In the development of prediction models, unsupervised Ai/ML systems are particularly susceptible to over fitting, tailoring to the build data set, and are insufficiently robust when tested on live, and not strictly parameter controlled, inputs. This is acutely seen in IVF Embryo selection tests, where group classification of success and failure can be inaccurate and are blurred. i.e. failure can be non-implantation, non-implantation due endometrial receptivity, biochemical pregnancy and first-trimester implantation loss. Based on our previous studies of metabolomic analysis and utilisation in Ai/ML prediction model algorithms, mass peak feature variables in spent blastocyst medium (SBM) predict which IVF blastocyst-embryo had the greatest chance of success. To operationalise, we took a probabilistic based prediction algorithm approach to create a simpler and more informative scoring system. Generated from a cohort of 385 individual MALDI-ToF Mass Spectrometry SBM sample spectra, we developed a more statically robust Bayesian based algorithm approach for a preimplantation metabolomic test for blastocyst metabolic competence (PMT-BC). This system results in direct input of analysed spectra, to output clinically informative probability scores and in a stratified probability format requested by embryologists. Visual user friendly, results are displayed in the form of a battery charge scaling and relates to metabolic systems being found in optimum ratio’s. The more metabolite ratio/systems found in the optimum the greater the probability of viable implantation. This opens up accessible metabolic profiling to the non-biochemist embryologist and implementation of metabolomic, PMT-BC, testing in the Clinical IVF centre. Furthermore, failure of high PMT-BC scoring IVF Blastocyst-Embryos will help define and quantify, failed fertility due to endometrial receptivity issues.

We developed a MALDI-TOF mass spectrometry method for the detection of the SARS-CoV-2 virus in saliva-gargle samples using Shimadzu MALDI-TOF mass spectrometers in the UK. This was validated in the USA to CLIA-LDT standards for asymptomatic infection detection remotely via sharing protocols, shipping key reagents, video conferencing, and data exchange. In Brazil, more so than in the UK and USA, there is a need to develop non-PCR-dependent, rapid, and affordable SARS-CoV-2 infection screening tests that also identify variant SARS-CoV-2 and other virus infections. In addition, travel restrictions necessitated remote collaboration with validation on the available clinical MALDI-TOF-the Bruker Biotyper (microflex® LT/SH)-and on nasopharyngeal swab samples, as salivary gargle samples were not available. The Bruker Biotyper was shown to be almost log103 more sensitive at the detection of high molecular weight spike proteins. A protocol for saline swab soaks out was developed, and duplicate swab samples collected in Brazil were analyzed by MALDI-TOF MS. The swab collected sample spectra that varied from that of saliva-gargle in three additional mass peaks in the mass region expected for IgG heavy chains and human serum albumin. A subset of clinical samples with additional high mass, probably spike-related proteins, were also found. Further, spectral data comparisons and analysis, subjected to machine learning algorithms in order to resolve RT-qPCR positive from RT-qPCR negative swab samples, showed 56-62% sensitivity, 87-91% specificity, and a 78% agreement with RT-qPCR scoring for SARS-CoV-2 infection.

Glycosylation of antibodies and the effects this has on inflammatory responses has concentrated predominately on the study of glycosylation moieties found in the Fc region of heavy chains. Light chain glycosylation and their ratios are relatively understudied. Nevertheless, variable glycosylation and ratio of κ and λ light chains have been associated with worse prognosis in myeloma and in tissue deposition – amyloidosis.The κ & λ light chains, of antibodies binding to SARS-CoV2 nucleocapsid and spike protein were analysed, using MALDI-ToF MS, in respect to their intensity, ratios, glycosylation patterns and any pattern changes correlating with COVID-19 severity. The molecular masses and signal intensity of κ and λ glycosylated and non-glycosylated light chains were measured for immunoglobulins isolated from plasma of sero-positive and sero-negative health care workers (HCW), and convalescent patients who had suffered from acute respiratory distress syndrome (ARDS).Overall, there was no significant changes in κ to λ ratio of total IgG (via protein G capture) antibodies between the groups. A non-statistically significant trend towards λ light chains was found in antibodies against SARS CoV-2 Nucleocapsid and Spike proteins. However, detailed analysis of the molecular forms found a significant increase and bias towards un-glycosylated light chains and in particular un-glycosylated κ light chains, in antibodies against SAR-CoV-2 spike protein, from convalescent COVID-ARDS patients.Here we have demonstrated a bias towards un-glycosylated κ chains in anti-spike antibodies in those who suffered from ARDS as a result of SARS-CoV2 infection 3 months after recovery. How this relates to the immunopathology of COVID-19 requires further study.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementUKRI and NIHR; grant number G107217 (COV0170 - HICC: Humoral Immune Correlates for COVID-19) for providing samples and part funding the study.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Study approved by Research Ethics Committee Wales, IRAS: 96194 12/WA/0148. Amendment 5I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors* HCW : Health care workers ARDS : Acute respiratory distress syndrome ITU : Intensive Therapy unit AGE : advanced glycation end products MALDI-ToF : Matrix assisted laser desorption ionization – time of flight.

Objective: The aim of the current study was to examine the potential relationship between sleep patterns, cortisol levels, and anxiety profiles in adolescents with Williams Syndrome (WS) compared to typically developing adolescents. Method: Thirteen adolescents with WS and thirteen TD adolescents (age range 12–18 years) were recruited. Participants were provided with a “testing kit”, containing instructions for collecting data through a sleep diary, MotionWare actigraphy, the Childhood Sleep Habits Questionnaire (CSHQ), and the Spence Children’s Anxiety Scale, and a salivary cortisol collection kit. Results: Adolescents in the WS group did not show diurnal variation in salivary cortisol. Significantly higher scores were reported for two CSHQ subsections, night wakings and parasomnias, in the WS group. Regarding the actigraphy, only significantly longer sleep latency was observed in the WS group. In comparison to the TD group, the WS group had significantly higher anxiety. As expected, the TD group showed typical diurnal variation in cortisol, whereas the WS group showed a flattened cortisol profile throughout the day. Conclusions: From the developmental perspective, this study provides new data supporting the conclusion that sleep problems are not transient but continue to persist into adolescence in WS. Future studies ought to consider examining the role of cortisol and its interplay with anxiety levels and sleep problems across the lifespan in individuals with WS.

Applying MALDI-TOF mass spectrometry as a clinical diagnostic test for viruses is fundamentally different from that of bacteria, fungi, and other microorganisms. This is because of the systems biology of viral infections, and the size and chemical nature of specific viral proteins that may be identified by mass spectrometry. The analytical challenges to overcome when developing a clinical MALDI-TOF mass spectrometry tests for a virus, particularly human pathogenic enveloped viruses, are: Sample enrichment Virus envelope disruption Optimal matrix formulation Optimal MALDI TOF MS performance. Optimal data processing. These all require corresponding changes to the operational parameters of the mass spectrometer, which are not compatible with settings established when testing for bacterial and many other microorganisms. These instrument operating specifications must be optimized and this often stretches the instruments' capacity from being a virus research tool to being a viral infection clinical diagnostic instrument. Finally, the entire operational procedures for the technique must meet the legal and operational requirements for (often) high-throughput, clinical testing.

The prefusion spike protein of SARS-CoV-2 binds advanced glycation end product (AGE)-glycated human serum albumin (HSA) and a higher mass (hyperglycosylated/glycated) immunoglobulin (Ig) G3, as determined by matrix assisted laser desorption mass spectrometry (MALDI-ToF). We set out to investigate if the total blood plasma of patients who had recovered from acute respiratory distress syndrome (ARDS) as a result of COVID-19, contained more glycated HSA and higher mass (glycosylated/glycated) IgG3 than those with only clinically mild or asymptomatic infections. A direct serum dilution, and disulphide bond reduction, method was developed and applied to plasma samples from SARS-CoV-2 seronegative (n = 30) and seropositive (n = 31) healthcare workers (HCWs) and 38 convalescent plasma samples from patients who had been admitted with acute respiratory distress (ARDS) associated with COVID-19. Patients recovering from COVID-19 ARDS had significantly higher mass AGE-glycated HSA and higher mass IgG3 levels. This would indicate that increased levels and/or ratios of hyper-glycosylation (probably terminal sialic acid) IgG3 and AGE glycated HSA may be predisposition markers for the development of COVID-19 ARDS as a result of SARS-CoV2 infection. Furthermore, rapid direct analysis of serum/plasma samples by MALDI-ToF for such humoral immune correlates of COVID-19 presents a feasible screening technology for the most at risk; regardless of age or known health conditions.

Background: Neurocognitive impairment is common in people with Sickle Cell Disease (SCD) and evidence is accumulating that sleep disturbances play a role. The interaction between cortisol and sleep in the general population is associated with cognition as well as general wellbeing but there are few data in SCD. We aimed to understand the relationship between cortisol and sleep in individuals with SCD and explored associations with cognition. Methods: Forty-five participants of black heritage (SCD: N = 27, 9-29 years, 16 females; Controls: N = 18, 11-25 years, 13 females) were recruited from the community between 2018 -2020. Participants completed stan-dardized questionnaires about their sleep behaviour and wore actigraphy MotionWatch8 for 7 nights to assess nocturnal sleep patterns. Salivary cortisol samples were taken on wakening and 3 times after 14:00. Cognition was assessed using the Wechsler Intelligence Scales for children and adults. Results: People with SCD took longer to fall asleep and experienced greater wake bouts, mobile minutes and fragmented sleep compared to controls. Although non-significant, people with SCD experienced lower morning cortisol, with a flattened diurnal cortisol ratio compared to controls. Interestingly, SCD participants, but not controls, with low diurnal variation scored lowest on processing speed (PSI) and perceptual reasoning index (PRI). A moderator analysis revealed that the effect of morning cortisol and diurnal cortisol ratio on PRI by group health (i.e., SCD and healthy controls) depended on sleep quality. Discussion: Sleep and cortisol may play a crucial role in the expression of cognitive difficulties seen in SCD. This should be considered for the development of interventions to optimise cognitive functioning and sleep. This, in turn, could positively impact on secretion of cortisol and general health in SCD.

Objectives:To characterise the prevailing pharmacological and non-pharmacological pain management strategies among adults with chronic pain, comparing these against the newly published NICE guidelines NG-193, and examine these pre-NG-193 pain management strategies in relation to pain severity, pain interference, sleep quality and mental health outcomes.Design:This study was conducted using a cross-sectional online survey study design.Setting:This study was conducted on a community-dwelling cohort.Participants:Adults aged 18+, living in the UK, with diagnosis of chronic pain by a health care professional.Main outcome measures:Primary outcomes were characterisation of the pain management strategies utilised. Secondary outcomes were related to pain severity, pain interference, sleep quality, depression and anxiety via validated self-report measures.Results:Several strategies were employed by respondents to manage their chronic pain condition including physical therapy, exercise, psychological therapy and pharmacological therapy. The data also indicated a high level of joint-care planning among patients and their clinicians. Some group differences were found in relation to pain, sleep and mental health outcomes.Conclusion:This study set a comparative starting baseline to which the efficacy of the NG-193 may be compared in future years. There is evidence that NICE recommendations are being followed for the management of chronic primary pain conditions; however, pharmacological use of opioid drugs is still reported by 47%. Despite the confirmed evidence in this study of small efficacy of chronic pain by pharmacological agent, the reduction in the use of pain relief medications be it over the counter medications or prescription opioids, as recommended by NG-193, may be slow to be adopted. The data suggest that more care provision is needed to meet the recommendations around pharmacological management and review.

Ovarian cancer is a worldwide health issue that grows at a rate of almost 250,000 new cases every year. Its early detection is key for a good prognosis and even curative surgery. However, current medical examination methods and tests have been inefficient in detecting ovarian cancer at the early stage, leading to preventable death. So far, new screening tests based on molecular biomarker analysis techniques have not resulted in any substantial improvement in early-stage diagnosis and increased survival. Thus, whilst there remains clear potential to improve outcomes through early detection, novel approaches are needed. Here, we postulated that MALDI-ToF-mass-spectrometry-based tests can be a solution for effective screening of ovarian cancer. In this retrospective cohort study, we generated and analyzed the mass spectra of 181 serum samples of women with and without ovarian cancer. Using bioinformatics pipelines for analysis, including predictive modeling and machine learning, we found distinct mass spectral patterns composed of 9–20 key combinations of peak intensity or peak enrichment features for each stage of ovarian cancer. Based on a scoring algorithm and obtained patterns, the optimal sensitivity for detecting each stage of cancer was 95–97% with a specificity of 97%. Scoring all algorithms simultaneously could detect all stages of ovarian cancer at 99% sensitivity and 92% specificity. The results further demonstrate that the matrix and mass range analyzed played a key role in improving the mass spectral data quality and diagnostic power. Altogether, with the results reported here and increasing evidence of the MS assay’s diagnostic accuracy and instrument robustness, it has become imminent to consider MS in the clinical application for ovarian cancer screening.

Applying MALDI-ToF mass spectrometry as a clinical diagnostic test for viruses is different from that of bacteria, fungi and other micro-organisms. This is because the systems biology of viral infections, the size and chemical nature of specific viral proteins and the mass spectrometry biophysics of how they are quantitated are fundamentally different. The analytical challenges to overcome when developing a clinical MALDI-ToF mass spectrometry tests for a virus, particularly human pathogenic enveloped viruses, are sample enrichment, virus envelope disruption, optimal matrix formulation, optimal MALDI ToF MS performance and optimal spectral data processing/bioinformatics. Primarily, the instrument operating settings have to be optimized to match the nature of the viral specific proteins, which are not compatible with setting established when testing for bacterial and many other micro-organisms. The capacity to be a viral infection clinical diagnostic instrument often stretches current mass spectrometers to their operational design limits. Finally, all the associated procedures, from sample collection to data analytics, for the technique have to meet the legal and operational requirement for often high-throughput clinical testing. Given the newness of the technology, clinical MALDI ToF mass spectrometry does not fit in with standard criteria applied by regulatory authorities whereby numeric outputs are compared directly to similar technology tests that have already been authorized for use. Thus, CLIA laboratory developed test (LDT) criteria have to be applied. This article details our experience of developing a SAR-CoV-2 MALDI-ToF MS test suitable for asymptomatic carrier infection population screening.

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype’s differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization–time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.

The immune response to SARS-CoV-2 infection requires antibody recognition of the spike protein. In a study designed to examine the molecular features of anti-spike and anti-nucleocapsid antibodies, patient plasma proteins binding to pre-fusion stabilised complete spike and nucleocapsid proteins were isolated and analysed by matrix-assisted laser desorption ionisation–time of flight (MALDI-ToF) mass spectrometry. Amongst the immunoglobulins, a high affinity for human serum albumin was evident in the anti-spike preparations. Careful mass comparison revealed the preferential capture of advanced glycation end product (AGE) forms of glycated human serum albumin by the pre-fusion spike protein. The ability of bacteria and viruses to surround themselves with serum proteins is a recognised immune evasion and pathogenic process. The preference of SARS-CoV-2 for AGE forms of glycated serum albumin may in part explain the severity and pathology of acute respiratory distress and the bias towards the elderly and those with (pre)diabetic and atherosclerotic/metabolic disease.

e17046 Background: Sleep disturbances and cancer related fatigue are commonly associated. Prostate cancer patients may suffer from disturbed sleep as a result of their diagnosis and following treatment, especially with androgen deprivation therapy (ADT). Wrist actigraphy is a non-invasive objective method of sleep data collection. This feasibility study compares sleep data obtained by actigraphy with subjective data from sleep questionnaires in order to determine the nature and severity of sleep disturbances in patients with and without ADT use. Methods: A prospective cross-sectional pilot study was conducted on 74 patients with prostate cancer attending a regional oncology clinic. Two validated subjective sleep questionnaires namely the Pittsburgh Sleep Quality Index [PSQI] and the Epworth Sleepiness Scale [ESS] were used. Patients wore actigraphy watches for a minimum of five consecutive days. The parameters of interest included: actual sleep time, sleep efficiency, fragmentation index, daytime napping frequency and duration. The questionnaire and actigraphy data were compared between 20 patients receiving ADT and 41 who were treatment-naive. Results: The compliance rate for completed actigraphy was 85%. Complete data sets with actigraphy and questionnaires were available from 61 patients. Those already receiving ADT were on LHRH analogues for a median duration of 2.35 years. Poor sleep quality as self-identified by patients from the PSQI (cut-off > 5) was 49% in the treatment-naive group which increased to 70% for those on ADT. For daytime sleepiness as assessed by ESS (cut-off > 10) this was 16% and 20% respectively. Actigraphy showed that patients on ADT reported longer sleep duration (7.4 vs 6.5 hours, p = 0.02), higher levels of nocturnal wakings (51.1% vs 36.7%, p = 0.002), with greater daytime napping duration (80.7mins vs 53.0mins, p = 0.04), and frequency (8.6 vs 5.6, p = 0.02) compared to treatment-naive patients. Conclusions: Self-reported poor sleep quality is common in prostate cancer patients, which appears worse for those receiving ADT. In patients receiving ADT, data derived from actigraphy suggests that although they were sleeping for longer at night, the quality of sleep was poor which, in turn, may be responsible for an increase in the frequency and duration of daytime napping. Based on the current findings, we recommend the use of actigraphy to characterise patients’ sleep patterns and to assess if sleep treatment is needed. Actigraphic data may allow for direct comparisons of different hormonal agents on sleep whilst identifying those with specific sleep disorders amenable to therapeutic intervention.

The prefusion Spike protein of SARS-CoV2 binds advanced glycation end product (AGE) glycated human serum albumin (HSA) and a higher mass, hyperglycosylated/glycated, IgG3, as determined by matrix assisted laser desorption mass spectrometry (MALDI-ToF MS). We set out to investigate if the total blood plasma of patients who had recovered from acute respiratory distress as a result of COVID-19, contained more glycated HSA and higher mass (glycosylated/glycated) IgG3 than those with only clinically mild or asymptomatic infections. A direct dilution and disulphide bond reduction method was development and applied to plasma samples from SARS-CoV2 seronegative (N = 30) and seropositive (N = 31) healthcare workers and 38 convalescent plasma samples from patients who had been admitted with acute respiratory distress syndrome (ARDS) associated with COVID-19. Patients recovering from COVID-19 ARDS had significantly higher mass, AGE-glycated HSA and higher mass IgG3 levels. This would indicate that increased levels and/or ratios of hyper-glycosylation (probably terminal sialic acid) IgG3 and AGE glycated HSA may be predisposition markers for development of ARDS as a result of COVID-19 infection. Furthermore, rapid direct analysis of plasma samples by MALDI-ToF MS for such humoral immune correlates of COVID-19 presents a feasible screening technology for the most at risk; regardless of age or known health conditions.

There have been over 8 million babies born through in vitro fertilization (IVF) and this number continues to grow. There is a global trend to perform elective single embryo transfers, avoiding risks associated with multiple pregnancies. It is therefore important to understand where current research of noninvasive testing for embryos stands, and what are the most promising techniques currently used. Furthermore, it is important to identify the potential to translate research and development into clinically applicable methods that ultimately improve live birth and reduce time to pregnancy. The current focus in the field of human reproductive medicine is to develop a more rapid, quantitative, and noninvasive test. Some of the most promising fields of research for noninvasive assays comprise cell-free DNA analysis, microscopy techniques coupled with artificial intelligence (AI) and omics analysis of the spent blastocyst media. High-throughput proteomics and metabolomics technologies are valuable tools for noninvasive embryo analysis. The biggest advantages of such technology are that it can differentiate between the embryos that appear morphologically identical and has the potential to identify the ploidy status noninvasively prior to transfer in a fresh cycle or before vitrification for a later frozen embryo transfer.

Current methods for diagnosing human disease are still incapable of rapidly and accurately screening for multiple diseases simultaneously on a large scale, and at an affordable price. MALDI-ToF mass spectrometry is an ultra-sensitive, ultra-fast, lowcost, high-throughput technology that has the potential to achieve this goal, allowing human phenotype characterization and thus phenomic screening for multiple disease states. In this review, we will discuss the main advances achieved so far, putting forward targeted applications of MALDI-ToF mass spectrometry in the service of human disease detection. This review focuses on the methodological workflow as MALDI-ToF data processing for phenomic analysis, using state-of-the-art bioinformatic pipelines and software tools. The role of mathematical modelling, machine learning, and artificial intelligence algorithms for disease screening are considered. Moreover, we present some previously developed tools for disease diagnostics and screening based on MALDI-ToF analysis. We discuss the remaining challenges that are ahead when implementing MALDI-ToF into clinical laboratories. Differentiating a standard profile from a single disease phenotype is challenging, but the potential to simultaneously run multiple algorithm screens for different disease phenotypes may only be limited by computing power once this initial hurdle is overcome. The ability to explore the full potential of human clinical phenomics may be closer than imagined; this review gives an insight into the benefits this technology may reap for the future of clinical diagnostics.

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has stretched national testing capacities to breaking points in almost all countries of the world. The need to rapidly screen vast numbers of a country’s population in order to control the spread of the infection is paramount. However, the logistical requirement for reagent supply (and associated cost) of RT-PCR based testing (the current front-line test) have been hugely problematic. Mass spectrometry-based methods using swab and gargle samples have been reported with promise, but have not approached the task from a systematic analysis of the entire diagnostic process. Here, the pipeline from sample processing, the biological characteristics of the pathogen in human biofluid, the downstream bioand physical-chemistry and the all-important data processing with clinical interpretation and reporting, are carefully compiled into a single high-throughput and reproducible rapid process. Utilizing MALDI-ToF mass spectrometric detection to viral envelope glycoproteins in a systems biology-multidisciplinary team approach, we have achieved a multifaceted clinical MALDI ToF MS screening test, primarily (but not limited to) SARS-CoV-2, with direct application to other future epidemics/pandemics that may arise. The clinical information generated not only includes SARSCoV-2 coronavirus detection–(Spike protein fragments S1, S2b, S2a peaks), but other respiratory viral infections detected as well as an assessment of generalised oral upper respiratory immune response (elevated total Ig light chain peak) and a measure of the viral immune response (elevated intensity of IgA heavy chain peak). The advantages of the method include; (1) ease of sampling, (2) speed of analysis, and much reduced cost of testing. These features reveal the diagnostic utility of MALDI-ToF mass spectrometry as a powerful and economically attractive global solution

PURPOSE:Embryo genotyping in IVF clinics aims to identify aneuploid embryos, and current methodologies rely on costly, invasive and time-consuming approaches such as PGT-A screening. MALDI-ToF-based mass spectral analysis of embryo culture has been demonstrated to be a non-invasive, affordable and accurate technique that is able to capture secretome profiles from embryo culture media extremely quick. Thus, aneuploid embryo genotypes can be distinguished from euploids from these profiles towards the development of novel embryo selection tools.METHODS:A retrospective cohort study, including 292 spent media samples from embryo cultures collected from a single IVF clinic in USA. There were 149 euploid and 165 aneuploid embryos previously analysed by PGT-A next-generation sequencing techniques. Secretome mass spectra of embryos were generated using MALDI-ToF mass spectrometry in the UK. Data was systematically analysed using a fully automated and ultra-fast bioinformatic pipeline developed for the identification of mass spectral signatures.RESULTS:Distinct spectral patterns were found for euploid and aneuploid genotypes in embryo culture media. We identified 12 characteristic peak signatures for euploid and 17 for aneuploid embryos. Data analysis also revealed a high degree of complementarity among regions showing that 22 regions are required to differentiate between genotypes with a sensitivity of 84% and a false positive rate of 18%.CONCLUSION:Ultra-fast and fully automated screening of an embryo genotype is possible based on multiple combinations of specific mass spectral peak signatures. This constitutes a breakthrough towards the implementation of non-invasive and ultra-fast tools for embryo selection immediately prior to transfer.

Background: Human chorionic gonadotropin (hCG) is a glycoprotein hormone with a high degree of molecular microheterogeneity from variable glycosylation and peptide nicking. However, only one accepted structure exists, and all immunoassay epitope mappings are modeled against this structure. Significant microheterogeneity can give rise to charge fluctuations across the molecule resulting in epitope shifts and influence antibody avidity/affinity and consequently immunoassay performance. Methods: Using bioinformatic molecular modeling tools, we have modeled the effects of variable carboxyl terminal peptide positioning, glycosylation, and nicking, and we have characterized potential changes to antigenic regions of hCG. Results: The core epitope regions of the β-subunit of hCG assigned to the original model are distorted by glycosylation and nicking. In particular, hyperglycosylation of hCG significantly affects epitopes β2, β4, and β5 through a displaced Arg68. The presence of one to six fucose to the basal GlcNac of ASN N-linked glycosylations stabilizes the 3D structure regardless of the extent of glycosylation, and even nicking, where the moiety appears to interact with local side chain groups of adjacent amino acids. Modeling the carboxyl terminal peptide has established several likely conformations based on thermodynamic stability. In one of these models, two regions of β-pleated sheeting correspond to critical epitopes, β8 and β9, commonly utilized in commercial immunoassays. Conclusions: The native folding patterns of hCG are affected by glycosylation and nicking to an extent which may affect the orientation of well-characterized β-subunit epitopes and affect the ability of immunoassays to recognize glycovariants commonly seen in early pregnancy, trophoblast diseases, and cancer.

Human chorionic gonadotropin β-core fragment (hCGβcf) is a polypeptide considered to be ubiquitously present in urine regardless of race, sex, and age. This fragment is mainly produced by the degradation of human chorionic gonadotropin in the kidney and excreted in the urine, representing around 80% of total hCG in urine. The presence of hCGβcf in urine has been extensively used for noninvasive clinical diagnostics assays associated with pregnancy. Matrix-Assisted Laser Desorption Ionization Time of Flight (ToF) mass spectrometry (MS) is a rapid, affordable, and powerful diagnostic technique that is starting to find an increasing presence in clinical laboratories. hCGβcf has been used as biomarker of pregnancy and cancer, and, in this chapter, we discuss its potential as quality control marker for urine analysis using MALDI-ToF mass spectrometry. We focus on hCGβcf spectral properties and discuss the advantages and disadvantages to its use as quality control calibrator for MS analysis of urine. Further, we will present how can hCGβcf be implemented as quality control measure in a routine laboratory analysis and in high-throughput analysis of mass spectral data.