statistical software R to clean and reconcile EM data according to patient’s report and patient’s electronic medical record.Finally, some patients actively participate in shared decision-making about their treatment management . The way to consider them needs to be defined before analysing medication implementation (and adherence) either by considering it as a treatment nonimplementation as it deviates from the recommended regimen or as an optimal treatment implementation as the new regimen relies on a shared-decision process.This study has several strengths. First, medication adherence was evaluated by EM, which is considered one of the gold standard methods to measure adherence and especially implementation ,44,45. ASome limitations should be acknowledged. First, palbociclib implementation in control patients seems to increase towards the study end. This phenomenon was already observed in a study depicting patient implementation to antiretrovirals in patients with human immunodeficiency virus (HIV)  and was The interprofessional medication adherence program shows that the intervention had a larger effect in patients older than 65 and those with longer treatment and disease experience. Various clinical, administrative or patients’ personal events can alter the dates of ON and OFF phases. OFF-period reduction due to missed doses that are caught up at cycle end increases the risk of severe neutropenia in the next cycle, which may lead to inappropriate dose reduction. Currently, two CDK4/6i are administered cyclically, palbociclib and ribociclib. Well-designed education programs and monitoring of patient cycle management by an interprofessional team along with PK-piloted dose reduction in case of side effects can help clinicians improve prescriptions and better prevent toxicity. The impact of adherence on CDK4/6i effectiveness should be further investigated.