This cross-sectional study examines the association between use of an insurance-owned patient-facing price estimator tool by pregnant individuals and out-of-pocket childbirth spending. What are the patterns and characteristics of price transparency tool use among pregnant individuals with commercial insurance?In this cross-sectional study of 253 606 pregnant individuals, price transparency tool use increased from 5.9% in the 2011 to 2012 study period to 13.0% in the 2015 to 2016 study period. Higher coinsurance was associated with more price transparency tool use, whereas a previous cesarean delivery was associated with less use.Use of the price transparency tool was associated with higher childbirth spending; these tools may be used for informational and planning purposes by pregnant individuals. When introduced a decade ago, patient-facing price transparency tools had low use rates and were largely not associated with changes in spending. Little is known about how such tools are used by pregnant individuals in anticipation of childbirth, a shoppable service with increasing out-of-pocket spending.To measure changes over time in the patterns and characteristics of use of a price transparency tool by pregnant individuals, and to identify the association between price transparency tool use, coinsurance, and childbirth spending.This descriptive cross-sectional study of 2 cohorts used data from a US commercial health insurance company that launched a web-based price transparency tool in 2010. Data on all price transparency tool queries for 2 periods  were obtained. The sample included enrollees aged 19 to 45 years who had a delivery episode during 2 periods  and were continuously enrolled for the 10 months prior to delivery (N = 253 606).Access to a web-based price transparency tool that provided individualized out-of-pocket price estimates for vaginal and cesarean deliveries.The primary outcomes were searches on the price transparency tool by delivery mode , timing , and individual characteristics . Another outcome was the association of out-of-pocket childbirth spending with price transparency tool use.The sample included 253 606 pregnant individuals, of whom 131 224 (51.7%) were in the 2011 to 2012 cohort and 122 382 (48.3%) were in the 2015 to 2016 cohort. In the 2015 to 2016 cohort, the mean (SD) age was 31 years (5.2 years) and most individuals had coinsurance for delivery (94 251 [77.0%]). Price searching increased from 5.9% in the 2011 to 2012 cohort to 13.0% in the 2015 to 2016 cohort. In the 2015 to 2016 cohort, 43.9% of searchers’ first price query was in their first trimester. The adjusted probability of searching was lower for individuals with a high-risk pregnancy due to a previous cesarean delivery  vs individuals with low-risk pregnancy . Use increased monotonically with coinsurance, from 9.2%  among individuals with no coinsurance to 15.0%  among individuals with 11% or higher coinsurance. After adjusting for covariates, searching was positively associated with out-of-pocket delivery episode spending. Among patients with 11% coinsurance or higher, early and late searchers spent more out of pocket , compared with never searchers.The results of this cross-sectional study indicate that the proportion of pregnant individuals who sought price information before childbirth more than doubled within the first 6 years of availability of a price transparency tool. These findings suggest that price information may help individuals anticipate their out-of-pocket childbirth costs. In this cross-sectional study, we used data spanning the 6 years after the introduction of an insurer’s price transparency tool to examine how pregnant individuals used price information on delivery and to add to our understanding of the function of price transparency for shoppable services. Specifically, we sought to measure changes over time in the patterns and characteristics of use of a price transparency tool by pregnant individuals and to identify the association between price transparency tool use, coinsurance, and delivery spending.STROBE) reporting guideline. This study was determined to be exempt by the institutional review board at the Harvard T.H. Chan School of Public Health. This institutional review board also waived informed consent because the study was determined to be of minimal risk to participants.We followed the Strengthening the Reporting of Observational Studies in Epidemiology  and median household income in the area . We used the individual’s zip code to identify their hospital referral region (HRR) and whether they lived in an area that was urban, nonurban- and urban-adjacent, or rural.International Classification of Diseases, Ninth Revision and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes from established methods.20 We identified whether an individual had a previous cesarean delivery through diagnosis and procedure codes (eTable 1 in the We categorized pregnancy risk as follows: (1) high risk for cesarean delivery attributed to previous cesarean delivery, (2) high risk for cesarean delivery without previous cesarean delivery, (3) presence of other obstetric comorbidities, and (4) low risk. These mutually exclusive risk categories were constructed with 19 Individuals who did not meet these criteria but had 1 or more conditions that were captured in the Obstetric Comorbidity Index, including maternal age of 35 years or older, during the 10 months before delivery composed the third risk category group.20 The remaining individuals were classified under the fourth risk category of low-risk pregnancy.The second risk category included individuals who met the Society for Maternal-Fetal Medicine’s definition of high risk for cesarean delivery but who had not had a previous cesarean delivery.We calculated total delivery episode spending as the sum of the allowed amount on each claim during the delivery episode and out-of-pocket spending as the sum of the deductible, coinsurance, and copayment amounts during the delivery episode. We measured coinsurance exposure during delivery as the sum of all coinsurance payments during the delivery episode divided by total delivery episode spending minus any deductible spending.2 test for equality of proportions.We calculated the percentage of the study sample who queried the price transparency tool overall and by delivery mode in 2011 to 2012 and in 2015 to 2016. We compared the differences between the 2 cohorts using a 2-sample χThe remaining analyses were conducted on each cohort separately. Results from the 2015 to 2016 cohort are reported in the main text given its relative recency and to provide new evidence on the function of price transparency for this shoppable service. Results from the 2011 to 2012 cohort are provided in eFigure 2, eFigure 3, and eTable 5 in the We examined patterns of price transparency tool use, including whether the delivery mode searched aligned with the delivery outcome, and the timing of searching relative to delivery. We performed multivariable logistic regression to analyze whether rates of searching varied by individual characteristics such as age at delivery, rurality, pregnancy risk status, coinsurance exposure, and the educational attainment quartile and median household income quartile associated with zip codes. The multivariable logistic regression model included fixed effects for month of delivery and HRR and clustered SEs at the HRR level.We estimated the association between price transparency tool use and delivery spending by running linear regression models with log-transformed total and out-of-pocket delivery episode spending as the dependent variables. The key independent variable was a categorical variable that indicated searcher type: never searcher, early searcher (first searched before the third trimester), or late searcher (first searched during the third trimester). Analysis of total delivery episode spending was stratified by delivery mode. Analysis of out-of-pocket delivery episode spending controlled for delivery mode and was stratified by coinsurance for delivery . Individuals with 0% coinsurance were excluded from the out-of-pocket spending analysis because nearly half of these individuals (46.3%) had no out-of-pocket spending on the delivery episode. Linear regression models controlled for the individual characteristics that were associated with searching in the multivariable logistic regression analysis . The SEs were clustered at the HRR level. Results of these models are presented as percentage differences in spending and mean dollar differences relative to the reference group (never searchers). We used likelihood ratio tests to compare the estimated coefficients for early and late searchers.We estimated whether searcher type was associated with delivering at a facility with case-mix and market-adjusted total delivery episode spending above the 50th percentile of all hospitals within the individual’s HRR (a higher-cost facility). Logistic regression was performed for this analysis .The sample comprised 253 606 pregnant individuals, of whom 131 224 (51.7%) were in the 2011 to 2012 cohort and 122 382 (48.3%) were in the 2015 to 2016 cohort. The demographic characteristics were similar across the 2 cohorts. In the 2015 to 2016 cohort, the mean (SD) age was 31 years (5.2 years) and most individuals had coinsurance for delivery (94 251 [77.0%]). Most individuals in both cohorts lived in areas that were urban, were in the top 2 quartiles of educational attainment, and were in the top 2 quartiles of median household income . ApproxiP < .001) (P < .001) and for cesarean delivery .Use of the price transparency tool before delivery increased from the 2011 to 2012 cohort to the 2015 to 2016 cohort  . Rates oMost of the results from the 2015 to 2016 cohort were qualitatively consistent with the results from the 2011 to 2012 cohort. We present results from the 2015 to 2016 cohort; the 2011 to 2012 cohort results are shown in eFigure 2, eFigure 3, and eTable 5 in the Searchers queried price estimates for vaginal delivery only (56.8% of searchers), cesarean delivery only (17.3%), or both delivery modes (25.9%). Searchers and never searchers had similar delivery outcomes   of searchers first queried the price transparency tool in the first trimester of pregnancy . First-tIndividuals in the middle of the age distribution (aged 25-34 years) were more likely to search for delivery prices than younger (aged 19-24 years) or older (aged 35-45 years) individuals  (eFigure 4 in the 14 and this finding is consistent with patient surveys reporting that awareness of price transparency tools improved between 2010 and 2017.22Among a national sample of commercially insured pregnant individuals, use of a price transparency tool for delivery doubled in the first 6 years the tool was available, from 5.9% in 2011 to 2012 to 13.0% in 2015 to 2016. This 6-year period corresponded with increasing out-of-pocket costs for maternity services,Use of the price transparency tool appeared responsive to clinical information about delivery mode. Much higher rates of vaginal delivery were found among individuals who searched only for vaginal delivery price estimates compared with those who searched only for cesarean delivery price estimates. We observed an increase in first-time searches for cesarean delivery in the third trimester; this time is when individuals may receive clinical information about a likely cesarean delivery, such as fetal presentation.23Individuals with higher levels of coinsurance were more likely to use the price transparency tool. These individuals generally have a stronger incentive to obtain price estimates because they pay a higher proportion of total delivery costs than other individuals do. Searching may also be affected by the number of in-network childbirth facility options that pregnant individuals have. We observed lower rates of searching in rural areas and areas with lower median household income, which are more likely to have fewer facilities offering hospital-based obstetric services.24We observed that individuals who had a previous cesarean delivery were less likely to conduct a price search. These individuals may have an existing relationship with a clinician or may be committed to delivering at a particular facility that they perceive to be suited to caring for more complex cases. Most price transparency tools display limited, if any, information on quality of care that could assist patients in evaluating cost and quality together to choose a higher-value, rather than just lower-priced, clinician or facility. Studies of clinician selection among pregnant individuals have reported that most of these individuals select their clinician, obstetrician, or midwife and then deliver at whichever hospital or birthing center the clinician has admitting privileges.26We found a positive association between using the price transparency tool and delivery spending. Although this association cannot be interpreted as a causal relationship between searching and spending, it raises important hypotheses about the unobserved characteristics of searchers that are also associated with higher delivery spending. The results of this study suggest that this association is unlikely to be attributed to searchers selecting higher-cost facilities compared with nonsearchers. Searching late in the pregnancy, but not searching before the third trimester, was associated with delivering at a high-cost facility. By the time they conducted a search in the third trimester, late searchers were likely to have already selected a facility for childbirth. Instead, those who had planned to give birth at a relatively high-cost facility may have been aware of the expected costs and may have used the price transparency tool for financial planning. We cannot rule out the possibility that late searchers preferred higher-cost facilities because they equated higher cost with higher quality. However, surveys have found that most people in the United States understand that health care costs and quality are not necessarily correlated.11; more studies are needed to trace whether this finding would continue to be true over time.Early searchers, who were not more likely than never searchers to deliver at a high-cost facility, may be different from never searchers in some way that is associated with delivery cost within a facility. Early searchers may have had higher costs because of having a higher risk pregnancy in ways that we could not measure in our data. Perhaps they preferred more health care services within the delivery episode. Previous research has generally shown that price transparency tools are not associated with selection of lower-priced facilities27 Individuals may also perform a price search to verify that a facility is within their network to protect against high out-of-pocket costs or surprise bills.28 Future research can examine whether price transparency has helped patients prepare for their out-of-pocket costs and improved downstream financial outcomes.Use of price transparency tools that is not associated with selection of lower-priced clinicians or facilities does not necessarily mean the tools are of no value. Individuals may use the tool for informational or financial planning purposes. Health care price information can help patients anticipate their out-of-pocket expenditures.29 Fifth, there are likely unobserved differences between searchers and never searchers that may be associated with delivery spending; therefore, these findings cannot be interpreted as causal.This study has several limitations. First, we examined only 1 price transparency tool, and the results may not be generalizable to tools that may be designed differently or marketed and offered by other insurers, employers, government, or third parties. Second, the data we obtained did not capture whether individuals in the sample used other price transparency tools. Third, the results may not be generalizable to services other than delivery, although focusing on this shoppable service provides insights into price transparency in the context of high out-of-pocket costs. Fourth, the results may not be generalizable to pregnant individuals who are uninsured, are covered by Medicaid, use the price transparency tool to select a nonhospital setting for delivery, or experience insurance churn during pregnancy.Searching for childbirth price information increased over the first 6 years after the launch of a price transparency tool but remains uncommon. Interest in price estimates was highest early in pregnancy, although early searchers were not more likely than never searchers to deliver at a lower-cost facility. The findings of this cross-sectional study suggest that price transparency tools can be used for informational or financial planning purposes in addition to clinician and facility selection. Promoting the use of price transparency tools for these purposes may increase their use and could help individuals plan for high out-of-pocket costs.


Mitochondria are critical for several cellular functions as they control metabolism, cell physiology, and cell death. The mitochondrial proteome consists of around 1500 proteins, the vast majority of which (about 99% of them) are encoded by nuclear genes, with only 13 polypeptides in human cells encoded by mitochondrial DNA. Therefore, it is critical for all the mitochondrial proteins that are nuclear-encoded to be targeted precisely and sorted specifically to their site of action inside mitochondria. These processes of targeting and sorting are catalysed by protein translocases that operate in each one of the mitochondrial sub-compartments. The main protein import pathway for the intermembrane space (IMS) recognises proteins that are cysteine-rich, and it is the only import pathway that chemically modifies the imported precursors by introducing disulphide bonds to them. In this manner, the precursors are trapped in the IMS in a folded state. The key component of this pathway is Mia40 , which itself contains cysteine motifs and is subject to redox regulation. In this review, we detail the basic components of the MIA pathway and the disulphide relay mechanism that underpins the electron transfer reaction along the oxidative folding mechanism. Then, we discuss the key protein modulators of this pathway and how they are interlinked to the small redox-active molecules that critically affect the redox state in the IMS. We present also evidence that the mitochondrial redox processes that are linked to iron–sulfur clusters biogenesis and calcium homeostasis coalesce in the IMS at the MIA machinery. The fact that the MIA machinery and several of its interactors and substrates are linked to a variety of common human diseases connected to mitochondrial dysfunction highlight the potential of redox processes in the IMS as a promising new target for developing new treatments for some of the most complex and devastating human diseases. Mitochondria play also a critical role in specific cell death modalities ,2,3. Surc .Saccharomyces cerevisiae (S. cerevisiae) and roughly 1400 in humans, yet the yeast and human mitochondrial genome encode for just seven and 13 proteins, respectively [S. cerevisiae [Mitochondria contain around 1000 different proteins in ectively . Thereforevisiae . We shourevisiae . The difrevisiae ,10.The MIA  pathway in the IMS is unique as it is the only pathway that involves chemical modification of cysteine residues and therefore is potentially subject to regulation through redox signalling. This potential is due to the sensitivity that the cysteine residues have to redox states due to the presence of anionic sulfur, which is reactive with different oxidising agents, resulting in multiple oxidised species . The IMSThe redox active environment of the IMS promotes the oxidation of cysteine residues, which ultimately leads to the formation of disulphide bonds. These disulphide bonds are formed in substrate proteins that are imported into the IMS, in a reaction where the thiol groups of two cysteine residues are oxidised, forming a covalently linked intramolecular disulphide. It is through the process of oxidative folding where a protein acquires its native disulphide bonds and therefore its native three-dimensional structure ,18. The Mia40 contains six strictly conserved cysteine residues that are organised as a redox-sensitive CPC motif and two CX9C motifs. These two CX9C motifs stabilise the protein and form a hydrophobic cleft of two anti-parallel helices to bind the substrate. The solvent-exposed CPC motif constitutes the redox-active cysteine pair of Mia40 that accepts electrons shuttled from the substrate protein and introduces the disulphide bond whilst switching between a reduced and oxidised state ,19,21,22Classical Mia40 substrates typically have a small mass of between 8 and 22 kDa, a hydrophobic intermembrane space targeting signal (ITS), and either twin CX3C or CX9C motifs, such as the family of Tim proteins ,24,25. OTo reach the IMS, proteins must pass first through the OMM and, in the MIA pathway protein, substrates are translocated through the TOM complex in an unfolded and reduced conformation which renders them import-competent . During This is where the second essential component of the MIA pathway Erv1 comes into play and re-oxidises Mia40 for another cycle of disulphide bond formation . Erv1 isc and complex IV of the respiratory chain  interacts with the hydrophobic binding groove of Mia40 in a similar manner to the ITS of the substrate, accepting the electrons via its N-terminal redox active CPC motif ,40. Thenry chain  [41,42,4ficiency .It is worth mentioning that Mia40 requires and interacts with endogenous Mia40 for its own import into the IMS. The import of Mia40 occurs in kinetically distinct steps; firstly, Mia40 translocates through the TOM complex, and it is then inserted into the IMM by the TIM23 complex . SecondlOverall, the MIA pathway is responsible for inserting disulphide bonds into proteins, trapping them in the IMS. Similar to the disulphide relay systems in the ER and bacterial periplasm, electrons are transferred from the substrate onto Mia40, then Erv1, and onto a final electron acceptor with Mia40 being re-oxidised in the process . HoweverAif or chchd4 genes [Mia40 exists in the IMS as either a soluble protein in higher eukaryotes or anchored through its N-terminal in fungi and yeast . The evolutionary conserved human homolog of yeast Mia40 is the small (16 kDa) IMS-soluble CHCHD4 (Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 4) ,49. Compd4 genes ,56.As mentioned previously, the MIA pathway is unique, as it is the only import pathway that chemically modifies its substrates, resulting in intramolecular disulphide bonds. This potentially opens up this pathway to regulation by the redox environment of the cell, which is determined by all the oxidative and reductive reactions that occur. Redox reactions produce reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are beneficial for cell signalling at low levels but damaging at high levels. These damaging effects can include DNA modification, protein oxidation, and lipid oxidation, all of which are detrimental to cell function and can lead to cell death ,57. The Redox signalling is crucial, as it allows cells to rapidly adapt to a range of environmental stimuli. The cellular redox status is maintained by several antioxidant systems, primarily the thioredoxin (Trx) and glutaredoxin (Grx) systems. Redox signals produced by the essential functions of mitochondria such as oxidative phosphorylation (OXPHOS), fatty acid oxidation, and the synthesis of iron–sulfur clusters allow mitochondria to be integrated into the wider cellular context through the free diffusion of small molecules between the IMS and the cytosol via the semi-permeable OMM . It is a2O2 within the cell and promotes the oxidation of the transcription factor Yap1 [2O2 that directly oxidises the Cys36 of Gpx3. Then, Gpx3 forms a transient intermolecular disulphide with Cys598 of Yap1, which is then oxidised and activated. Then, Yap1 translocates to the nucleus and induces the anti-oxidant response, which leads to the transcription of anti-oxidant response genes. This pathway is attenuated by thioredoxin, which reduces the disulphide in Gpx3 that is formed by an increase in H2O2 levels [Glutathione peroxidase 3  is a thiol peroxidase and was one of the redox regulatory protein identified in the 2012 yeast IMS proteome study . Gpx3 actor Yap1 . This le2O2 stress to form an 18-amino acid N-terminal (N18)-extended version of the protein that was identified as being targeted to the mitochondrial IMS [Gpx3 has a molecular weight (MW) of ≈26 kDa and undergoes alternative translation under Hrial IMS ,61. Thist-butyl hydroperoxide and impaired import of Mia40 substrates, leading to suggestions that Hot13 plays a role in IMS redox regulation. Additionally, Hot13 mutants, when compared to wild-type cells, have a greater level of reduced Mia40 but no difference in overall levels of Mia40 and Erv1. This leads to a proposed model where Hot13 acts as a metal chaperone to transfer zinc ions from Mia40 allowing for re-oxidation by Erv1 [Another modulator of Mia40 is the soluble IMS protein Hot13, which is a non-essential player of the MIA pathway, instead having an ancillary role. Hot13 is conserved among eukaryotes, contains 10 cysteines, and has a CHY zinc-finger domain, which provides the capacity to bind zinc . Hot13 h by Erv1 . In concS. cerevisiae contains three thioredoxins  and two thioredoxin reductases (Trr1 and Trr2) [2C motif. The mechanism of the Trx system is similar to the Grx system with electrons being transferred by Trx to the substrate through thiol–disulphide exchange reactions [The thioredoxin system is comprised of thioredoxin (Trx), thioredoxin reductase (TrR), and nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. nd Trr2) . Trx3 aneactions .Interestingly, Trx1 has previously been shown to directly interact with Mia40 in a large proteomic screen . Trx1 alThe process of disulphide bond formation is error-prone, and therefore, it seems logical that a reductase system would be present in this constricted sub-compartment in yeast mitochondria to allow for disulphide isomerisation as a means to achieve the correct disulphide bonds in IMS proteins. The reducing enzyme glutaredoxin 1 (GRX1) in the IMS was shown to modulate the oxidation of COX17 (a copper chaperone for the Cytochrome c Oxidase or COX complex), which is a mammalian Mia40 (CHCHD4) substrate . Trx1 coFurther research is required to identify the interplay and communication of the mitochondrial-targeted Gpx3 and the levels of Trx1 in the IMS. It will be interesting to see how these two proteins act to influence the redox environment of this sub-compartment, especially as Erv1 has recently been identified to not be as active as previously thought . A summa2O2) and glutathione (GSH), which act as signals and are important for initiating the redox response of the cell. H2O2 is classified as a reactive oxygen species (ROS), which are produced through normal cell metabolism and also include the superoxide anion (O2−) and the hydroxyl radical (OH−), which are all associated with oxidative stress [2O2 has oxidising activities, and they both have a role in maintaining the redox environment of the IMS. In this section of the review, we will discuss potential dialogues between these molecules and the MIA pathway  because it does not contain unpaired electrons. Instead the reactivity of H2O2 is provided by its relatively weak O–O bond, which is susceptible to decomposition, resulting in the formation of a hydroxyl radical (OH−), which is very reactive [Reactive oxygen species are normally produced as by-products of cellular respiratory metabolism and can be harmful to cells when their concentrations are very high. Here, we focus on Hreactive ,69.2O2 (and/or O2−) are produced by up to eleven distinct sites in mammalian mitochondria. These sites include processes such as the electron transport chain (ETC) and substrate oxidation, which have been covered extensively in a review elsewhere [2O2 is generated in places where there is a leakage of pairs of electrons, and there are various antioxidant defence systems in place to consume H2O2 and prevent damage to the cell. Additionally, H2O2 can easily pass through mitochondrial membranes, meaning that H2O2produced in the matrix can travel to the IMS through the IMM. This both increases the level of H2O2 in the IMS and may have a role in signalling to the cell through the buffering function of the IMS. H2O2 is generated in the IMS several ways. The copper–zinc isoform of Superoxide dismutase 1 (SOD1), which catalyses the rapid dismutation of two molecules of O2− to produce H2O2 and O2, is present in the IMS. Superoxide anions are released to the IMS side of the IMM by various enzymes of the respiratory chain, notably complex III [2O2. During this process, Erv1 can pass the electrons directly to molecular oxygen with a concomitant production of H2O2. Daithankar et al. (Biochemistry 2009) first showed that Erv1 interacts with cytochrome c in vitro [c (cyt c) is the in vivo oxidase for Erv1, as yeast cells mutated in cyt c cannot grow under anaerobic conditions. This demonstrated that Erv1 functionally links the Mia40-dependent import pathway to the Mia40-independent cyt c import pathway by transferring electrons from the incoming precursors to cyt c as an acceptor [c may protect the cell from oxidative damage, as avoiding passing the electrons to molecular oxygen prevents H2O2 formation. It was concluded that this interaction allowed for electrons to be passed to the respiratory chain, which led to the production of water instead of H2O2 and simultaneously connected the ETC to the redox state of the IMS [2O2 in the IMS, cytochrome c peroxidase (Ccp1) reduces the harmful H2O2 to water [2O2as thiol peroxidases are major antioxidants in cells and protect against H2O2 caused oxidative stress [2O2 from this process and the IMS [Hlsewhere . Generalplex III . Additioin vitro . Allen eacceptor . Later,  the IMS . To remoto water . Additioe stress . Both Cc the IMS .2O2 can directly oxidise select cysteine residues to induce signalling and is thought to be broadly non-specific. This oxidation of cysteine thiol groups can lead to structural changes in the protein, which can lead to a change of function. As mentioned above, Trx1 is capable of reducing these disulphide bonds with both Trx1 and Trr1 being identified in the IMS [2O2 to manifest both from invaginations of the IMM and from the presence of respiratory chain enzymes [2O2 in mitochondria, and any effects of H2O2 diffusion through pores in the OMM may have on signalling between mitochondria and the cell. Interestingly, it has been reported that H2O2 signalling may be under the control of a signal transduction pathway, which links the ETC to the IMS-localised Syk pathway, with H2O2 inducing the phosphorylation of Syk. The authors suggest that this connection between the respiratory chain and the conserved Syk pathway produces a focused signalling response to the cell; however, only extracellular H2O2 was used in this study [2O2 and not just the potential damage it can cause in cells.In terms of signalling, H the IMS . In the  enzymes . Furtheris study . Nonethe2− and H2O2 or disrupting mitochondrial thiol homeostasis (mitoParaquat and MitoChlorodinitrobenzoic acid respectively) will allow for further study into the effects of these process in the mitochondria [2O2 has an oxidising effect in the IMS, with signalling functions between the mitochondria and the wider cell. In the future, it will be interesting to look at any interplay between a potential reducing system in the IMS (Trx1 and Trr1) and H2O2 for maintaining the redox status of the IMS for optimal oxidative folding conditions  and GSH reductases (GR). The glutaredoxin system is one of the major pathways that controls cellular redox homeostasis, in addition to the thioredoxin pathway. GSH has various key roles in reducing disulphide bonds, modulating iron–sulfur clusters biogenesis, and detoxifying ROS. Participating in these reactions frequently leads to the oxidation of GSH to GSSG, which contains a disulphide bond that links two molecules of GSH together, whilst the targets of GSH are left reduced. GSSG is reduced back to GSH by glutathione reductase (Glr) using electrons supplied by NADPH ,72. It iGSH) is key to further untangling the influence of redox processes. In 2012, Kojer et al. demonstrated that the GSH redox potential in the IMS is linked to the cytosol, with both compartments having a similar EGSH. They found that EGSH in the IMS is determined by the glutathione reductase system in the cytosol with the two pools of GSH being kinetically connected. Furthermore, EGSH in the matrix is regulated separately from the cytosol and the IMS. Interestingly, they reported that the local EGSH has a role in maintaining the partially reduced redox state of Mia40, which is found to be around 70% oxidised. When Erv1 was depleted in cells, Mia40 was found to be increasingly reduced, which could be due to the GSH pool in this compartment [GSH.In cells, the majority of glutathione is in the reduced form GSH, and the ratio of GSH to GSSG (GSH:GSSG) is an indicator of oxidative stress, with a greater level of stress increasing the ratio of GSSG:GSH. This ratio varies throughout the cell, and understanding the local glutathione redox potential c . Howeverc .+), reduced Nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP+), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) are now well established as important enzyme cofactors and signalling mediators of fundamental metabolic and biosynthetic processes and antioxidant functions in the cell [Nicotinamide adenine dinucleotide . The function of Ary1 remains unknown, including what the channel is responsible for transporting. The presence of NADPH was reported to stimulate the frequency of channel gating between different states, and in the absence of NADPH, the potential of the channel was reversed . Therefo+/NADH binding segment, exists both in monomer and dimer forms in the mitochondrion [+/NADH-binding domain and impairs the interaction with CHCHD4, is an indicator of the relevance of NADH/NADPH homeostasis in the physiological functioning of the MIA import pathway [In human cells, an NAD(P)H-dependent redox process was also found to influence the interaction of the human homolog of Mia40 (CHCHD4) with its regulatory partner AIF . The flahondrion ,85,86,87hondrion ,85,86,87hondrion . Althoug pathway ,88.A summary of the important small redox-active molecules associated with redox regulation in the mitochondrial IMS discussed in this section is given in As the IMS is a constricted compartment, there is likely to be some communication between the different proteins involved in the IMS machinery and other IMS-related pathways, particularly signalling pathways. It has previously been suggested that there is crosstalk between the mitochondrial GSH and Trx systems in the matrix, which does not occur in the cytosol . We prop2O2 (which oxidises Mia40) leads to the disassembly of the ISC [2O2 lead to ISC dissociation [Iron–sulfur clusters (ISCs) are cofactors, specifically metal prosthetic groups that are involved in a wide range of cellular pathways such as protein translation, energy production, and DNA replication and repair ,90. The  the ISC . Additioociation . Interesociation . HoweverS. cerevisiae have lost MCU and instead contain MCU homologs [Calcium uptake into mitochondria of higher eukaryotes is mediated by the mitochondrial calcium uniporter (MCU), which is an IMM-anchored calcium-conducting protein that functions in concert with regulatory proteins MICU1  and MICU2 . The MCU-mediated uptake of calcium is driven by the membrane potential of the IMM . During homologs . Tight chomologs . Recentlhomologs . Intereshomologs . MICU1 rhomologs . MICU1 chomologs . OverallIn this review, we have highlighted some critical aspects of how redox regulation and signalling converge in the mitochondrial intermembrane space at the MIA pathway. Optimal operation of these processes requires a finely tuned interplay between proteins and small molecules. Despite several important advances in identifying the important players and in elucidating some of the key mechanisms, our understanding is not yet complete. Several proteins components that we have discussed in this review have been identified as regulators or potential interactors of the disulphide relay machinery, but it is not clear whether they operate in a constitutive manner or they are part of mechanisms activated in response to metabolic changes or stress conditions. Furthermore, small molecules and ROS, which can also influence the function of the MIA machinery need to be kept under balance and detoxified to avoid deleterious effects and keep them within a concentration regime that is beneficial for signalling and not detrimental. Furthermore, the influence of the most damaging ROS generated in mitochondria (the super oxide anion), which has at least eight sites of production , is unclThe critical link of the MIA machinery to generalised mitochondrial dysfunction and human disease is exemplified by at least three different axes. First, the mutation or abnormal expression of components of the MIA machinery and its interactors such as AIF are involved in several human diseases ,55. Seco


Capacitive proximity sensing is widespread in our everyday life, but no sensor for biomedical optics takes advantage of this technology to monitor the probe attachment to the subject’s skin. In particular, when using optical monitoring devices, the capability to quantitatively measure the probe contact can significantly improve data quality and ensure the subject’s safety. We present a custom novel optical probe based on a flexible printed circuit board which integrates a capacitive contact sensor, 3D-printed optic fiber holders and an accelerometer sensor. The device can be effectively adopted during continuous monitoring optical measurements to detect contact quality, motion artifacts, probe detachment and ensure optimal signal quality. The interest towards non-invasive optical probing of human tissues in depth has constantly increased in recent years, with near-infrared optical spectroscopy techniques finally reaching both the clinical and commercial fields ,2. DiffuOptical probes for fiber-based NIRS systems are typically made of flexible 3D-printed materials that incorporate the injection and detection fibers and accommodate for curved surfaces ,8,9. SysCapacitive sensing is a well-known proximity measurement technique that is widespread in our everyday life and that relies on measuring the capacitance variation induced by capacitive coupling between electrically conductive electrodes and an object in their proximity ,14. The Here, we present a novel sensor based on capacitive proximity sensing, which can be integrated into optical probes to continuously monitor the contact with the subject and promptly alert users/operators in case of an unanticipated detachment, ensuring patient safety and data quality, especially in continuous monitoring applications. The contact sensor is based on a capacitive sensitive electrode integrated in a flexible PCB, and commercially available driving and readout integrated circuit (IC). Human contact with the probe adds a macroscopic amount of additional capacitance to the sensing network, and variation in the overall sensing network capacitance can be detected by the readout electronics. By periodically measuring the probe capacitance, it is possible to constantly monitor the probe attachment to the skin and output a contact quality signal in real time. An additional accelerometer sensor integrated on the probe PCB allows for the detection of motion artifacts in the acquired optical data . The preThe fully custom instrument is based on: (i) a flexible probe-shaped PCB which hosts a contact-sensing electrode, an accelerometer sensor, and the 3D-printed fiber holders for diffuse optics measurements; (ii) a control board PCB with the acquisition electronics. The control board is manufactured on standard FR4 substrate and is battery-operated to minimize the hazard-risk in clinical environments.Several electrode shapes for capacitive sensing are present in the literature, consisting of different sizes, performance capabilities, and operating principles , with thIn the interdigitated comb electrode, the contact measurement takes advantage of the fringing electric field between two adjacent copper strips, which act as the two plates of a planar capacitor . Any matThe single-electrode sensor relies on the same fringing capacitance effect of the parallel strips but makes use of a single plate of a planar capacitor and the object in its proximity acts as grounded secondary plate .The mutual-capacitance sensor is made of two interdigitated comb electrodes, each one made of 300 μm copper strips with 1.4 mm pitch, leading to a 400 μm spacing between two adjacent strips. The electrode of the self-capacitance sensor covers all the available surface of the PCB.The presented flexible probe PCB (pictures in 2) when powered at 2.5 V, with 1.6 kHz bandwidth, and provides three independent analog outputs. To further reduce risks of hazard related to electronic equipment, the maximum current that can be drawn from the accelerometer dedicated 2.5 V power supply is limited to 30 mA.Mounted on the top layer of the sensor probe PCB, a compact and low-power 3-axis MEMS accelerometer IC  allows the probe movement to be detected and motion artifacts in the acquired data to be identified. The sensor measures the acceleration along three axes with a full-scale range of ±2 g and a sensitivity of 355 mV/g  holographic diffuser with 40° diffusing angle is integrated with the injection fiber holder to increase the surface area illuminated by the injection fiber.2 and 36.5 × 15.2 mm2 for the single-electrode sensor, and a narrow 3.5 × 200 mm2 PCB strip distances the probe sensing area from the cable connection. A 1.5 m extremely flexible flat cable connects the probe to the control board, minimizing the bulk on the subject side and guaranteeing optimal flexibility in probe positioning. It is important to note that the dimensions of the sensing electrodes presented in this work are determined by the probe optical geometry, and sensors with a smaller sensitive surface can still guarantee sufficient performance if probe miniaturization is needed.The sensing area for the comb-electrode probe is equal to 44 × 1 mSENS) as a ratio with respect to an external reference clock source (see FREF in SENS/FREF) × 228. FREF in the presented design is 40 MHz, as this value allows for a wide capacitance measurement range and the finest resolution allowed by the sensing IC, i.e., 40 MHz / 228 = 0.149 Hz. From the measured oscillation frequency of the resonant network, it is possible to retrieve the total capacitance C by reversing the formula FSENS = 1/2π(LC)1/2 [A simplified block diagram of the instrument is shown in π(LC)1/2 ,31, wherThe three accelerometer signals are independently buffered, low-pass filtered, and then sampled through a 3-channel Analog-to-Digital Converter  the output of the contact-sensing IC proportional to the measured oscillation frequency of the resonant network; (ii) the 3 accelerometer signals, proportional to the acceleration measured on the 3-reference axis of the device. The modulus of the variations in the three accelerations are calculated with respect to baseline values and are summed into a single value, the motion signal. The variation with respect to the baseline value is also calculated for the frequency (thus the capacitance) measured by the contact sensor to obtain the contact signal. These two digital values are converted into analog signals by a two-channel 12-bit Digital-to-Analog Converter, buffered through a dual-channel high-current OP-AMP with 0–2.5 V output range and provided at instrument output through two standard BNC connectors. This easily integrates the instrument as a stand-alone device in existing measurement setups. Once the button is pressed, the contact analog output is initialized to 1.25 V (VDD/2) and is then driven with a signal directly proportional to the variation in the sensor oscillation frequency from the baseline: a reduction in the overall capacitance on the network (from contact to no contact) results in higher oscillation frequency, and subsequently results in a positive variation in the output. Inversely, when contact is applied on the sensing electrode, it will cause a reduction in the oscillation frequency and a lower output value. The motion analog signal is initialized at 0 V and any positive variation in the signal from the baseline implies a movement detected by the device, whereas a steady difference from the baseline implies a different probe angle.The microcontroller also communicates with an external PC through a USB 2.0 link, used for instrument debugging, testing and signal acquisition during experimental characterization. A specific LabVIEW  software interface was designed for data acquisition and to adjust the measurement parameters of the contact-sensing network (not shown). The overall data sampling and output rate is 54 Hz during normal operation with analog output.3) and a panel LED is used to inform the user about the battery level and the baseline acquisition through the button interface.Finally, a battery management IC operates the instrument from a standard 3.7 V Lithium-Polymer (LiPo) rechargeable battery, and the USB 5 V DC power supply input can be used to recharge it, even during device operation. The adopted LiPo battery features an integrated short-circuit protection and provides up to 600 mAh charge, which leads to a continuous acquisition longer than 24 h. The control board and battery are enclosed into a compact plastic box  on the sensing surface to validate its performance and applicability in a clinical environment. After an extensive validation of the device in time-varying ambient conditions, we adopted the presented flexible probe with integrated motion and contact sensing in a clinical measurement campaign with diffuse optics on extremely low gestational age  babies in a neonatal intensive care unit (NICU).SENSE 1 and CSENSE 2) on two test points on the control PCB, for the two probes V1 and V2. The ground of the oscilloscope probe was shorted to the control PCB ground.We validated the performance of the capacitive sensing network by initially testing the capability of the system in measuring the value of a known capacitance added to the resonant network. For this test, an oscilloscope passive probe (Tektronix P6501R) with 12 pF nominal capacitance was placed in contact with the two resonant network sensing nodes  for non-invasive and continuous monitoring of cerebral hemodynamics in extremely low gestational age infants to find association between dysregulation of cerebral blood flow and intraventricular hemorrhage. For these measurements, only the probe V2 with a hydrogel layer was used. The hydrogel layer ensures that the probe is never in direct contact with the delicate infant’s skin to avoid any damage or irritation, while guaranteeing a secure probe positioning. The motion and contact analog output signals were acquired simultaneously with the optical acquisition through the auxiliary inputs of the DCS system used for the measurement . Analog The steady-state overall capacitance measured by the sensing network with the two probes with/without a hydrogel layer is 279.6/184.1 pF for probe V1 and 161.2/160.7 pF for probe V2. The values of the measured capacitance were calculated in post-processing, starting from the digitized value of the resonant network frequency measured by the contact-sensing IC, assuming all the parameters are constant except for the network capacitance.SENSE 1  varied from 18.6 to 26.6 °C (orange-shaded area), which led to a variation in the overall measured capacitance of 2 pF , leading to a thermal drift equal to 0.25 pF/°C. The probe V1 demonstrated a worse performance against ambient temperature variations, with a coefficient of variation equal to 0.55 pF/°C (data not shown). No effect of the hydrogel layer was found during the experiments.The contact and accelerometer sensor probe for biomedical optics devices presented here allows the user to constantly monitor the probe attachment to the subject’s skin during optical measurements. Proper probe attachment is fundamental to ensure subject eye safety and to acquire quality data. The ability to promptly detect a probe-detachment event and immediately shutdown the light emission can dramatically change the outcome of these events, especially in high-risk applications, such as optical measurements on premature babies or unconscious subjects. While an accelerometer signal can effectively detect motion artifacts in the acquired optical data during post-processing, the contact signal provides additional user safety and data quality metrics in real time.The two designed electrodes—the interdigitated comb electrode (V1) and the single electrode (V2)—demonstrated strong performance in detecting contact variations on the probe sensitive surface. The single electrode probe proved better operation in terms of skin-contact sensing when a hydrogel layer is applied on the sensor surface, and in terms of thermal stability. Other commercially available adhesive materials  were tested, with no losses in performance. An overall variation of only 2 pF over 8 °C ambient temperature variation can be negligible compared to the >20 pF capacitance variation experienced when skin is put in contact with the device. Furthermore, the capability to detect contact variations in a 100% relative humidity environment enables the use of the device in high-humidity environments such as Giraffe incubators in NICUs.The interdigitated comb electrode measured a higher capacitance variation when put in direct contact with the skin, but the addition of a hydrogel layer resulted in a dramatic worsening of the contact-sensing performance. This may be due to limited depth resolution of the interdigitated comb electrode, with the hydrogel layer adhering to the sensor surface resulting in it becoming the main source for the dielectric variation. This is confirmed by the strong difference in steady-state capacitance measured by the sensing IC for the probe V1 with and without hydrogel layer, i.e., 279.6 and 184.1 pF, while the variation experienced by V2 with the addition of the hydrogel layer is negligible. A redesign of the interdigitated comb electrode with increased spacing between adjacent copper strips may help to reduce the crosstalk between the electrodes, and subsequently, the stray capacitance induced by the hydrogel layer.The capacitance measurement over relative ambient humidity variations for probe V1 demonstrated a performance comparable to that of probe V2, and the measurement over ambient temperature exhibited a similar temporal profile as well, despite a higher sensitivity to temperature variations. Humidity and temperature dependence for the probe V1 are not presented at this time, since this probe was not selected for the clinical application due to worse contact measurement performance in presence of additional adhesive layers. Thus, the probe V1 measurement stability results add limited additional information to the scope of this work.Finally, it must be noted that the capacitance measurements presented in We presented a novel optical probe for diffuse optics measurements based on flexible PCBs, which integrates an accelerometer and a contact sensor based on capacitive proximity sensing. The unique features of flexible PCBs allow for the possibility of building complex optical probes, which provide a direct signal of the quality of the sensor attachment to the skin and its motion, while offering support for the fiber-holders. Constant monitoring of the probe attachment using the proposed design can be effectively used to guarantee subject safety in high-risk applications, such as optical measurements on infants and can finally foster continuous-monitoring acquisition with NIRS devices without the need of a dedicated operator. The presented design can be customized in terms of probe shape, dimensions and equipped sensors based on the specific application, due to the use of a low-cost, commercially accessible technology, flexible PCBs, and can be easily integrated into preexisting measurement setups thanks to analog outputs, USB communication, and battery-powered operation.A patent on the presented contact sensor has been filed by the authors.


To the Editor,1]. We read with interest the article by Shah YS et al. “Delayed diagnoses of Acanthamoeba keratitis at a tertiary care medical center” . We have had same and similar issues . The article well describes difficulties to diagnose microbial keratitis. As reported in 4]. Timely diagnosis is the key for an effective treatment. However, the current conventional diagnostic methods (CDM) of stain and culture are time consuming and often with no clinically useful results .In recent years, we have started to evaluate the analysis of the metagenome [7]. Nowadays, with some sets, it is possible to have results in 24-36 hours [6] with reduced costs than in the past [8]. Metagenomics analysis with next-generation sequencers is becoming more common. The first difficulties to such a new approach improved during time [Acanthamoeba and the intracellular microbiome [9]. This way, metagenomics analysis will move the investigator from a hypothesis guided approach of CDM to a hypothesis-free approach, with a more comprehensive evaluation of samples .Additionally, as recently reported by Yu-Jen et al., bacterial 16S ribosomal RNA sequencing techniques have the potential to profile the microbiome in contact lens associated with AK to characterize the association between Therefore, we hope that a widespread use of metagenomics analysis could improve the diagnosis of AK and reduce delays.Conflict of Interest statementAuthors state no conflict of interest.AcknowledgementsNone.Sources of FundingNone.DisclosuresNone.


More than six million children have been diagnosed with COVID-19 in the United States alone1, over 4,000 children have been hospitalized2 and over 600 children have died.1 Globally, there have been over ten million COVID-19 cases and over 4000 deaths in persons 19 years of age and younger.3 The number of pediatric COVID-19 cases may be underestimated because children tend to have milder symptoms from infection and may be less likely to be tested than adults, particularly in low- and middle-income countries where access to testing may be limited. and COVID-19 case data are not always reported by age group.  Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are at risk of postinfectious complications including Multi-system Inflammatory Syndrome in Children4 and “long-COVID.”5 Pandemic mitigation measures such as school closures and cancellation of athletic activities have been associated with increased mental health difficulties and obesity rates in children, and have widened health disparities related to race/ethnicity and socioeconomic status.6 Beyond COVID-19 infection, the impact of the pandemic on children’s mental and physical wellbeing and educational progress has been far-reaching.7The importance of coronavirus disease 2019 (COVID-19) vaccines in children has been debated during the pandemic because the incidence of COVID-19 in children is lower than in adults, with particularly low rates in children <5 years of age.3 During the week of November 11 2021, children accounted for over 120,000 new COVID-19 cases in the United States and represented 27% of the weekly COVID-19 cases.8 Until a greater proportion of the overall global population is vaccinated, new variants will continue to emerge.More recently, the emergence of the more transmissible Delta variant has magnified the threat to children globally. The proportion of new COVID-19 cases in children has increased substantially from May through November  2021.9 yet are largely unvaccinated against COVID-19, because until recently the vaccine had not been approved for children <12 years of age. Childhood vaccinations have lagged primarily because COVID-19 vaccines were first studied in adults, given the higher incidence and severity of illness in the older population. In the United States, the only vaccine to have received emergency use authorization for children as young as 5 years of age is the Pfizer-BioNTech vaccine11 and several vaccine manufacturers are studying COVID-19 vaccines in children as young as 6 months of age. Fortunately, pediatric COVID-19 vaccinations have been found to be safe and effective for children 5 years of age and older,14 with the benefits far outweighing risks.For these reasons and more, it is important for all children aged 6 months to 17 years of age to be vaccinated against COVID-19 when vaccine efficacy and safety data are available and rigorously evaluated. Children constitute over 30% of the world’s population,15 There are reports of infected children who have been vectors of transmission to their vaccinated or unvaccinated parents and this risk will likely increase as new variants arise and schools reopen.16 Although it is unlikely that COVID-19 will be eradicated given the high transmissibility of the virus and ability to mutate, vaccinating children can have a large impact on reducing transmission and severity of illness in children and adults while contributing to herd immunity. Pediatricians and other pediatric providers must play an active role in educating and providing vaccines to children as parents frequently cite their pediatric clinician as a trusted source of information regarding vaccines.17 Providing COVID-19 vaccines during well-child checks and sick visits may increase access to vaccines and ultimately increase vaccination rates in children. Pediatric practices are also an opportune location to offer vaccination to family members for multiple reasons, including: (1) infection transmission often occurs in families and vaccinating the entire family protects everyone; (2) pediatric clinicians are experts in discussing vaccines and vaccine hesitancy and are trusted; and (3) family members accompany children to pediatric visits where vaccines are available. Many practices have offered this approach as an efficient and effective mechanism to increase education and access to vaccines in their community. This is important as we know that the health of children is interdependent on the health of the family and their community. Finally, there also exists the opportunity to partner with schools, other educational institutions, and Pediatric-focused organizations to advocate for vaccines while educating families on the importance of vaccinating children of all ages when the vaccines are authorized for such use.In addition, children play a role in COVID-19 transmission to other children and adults including the most vulnerable immunocompromised individuals. As Pratico and Ruggieri state in their article, unvaccinated children could potentially lead to transmission events in adults, similar to prior measles outbreaks.18 Potential rare adverse events, such as myocarditis,19 should continue to be investigated thoroughly. The background rate of myocarditis in individuals in the United States is estimated to be 1 to 10 per 100,000 persons annually with variations by age group and gender20 and the risk increases after SARS-CoV-2 infection. The rate of myocarditis after SARS-CoV-2 infection is estimated to be 6–34 times greater than the risk of messenger RNA vaccine-associated myocarditis and vaccine experts largely agree that the benefits of vaccination far outweigh the risk of myocarditis in children age 12 years and above.22 Similar risk–benefit analyses should be conducted on children who are younger than 12 years of age and maybe at lower risk of severe COVID-19 infection.Researchers should continue to enroll young children in clinical trials aimed at evaluating the safety and efficacy of these vaccines. Vaccines should not be given to very young children until the efficacy, safety, and dosing have been thoroughly studied and vaccine experts have provided recommendations for administration.23 Since more data have emerged demonstrating the safety and efficacy of these vaccines in pregnant and lactating women, pediatric organizations should support the Society for Maternal Fetal Medicine and ACOG18 in encouraging all pregnant and lactating women to receive a COVID-19 vaccination. Pediatric practices should also consider providing COVID-19 vaccines to parents.Global organizations including the World Health Organization and COVAX should work with vaccine manufacturers to expeditiously facilitate vaccine trials in children globally. Emergency-use authorization and licensure of these vaccines globally should be held to the same standards as adult vaccines. In addition to children, it is essential that future clinical trials include pregnant and lactating mothers to establish safety and efficacy in this vulnerable population . High-risk conditions such as preterm labor and pre-eclampsia-like features have emerged in mothers infected with SARS-CoV-2 and their infants have suffered the consequences of prematurity and fetal inflammatory response syndrome.24 In contrast, low- and middle-income countries such as Haiti have yet to vaccinate 1% of their population.24 While some wealthy nations are providing supplemental and booster doses to their population, many healthcare workers in low-income countries have yet to receive a single vaccine dose. It is important for leaders of wealthy nations to improve vaccine equity outside their own borders. Administering the primary COVID-19 vaccine series to high-risk individuals in low- and middle-income countries should be a global priority and responsibility of all nations. Public health leaders should balance priorities in rolling out vaccines to low-risk children age <5 vs the possibility of a substantial impact on global vaccine availability.Access to pediatric vaccines must be equitable globally. Thus far, the disparities in vaccine distribution globally have been striking. High-income nations have vaccinated the majority of their population, including the United Arab Emirates, which has vaccinated 98% of their population.If adults and children in any global community are left behind in COVID-19 vaccine programs, it will be more difficult to mitigate the spread of SARS-CoV-2 for all, and pediatric hospitalizations and deaths will continue to increase globally.


Childhood obesity is an area of intense concern internationally and is influenced by events during antenatal and postnatal life. Although pregnancy complications, such as gestational diabetes and large-for-gestational-age birthweight have been associated with increased obesity risk in offspring, very few successful interventions in pregnancy have been identified. We describe a study protocol to identify if a reduced calorie diet in pregnancy can reduce adiposity in children to 3 years of age. The dietary intervention in gestational diabetes (DiGest) study is a randomised, controlled trial of a reduced calorie diet provided by a whole-diet replacement in pregnant women with gestational diabetes. Women receive a weekly dietbox intervention from enrolment until delivery and are blinded to calorie allocation. This follow-up study will assess associations between a reduced calorie diet in pregnancy with offspring adiposity and maternal weight and glycaemia. Anthropometry will be performed in infants and mothers at 3 months, 1, 2 and 3 years post-birth. Glycaemia will be assessed using bloodspot C-peptide in infants and continuous glucose monitoring with HbA1c in mothers. Data regarding maternal glycaemia in pregnancy, maternal nutrition, infant birthweight, offspring feeding behaviour and milk composition will also be collected. The DiGest follow-up study is expected to take 5 years, with recruitment finishing in 2026. Gestational diabetes, a common complication of pregnancy, is associated with short-term and long-term health implications for the baby ,2. InfanEvents in pregnancy, perinatal and early postnatal periods may be important for future childhood obesity, but are relatively understudied, particularly in specific high-risk populations . Babies It is therefore possible that an intervention which addresses maternal weight in pregnancy may reduce obesity rates in offspring. Family interventions (which target at least one parent to improve obesity rates in children) are already well-established in the prevention of childhood obesity . HoweverThe DiGest Study is a randomised, double-blind controlled trial of a reduced calorie diet using a novel dietary intervention to assess the benefits of controlling maternal weight gain in late pregnancy in gestational diabetes. The trial is described in detail elsewhere . BrieflyIn this manuscript we describe a follow up study to the DiGest trial which investigates the effect of the reduced calorie dietary intervention in pregnant women upon the development of obesity in a high-risk population of children from birth to 3 years of age. The hypothesis is that a reduced calorie diet in late pregnancy in women with gestational diabetes reduces offspring adiposity and improves maternal weight at 1, 2 and 3 years postpartum.Study design and ethical approval: The DiGest Follow up study is an observational study on the effects of a multicentre, prospective, randomised double-blind controlled dietary intervention trial conducted in late pregnancy. In summary, participants of the follow up study will have been exposed to either the intervention diet of 1200 kcal/day, or the control diet of 2000 kcal/day as part of the DiGest study. Macronutrient ratios were identical for each diet; 40% carbohydrate, 25% protein, 35% fat. Meals were prepared from the same recipes, with a factor of 1.667 used to convert portion size to obtain meals of two different sizes. This diet it provided from enrolment  to delivery of the infant. Participants will have attended 4 study visits in total to provide blood samples, blood pressure, body weight and anthropometry measurements, and to complete a series of questionnaires. Randomisation for the original DiGest study was stratified for centre. Throughout the DiGest intervention and Follow-up study, both mothers and children will receive standard NHS care, as described in the NICE guidelines . The stuRecruitment: The DiGest trial recruitment occurs at 5 hospital trusts in East Anglia, UK. The same study sites will be used for the follow-up study. At 3 months postpartum, DiGest trial participants will be given information about the follow-up study and invited to participate by the research midwives, nurses, clinical research staff or by their physician/obstetrician. For training purposes, students in healthcare disciplines  may also occasionally recruit patients under appropriate supervision. Informed consent will be obtained at the final visit of the DiGest dietary intervention, with the mothers providing consent on their infant’s behalf. Participants (or mother-infant dyad) can withdraw from the study at any time without reason without affecting their clinical care. There is no financial incentive for this study, but a small token of appreciation is provided for the child at each visit in line with guidelines of the Royal College of Paediatrics and Child Health .2 or above at enrolment) are eligible to enrol in the follow-up study, however, they must be recruited within 12 months of the baby’s birth. Mothers would be excluded if they are unwilling or unable to provide informed consent, if they experienced stillbirth, neonatal death or had an infant born with severe congenital anomaly.Eligibility criteria: All women from the DiGest cohort . Waist and hip circumference will be measured to the nearest 0.1 cm with a fibreglass tape, in accordance with the World Health Organisation criteria . Waist cMaternal Glucose Assessment: Due to the COVID-19 pandemic, a home-based OGTT using continuous glucose monitoring (CGM) will replace the gold standard OGTT for assessment of maternal glucose tolerance postnatally. An HbA1c will also be performed to replace mothers’ annual diabetes check in primary care. We have previously assessed the feasibility and efficacy of the home-based OGTT with good results . In brief, a Dexcom G6 CGM sensor is sited during the study visit with a masked receiver so participants do not see their glucose results in real time. On day 3, participants are asked to eat normally, and fast overnight for at least 10 h. On the morning of day 4, at 09.00, participants are asked to drink a sachet of Rapilose  containing 75 g of anhydrous glucose. Participants can have sips of water but are asked to consume no other foods or drinks for 3 h after the test. The timing of the home OGTT is chosen to coincide with peak sensor accuracy. Glucose readings are taken automatically every 5 min and transmit to the CGM receiver. Results from the OGTT at 0, 1 and 2 h are included in the analysis. Other CGM metrics will also be used to assess glycaemia as described in a recent CGM consensus statement. CGM metrics will be reported using both adult non-pregnant and pregnant ranges to allow comparison with pregnancy data gathered in the DiGest trial .Participants will be asked to wear a wrist-worn accelerometer continuously for 7 days concurrently with the CGM. The triaxial accelerometer is waterproof and does not have a visual display, nor any auditory or vibrational cues, which means that participants will not be able to influence their activity level based on what is recorded by the device and nor will they be prompted to move about during periods of inactivity. These accelerometers have been used in in women during and after pregnancy to assess their daily physical activity with high compliance and produce reliable estimates of energy expenditure, overall physical activity and moderate-vigorous intensity activity ,21,22. AOther Biochemistry samples: A blood spot sample will be taken from mothers and frozen at −80 °C for future batch analysis of C-peptide and metabolomics. An optional heelprick blood spot will also be taken from infants, for future batch analysis of C-peptide and metabolomics. If a genetic sample has not been taken already as part of the DiGest trial, a cheek swab will be taken from both mothers and infants. Mothers will be asked to provide a sample of milk (formula or breast) which will be collected onto filter paper for assessment of infant nutrition including lipidomic profiling. To protect participant’s privacy, this can be performed after the visit.Questionnaires: Mothers will be asked to complete validated questionnaires about quality of life (EuroQuol EQ5D), eating behaviour (three factor eating questionnaire—TFEQ-18) , physicaThe aim of the study is to investigate the effects of a reduced calorie diet in late pregnancy in women diagnosed with gestational diabetes upon longer-term maternal and offspring metabolic outcomes. The primary outcome for child health is standardised weight at 1, 2 and 3 years of age. The primary outcome for the maternal population is maternal weight at 1, 2 and 3 years postpartum.Offspring secondary outcomes at 1, 2 and 3 years of age: There are multiple secondary outcomes for children including weight, BMI, growth trajectory, and blood spot biomarkers such as C-peptide or metabolomics at 1, 2 and 3 years. Questionnaire data will be assessed to identify effects of the intervention in pregnancy upon child eating behaviour, with assessment for confounding factors including maternal BMI, maternal eating behaviour and parental feeding style.Maternal secondary outcomes at 1, 2 and 3 years postpartum: Maternal outcomes to be studied include maternal weight and weight change, BMI, anthropometry measures of adiposity, glycaemia  will be based on linear regression with adjustment for the stratification variable of study centre through a fixed effects model. The potential role of other explanatory variables such as pre-pregnancy BMI, infant nutrition, infant postnatal growth trajectory or information from the questionnaires will be investigated. A per protocol analysis will also be performed in participants with >80% compliance and at least 4 weeks’ exposure to the intervention. Secondary outcomes will also be examined through regression analyses (linear or logistic) appropriate for the type of outcome being considered.n = 250 women and their infants) and a 20% withdrawal rate. For the maternal primary endpoint, data from earlier work suggest that typical values for maternal BMI outside of pregnancy in women with a history of gestational diabetes is mean 28.7 kg/m2  and maternal postpartum HbA1c 37.5 mmol/mol  [2 difference in BMI  and a 3 mmol/mol difference in HbA1c postnatally while allowing for a 10–20% withdrawal rate. At 80% power, this sample size is sufficient to identify a 2 kg/m2 difference in BMI  and a 2 mmol/mol difference in HbA1c postnatally.Power calculation: All eligible women and their infants will be invited to join the follow-up study. However, calculations are based on assuming a 50% recruitment rate (n = 157) . Using tFor the offspring primary endpoint, assessment of infant weight will be based upon z-(SD) scores. At the sample size of 250 infants, there will be 90% power to identify a 0.45 SD increase in weight with 80% power to identify 0.4 SD increase in weight. At the age of 2 years old, a z-score of 0.4 is equivalent to 0.5 kg.This follow-up study of the DiGest randomised controlled trial provides a unique opportunity to assess the potential benefits of a dietary intervention in late pregnancy upon the development of obesity in children with multiple risk factors. The availability of data from mid pregnancy until the age of 3 years also allows detailed characterisation of the relative importance of pregnancy and postnatal risk factors in the development of adiposity in early childhood.Rates of maternal obesity are increasing in the antenatal population throughout the world, and pre-pregnancy BMI is a strong predictor of both birthweight and future childhood obesity. A recent metanalysis identified that maternal obesity was significantly associated with overweight/obesity in early, mid and late childhood with odds ratios 2.43, 3.12 and 4.47, respectively . Weight In addition to the effects of maternal obesity, exposure to intrauterine hyperglycaemia appears to further increase the risk of childhood obesity. There is evidence that maternal glycaemia in gestational diabetes is associated with childhood obesity at 10–14 years  and alteThe design of the DiGest and DiGest follow-up studies also allows longitudinal assessment of the effects of other pregnancy exposures upon longer-term offspring growth and health. For example, metformin use in pregnancy has been associated with lower birth weight but increased postnatal catch-up growth, but the consequences of this upon longer-term offspring cardiometabolic outcomes remain less clear ,40. The Serum and cord blood stored for biomarkers such as leptin, adiponectin and placental hormones provides opportunities to identify infants at an earlier stage who are at risk of obesity in childhood. Previous work has demonstrated that cord blood leptin levels are associated with pregnancy diet, physical activity and neonatal body composition in a comparable population ,42. CordAlthough maternal physical activity levels in pregnancy and postpartum are likely to be vital for determining offspring habitual exercise levels, relatively few modifiable factors have been identified in children’s physical activity levels in the very young ,48. FindInfant feeding and growth trajectory in the first year of life are also important. Although randomised studies of feeding modality in early life are not possible, observational analyses in unselected populations suggest consistent benefits of breastfeeding upon rates of childhood obesity ,50. TherThe aetiology of childhood obesity is therefore complex and multifactorial. In infants of mothers with gestational diabetes, multiple risk factors are often evident at birth. Successful interventions are urgently needed to reduce the risk of obesity and future metabolic disease in these high-risk children.The DiGest follow-up study provides the opportunity to assess pregnancy and postnatal risk factors for the development of childhood obesity, and to describe the potential impact of a dietary intervention in pregnancy. Early intervention in offspring with existing environmental and genetic susceptibilities to type 2 diabetes will be vital to break the intergenerational cycle of obesity.No patents are relevant to the study described in this manuscript.