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Introduction: Chatbots, which are also known as conversational or virtual agents, are digital programs that can interact with humans using voice, text, or animation. They have shown promise in providing preconception, pregnancy, and postpartum care. This review aims to consolidate the insights of parents and parents-to-be in using chatbots to improve their preconception, pregnancy, and postpartum health., Methods: Seven electronic databases were searched from their inception dates until April 2022 (PubMed, Embase, CINAHL, PsycINFO, Web of Science, Scopus, and ProQuest Dissertations and Theses Global) for relevant studies. English language primary studies that were conducted on parents or parents-to-be aged ≥18 years old who had undergone interventions involving the use of any type of chatbot were included in this review. The quality of included studies was appraised using the Mixed Methods Appraisal Tool. A convergent qualitative synthesis design for mixed studies reviews was used to synthesize the findings, and results were thematically analyzed., Results: Fifteen studies met the inclusion criteria: quantitative (n = 11), qualitative (n = 1), and mixed method (n = 3). Three main themes were identified: (1) welcoming a new health resource, (2) obstacles blocking the way, and (3) moving toward a digital health era., Discussion: Parents and parents-to-be appreciated the informational, socioemotional, and psychological support provided by chatbots. Recommendations for technological improvements in the functionality of the chatbots were made that include training sessions for health care providers to familiarize them with this new digital technology. Multidisciplinary chatbot development teams could also be established to develop more comprehensive chatbot-delivered health programs for more diverse populations., (© 2023 by the American College of Nurse-Midwives.)

Background: Early prediction of Gestational Diabetes Mellitus (GDM) risk is of particular importance as it may enable more efficacious interventions and reduce cumulative injury to mother and fetus. The aim of this study is to develop machine learning (ML) models, for the early prediction of GDM using widely available variables, facilitating early intervention, and making possible to apply the prediction models in places where there is no access to more complex examinations., Methods: The dataset used in this study includes registries from 1,611 pregnancies. Twelve different ML models and their hyperparameters were optimized to achieve early and high prediction performance of GDM. A data augmentation method was used in training to improve prediction results. Three methods were used to select the most relevant variables for GDM prediction. After training, the models ranked with the highest Area under the Receiver Operating Characteristic Curve (AUCROC), were assessed on the validation set. Models with the best results were assessed in the test set as a measure of generalization performance., Results: Our method allows identifying many possible models for various levels of sensitivity and specificity. Four models achieved a high sensitivity of 0.82, a specificity in the range 0.72-0.74, accuracy between 0.73-0.75, and AUCROC of 0.81. These models required between 7 and 12 input variables. Another possible choice could be a model with sensitivity of 0.89 that requires just 5 variables reaching an accuracy of 0.65, a specificity of 0.62, and AUCROC of 0.82., Conclusions: The principal findings of our study are: Early prediction of GDM within early stages of pregnancy using regular examinations/exams; the development and optimization of twelve different ML models and their hyperparameters to achieve the highest prediction performance; a novel data augmentation method is proposed to allow reaching excellent GDM prediction results with various models., (© 2023. The Author(s).)

Background: Intrauterine hematopoietic stem cell transplantation (IUT), potentially curative in congenital haematological disease, is often inhibited by deleterious immune responses to donor cells resulting in subtherapeutic donor cell chimerism (DCC). Microchimerism of maternal immune cells (MMc) trafficked into transplanted recipients across the placenta may directly influence donor-specific alloresponsiveness, limiting DCC. We hypothesized that dendritic cells (DC) among trafficked MMc influence the development of tolerogenic or immunogenic responses towards donor cells, and investigated if maternal DC-depletion reduced recipient alloresponsiveness and enhanced DCC., Methods: Using transgenic CD11c.DTR (C57BL/6) female mice enabled transient maternal DC-depletion with a single dose of diphtheria toxin (DT). CD11c.DTR females and BALB/c males were cross-mated, producing hybrid pups. IUT was performed at E14 following maternal DT administration 24 h prior. Bone marrow-derived mononuclear cells were transplanted, obtained from semi-allogenic BALB/c (paternal-derived; pIUT), C57BL/6 (maternal-derived; mIUT), or fully allogenic (aIUT) C3H donor mice. Recipient F1 pups were analyzed for DCC, while maternal and IUT-recipient immune cell profile and reactivity were examined via mixed lymphocyte reactivity functional assays. T- and B-cell receptor repertoire diversity in maternal and recipient cells were examined following donor cell exposure., Results: DCC was highest and MMc was lowest following pIUT. In contrast, aIUT recipients had the lowest DCC and the highest MMc. In groups that were not DC-depleted, maternal cells trafficked post-IUT displayed reduced TCR & BCR clonotype diversity, while clonotype diversity was restored when dams were DC-depleted. Additionally, recipients displayed increased expression of regulatory T-cells and immune-inhibitory proteins, with reduced proinflammatory cytokine and donor-specific antibody production. DC-depletion did not impact initial donor chimerism. Postnatal transplantation without immunosuppression of paternal donor cells did not increase DCC in pIUT recipients; however there were no donor-specific antibody production or immune cell changes., Conclusions: Though maternal DC depletion did not improve DCC, we show for the first time that MMc influences donor-specific alloresponsiveness, possibly by expanding alloreactive clonotypes, and depleting maternal DC promotes and maintains acquired tolerance to donor cells independent of DCC, presenting a novel approach to enhancing donor cell tolerance following IUT. This may have value when planning repeat HSC transplantations to treat haemoglobinopathies., (© 2023. The Author(s).)

Proof-of-principle disease models have demonstrated the feasibility of an intrauterine gene modification therapy (in utero gene therapy (IUGT)) approach to hereditary diseases as diverse as coagulation disorders, haemoglobinopathies, neurogenetic disorders, congenital metabolic, and pulmonary diseases. Gene addition, which requires the delivery of an integrating or episomal transgene to the target cell nucleus to be transcribed, and gene editing, where the mutation is corrected within the gene of origin, have both been used successfully to increase normal protein production in a bid to reverse or arrest pathology in utero. While most experimental models have employed lentiviral, adenoviral, and adeno-associated viral vectors engineered to efficiently enter target cells, newer models have also demonstrated the applicability of non-viral lipid nanoparticles. Amelioration of pathology is dependent primarily on achieving sustained therapeutic transgene expression, silencing of transgene expression, production of neutralising antibodies, the dilutional effect of the recipient's growth on the mass of transduced cells, and the degree of pre-existing cellular damage. Safety assessment of any IUGT strategy will require long-term postnatal surveillance of both the fetal recipient and the maternal bystander for cell and genome toxicity, oncogenic potential, immune-responsiveness, and germline mutation. In this review, we discuss advances in the field and the push toward clinical translation of IUGT., (© 2023 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

The options for prenatal genetic testing have evolved rapidly in the past decade, and advances in sequencing technology now allow genetic diagnoses to be made down to the single-base-pair level, even before the birth of the child. This offers women the opportunity to obtain information regarding the foetus, thereby empowering them to make informed decisions about their pregnancy. As genetic testing becomes increasingly available to women, clinician knowledge and awareness of the options available to women is of great importance. Additionally, comprehensive pretest and posttest genetic counselling about the advantages, pitfalls and limitations of genetic testing should be provided to all women. This review article aims to cover the range of genetic tests currently available in prenatal screening and diagnosis, their current applications and limitations in clinical practice as well as what the future holds for prenatal genetics.

The current gold standard for the definitive diagnosis of fetal aneuploidy uses either chorionic villus sampling (CVS) or amniocentesis, both of which are which are invasive procedures carrying a procedure-related risk of miscarriage of up to 0.1-0.2%. Non-invasive prenatal diagnosis using fetal nucleated red blood cells (FNRBCs) isolated from maternal peripheral venous blood would remove this risk of miscarriage since these cells can be isolated from the mother's blood. We aimed to detect whole-chromosome aneuploidies from single nucleated fetal red blood cells using whole-genome amplification followed by massively parallel sequencing performed on a semiconductor sequencing platform. Twenty-six single cells were picked from the placental villi of twelve patients thought to have a normal fetal genotype and who were undergoing elective first-trimester surgical termination of pregnancy. Following karyotyping, it was subsequently found that two of these cases were also abnormal (one trisomy 15 and one mosaic genotype). One single cell from chorionic villus samples for two patients carrying a fetus with trisomy 21 and two single cells from women carrying fetuses with T18 were also picked. Pooled libraries were sequenced on the Ion Proton and data were analysed using Ion Reporter software. We correctly classified fetal genotype in all 24 normal cells, as well as the 2 T21 cells, the 2 T18 cells, and the two T15 cells. The two cells picked from the fetus with a mosaic result by CVS were classified as unaffected, suggesting that this was a case of confined placental mosaicism. Fetal sex was correctly assigned in all cases. We demonstrated that semiconductor sequencing using commercially available software for data analysis can be achieved for the non-invasive prenatal diagnosis of whole-chromosome aneuploidy with 100% accuracy.

Competing Interests: Declaration of interests We declare no competing interests.

Raman spectroscopy (RS) is a widely used non-destructive technique for biosensing applications because of its ability to detect unique 'fingerprint' spectra of biomolecules from the vibrational bands. To detect these weak fingerprint spectra, a complex detection system consisting of expensive detectors and optical components are needed. As a result, surface enhanced Raman spectroscopy (SERS) method were used to increase the Raman signal multifold beyond 10 12 times. However, complexity of the entire Raman detection system can be greatly reduced if a short wavelength region/unique single spectral band can distinctly identify the investigating analyte, thereby reducing the need of multiple optical components to capture the entire frequency range of Raman spectra. Here we propose the development of a rapid, single peak Raman technique for the detection of epithelial ovarian cancers (EOC)s through haptoglobin (Hp), a prognostic biomarker. Hp concentration in ovarian cyst fluid (OCF) can be detected and quantified using Raman spectroscopy-based in vitro diagnostic assay. The uniqueness of the Raman assay is that, only in the presence of the analyte Hp, the assay reagent undergoes a biochemical reaction that results in product formation. The unique Raman signature of the assay output falls within the wavenumber region 1500-1700 cm -1 and can be detected using our single peak Raman system. The diagnostic performance of our Raman system had 100.0% sensitivity, 85.0% specificity, 100.0% negative predictive value and 84.2% positive predictive value when compared to gold standard paraffin histology in a proof-of-concept study on 36 clinical OCF samples. When compared to blood-based serum cancer antigen 125 (CA125) levels, the Raman system-based assay had higher diagnostic accuracy when compared to CA125, especially in early-stage EOCs., (© 2022. The Author(s).)

Objective: We conducted a survey-based discrete-choice experiment (DCE) to understand the test features that drive women's preferences for prenatal genomic testing, and explore variation across countries., Methods: Five test attributes were identified as being important for decision-making through a literature review, qualitative interviews and quantitative scoring exercise. Twelve scenarios were constructed in which respondents choose between two invasive tests or no test. Women from eight countries who delivered a baby in the previous 24 months completed a DCE presenting these scenarios. Choices were modeled using conditional logit regression analysis., Results: Surveys from 1239 women (Australia: n = 178; China: n = 179; Denmark: n = 88; Netherlands: n = 177; Singapore: n = 90; Sweden: n = 178; UK: n = 174; USA: n = 175) were analyzed. The key attribute affecting preferences was a test with the highest diagnostic yield (p < 0.01). Women preferred tests with short turnaround times (p < 0.01), and tests reporting variants of uncertain significance (VUS; p < 0.01) and secondary findings (SFs; p < 0.01). Several country-specific differences were identified, including time to get a result, who explains the result, and the return of VUS and SFs., Conclusion: Most women want maximum information from prenatal genomic tests, but our findings highlight country-based differences. Global consensus on how to return uncertain results is not necessarily realistic or desirable., (© 2022 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

The study aimed to investigate whether serum sFlt-1 (soluble fms-like tyrosine kinase-1) at 11-13 weeks' gestation in pregnancies that subsequently developed preeclampsia was different from those without preeclampsia and compare screening performance of the International Prediction of Pregnancy Complications (IPPIC) reported models, which include various combinations of maternal factors, systolic blood pressure, diastolic blood pressure, PlGF (placental growth factor) and sFlt-1 and the competing risk (CR) models, which include various combinations of maternal factors, mean arterial pressure (MAP) and PlGF for predicting any-onset, early-onset, and late-onset preeclampsia. This was a prospective multicenter study in 7877 singleton pregnancies. The differences of the predictive performance between the IPPIC and CR models were compared. There were 141 women (1.79%) who developed preeclampsia, including 13 cases (0.17%) of early-onset preeclampsia and 128 cases (1.62%) of late-onset preeclampsia. In pregnancies that developed preeclampsia compared to unaffected pregnancies, median serum sFlt-1 levels and its MoMs were not significantly different ( p >0.05). There was no significant association between gestational age at delivery and log 10 sFlt-1 and log 10 sFlt-1 MoM ( p >0.05). The areas under the curve of CR models were significantly higher than the IPPIC models for the prediction of any-onset and late-onset preeclampsia but not for early-onset preeclampsia. In conclusion, there are no significant differences in the maternal serum sFlt-1 levels at 11-13 weeks' gestation between women who subsequently develop preeclampsia and those who do not. Moreover, the CR models for the prediction of any-onset and late-onset preeclampsia perform better than the IPPIC reported model.

Introduction: Periodic benchmarking of the epidemiology of COVID-19 in the Association of Southeast Asian Nations (ASEAN) countries is critical for the continuous understanding of the transmission and control of COVID-19 in the region. The incidence, mortality, testing and vaccination rates within the ASEAN region from 1 January 2020 to 15 October 2021 is analysed in this paper., Methods: COVID-19 data on cases, deaths, testing, and vaccinations were extracted from the Our World in Data (OWID) COVID-19 data repository for all the ten ASEAN countries. Comparative time-trends of the epidemiology of COVID-19 using the incidence rate, cumulative case fatality rate (CFR), delay-adjusted case fatality rate, cumulative mortality rate (MR), test positivity rate (TPR), cumulative testing rate (TR) and vaccination rate was carried out., Results: Over the study period, a total of 12,720,661 cases and 271,475 deaths was reported within the ASEAN region. Trends of daily per capita cases were observed to peak between July and September 2021 for the ASEAN region. The cumulative case fatality rate (CFR) in Brunei, Cambodia, Indonesia, Laos, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam, was of 0.9% (N=68), 2.2% (N=2,610), 3.5% (N=142,889), 0.1% (N=36), 1.2% (N=27,700), 4.0% (N=18,297), 1.6% (N=40,424), 0.1% (N=215), 1.7% (N=18,123), and 2.6% (N=21,043), respectively. CFR was consistently highest between January-June 2020. The cumulative mortality rate (MR) was 9.5, 13.7, 51.4, 0.2, 80.3, 32.4, 34.5, 1.6, 23.9 and 19.7 per 100,000 population, respectively. The cumulative test positivity rate (TPR) was 8.4%, 16.9%, 4.6%, 7.5%, 11.1%, 12.9%, 0.5%, 11.7%, and 3.6%, with the cumulative testing rate (TR) at 25.0, 90.1, 27.4, 917.7, 75.8, 177.8, 3303.3, 195.2, and 224.9 tests per 1,000 population in Cambodia, Indonesia, Laos, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam, respectively. The percentage of population that completed vaccinations (VR) was 44.5%, 65.3%, 18.5%, 28.2%, 61.8%, 6.8%, 19.2%, 76.8%, 22.7%, and 10% in Brunei, Cambodia, Indonesia, Laos, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam, respectively., Conclusion: In 2020, most countries in ASEAN had higher case fatality rates but lower mortalities per population when compared to the third quarter of 2021 where higher mortalities per population were observed. Low testing rates have been one of the factors leading to high test positivity rates. Slow initiation of vaccination programs was found to be the key factor leading to high incidence and case fatality rate in most countries in ASEAN. Effective public health measures were able to interrupt the transmission of this novel virus to some extent. Increasing preparedness capacity within the ASEAN region is critical to ensure that any future similar outbreaks can be dealt with collectively.

There are over 50 SARS-CoV-2 candidate vaccines undergoing Phase II and III clinical trials. Several vaccines have been approved by regulatory authorities and rolled out for use in different countries. Due to concerns of potential teratogenicity or adverse effect on maternal physiology, pregnancy has been a specific exclusion criterion for most vaccine trials with only two trials not excluding pregnant women. Thus, other than limited animal studies, gradually emerging development and reproductive toxicity data, and observational data from vaccine registries, there is a paucity of reliable information to guide recommendations for the safe vaccination of pregnant women. Pregnancy is a risk factor for severe COVID-19, especially in women with comorbidities, resulting in increased rates of preterm birth and maternal morbidity. We discuss the major SARS-CoV-2 vaccines, their mechanisms of action, efficacy, safety profile and possible benefits to the maternal-fetal dyad to create a rational approach towards maternal vaccination while anticipating and mitigating vaccine-related complications. Pregnant women with high exposure risks or co-morbidities predisposing to severe COVID-19 infection should be prioritised for vaccination. Those with risk factors for adverse effects should be counselled accordingly. It is essential to support patient autonomy by shared decision-making involving a risk-benefit discussion with the pregnant woman., (© 2021 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Preeclampsia (PE) and Intrauterine Growth Restriction (IUGR) are two pregnancy-specific placental disorders with high maternal, fetal, and neonatal morbidity and mortality rates worldwide. The identification biomarkers involved in the dysregulation of PE and IUGR are fundamental for developing new strategies for early detection and management of these pregnancy pathologies. Several studies have demonstrated the importance of long non-coding RNAs (lncRNAs) as essential regulators of many biological processes in cells and tissues, and the placenta is not an exception. In this review, we summarize the importance of lncRNAs in the regulation of trophoblasts during the development of PE and IUGR, and other placental disorders.

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2 nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14 th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2 nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

Preterm birth (PTB) is a major cause of neonatal death and long-term consequences for the newborn. This review aims to update the evidence about the potential benefit of pharmacological supplementation with omega 3 fatty acids during pregnancy on the incidence of PTB. The Medline, Embase, Cochrane Library and Central databases were searched until 28 June 2020 for RCTs in which omega 3 supplementation was used versus placebo to reduce PTB risk. Data from 37 trials were analyzed. We found an 11% reduction in PTB risk (RR(risk ratios), 0.89; 95% CI (confidence intervals), 0.82 to 0.97) in trials using omega 3 supplements versus placebo. Regarding early PTB (ePTB), there was a 27% reduction in the risk of ePTB (RR, 0.73; 95% CI, 0.58 to 0.92). However, after sensitivity analyses, there were no significant differences in PTB and ePTB risk (PTB RR, 0.92; 95% CI, 0.83 to 1.01, ePTB RR, 0.82; 95% CI, 0.61 to 1.09). We conclude that omega 3 supplementation during pregnancy does not reduce the risk of PTB and ePTB. More studies are required to determine the effect of omega 3 supplementations during pregnancy and the risk of detrimental fetal outcomes.

Objectives: To conduct qualitative interviews with healthcare providers working in different countries to understand their experiences of dealing with uncertain results from prenatal chromosome microarray analysis (CMA) and exome sequencing (ES)., Methods: Semi-structured interviews with 31 healthcare providers who report or return prenatal CMA and/or ES results (clinicians, genetic counsellors and clinical scientists) in six countries with differing healthcare systems; Australia (4), Denmark (5), Netherlands (6), Singapore (4), Sweden (6) and United Kingdom (6). The topic guide explored the main sources of uncertainty and their management., Results: There was variation in reporting practices both between and across countries for variants of uncertain significance, however, there was broad agreement on reporting practices for incidental findings. There was also variation in who decides what results are reported (clinical scientists or clinicians). Technical limitations and lack of knowledge (to classify variants and of prenatal phenotypes) were significant challenges, as were turnaround times and lack of guidelines., Conclusion: Health professionals around the globe are dealing with similar sources of uncertainty, but managing them in different ways, Continued dialogue with international colleagues on ways of managing uncertain results is important to compare and contrast the benefits and limitations of the different approaches., (© 2021 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Successful intrauterine hematopoietic cell transplantation (IUT) for congenital hemoglobinopathies is hampered by maternal alloresponsiveness. We investigate these interactions in semi-allogenic murine IUT. E14 fetuses (B6 females × BALB/c males) were each treated with 5E+6 maternal (B6) or paternal (BALB/c) bone marrow cells and serially monitored for chimerism (>1% engraftment), trafficked maternal immune cells, and immune responsiveness to donor cells. A total of 41.0% of maternal IUT recipients (mIUT) were chimeras (mean donor chimerism 3.0 ± 1.3%) versus 75.0% of paternal IUT recipients (pIUT, 3.6 ± 1.1%). Chimeras showed higher maternal microchimerism of CD4, CD8, and CD19 than non-chimeras. These maternal cells showed minimal responsiveness to B6 or BALB/c stimulation. To interrogate tolerance, mIUT were injected postnatally with 5E+6 B6 cells/pup; pIUT received BALB/c cells. IUT-treated pups showed no changes in trafficked maternal or fetal immune cell levels compared to controls. Donor-specific IgM and IgG were expressed by 1%-3% of recipients. mIUT splenocytes showed greater proliferation of regulatory T cells (Treg) upon BALB/c stimulation, while B6 stimulation upregulated the pro-inflammatory cytokines more than BALB/c. pIUT splenocytes produced identical Treg and cytokine responses to BALB/c and B6 cells, with higher Treg activity and lower pro-inflammatory cytokine expression upon exposure to BALB/c. In contrast, naïve fetal splenocytes demonstrated greater alloresponsiveness to BALB/c compared to B6 cells. Thus pIUT, associated with increased maternal cell trafficking, modulates fetal Treg, and cytokine responsiveness to donor cells more efficiently than mIUT, resulting in improved engraftment. Paternal donor cells may be considered alternatively to maternal donor cells for intrauterine and postnatal transplantation to induce tolerance and maintain engraftment., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway, and its loss of function through polymorphisms is often associated with human conditions, including cancer, congenital heart disease, and Down syndrome. MTHFR is also required in the maintenance of heterochromatin, a crucial determinant of genomic stability and precise chromosomal segregation. Here, we characterize the function of a fission yeast gene met11 + , which encodes a protein that is highly homologous to the mammalian MTHFR. We show that, although met11 + is not essential for viability, its disruption increases chromosome missegregation and destabilizes constitutive heterochromatic regions at pericentromeric, sub-telomeric and ribosomal DNA (rDNA) loci. Transcriptional silencing at these sites were disrupted, which is accompanied by the reduction in enrichment of histone H3 lysine 9 dimethylation (H3K9me2) and binding of the heterochromatin protein 1 (HP1)-like Swi6. The met11 null mutant also dominantly disrupts meiotic fidelity, as displayed by reduced sporulation efficiency and defects in proper partitioning of the genetic material during meiosis. Interestingly, the faithful execution of these meiotic processes is synergistically ensured by cooperation among Met11, Rec8, a meiosis-specific cohesin protein, and the shugoshin protein Sgo1, which protects Rec8 from untimely cleavage. Overall, our results suggest a key role for Met11 in maintaining pericentromeric heterochromatin for precise genetic inheritance during mitosis and meiosis.

There remain unanswered questions concerning mother-to-child-transmission of SARS-CoV-2. Despite reports of neonatal COVID-19, SARS-CoV-2 has not been consistently isolated in perinatal samples, thus definitive proof of transplacental infection is still lacking. To address these questions, we assessed investigative tools used to confirm maternal-fetal infection and known protective mechanisms of the placental barrier that prevent transplacental pathogen migration. Forty studies of COVID-19 pregnancies reviewed suggest a lack of consensus on diagnostic strategy for congenital infection. Although real-time polymerase chain reaction of neonatal swabs was universally performed, a wide range of clinical samples was screened including vaginal secretions (22.5%), amniotic fluid (35%), breast milk (22.5%) and umbilical cord blood. Neonatal COVID-19 was reported in eight studies, two of which were based on the detection of SARS-CoV-2 IgM in neonatal blood. Histological examination demonstrated sparse viral particles, vascular malperfusion and inflammation in the placenta from pregnant women with COVID-19. The paucity of placental co-expression of ACE-2 and TMPRSS2, two receptors involved in cytoplasmic entry of SARS-CoV-2, may explain its relative insensitivity to transplacental infection. Viral interactions may utilise membrane receptors other than ACE-2 thus, tissue susceptibility may be broader than currently known. Further spatial-temporal studies are needed to determine the true potential for transplacental migration., (© 2020 John Wiley & Sons, Ltd.)

Introduction: COVID-19 has caused unprecedented public health concerns, triggering an escalated burden to health systems worldwide. The pandemic has altered people's living norms, yet coherently escalating countries' socioeconomic instability. This real-time consensus review aims to describe the epidemiological trends of COVID-19 pandemic across six South-East Asian nations, and countryspecific experiences on pandemic preparedness, responses and interventions., Methods: Consensus-driven approach between authors from the six selected countries was applied. Country specific policy documents, official government media statements, mainstream news portals, global statistics databases and latest published literature available between January-October 2020 were utilised for information retrieval. Situational and epidemiological trend analyses were conducted. Country-specific interventions and challenges were described. Based on evidence appraised, a descriptive framework was considered through a consensus. The authors subsequently outlined the lessons learned, challenges ahead and interventions that needs to be in place to control the pandemic., Results: The total number of people infected with COVID-19 between 1 January and 16 November 2020 had reached 48,520 in Malaysia, 58,124 in Singapore, 3,875 in Thailand, 470,648 in Indonesia, 409,574 in Philippines and 70,161 in Myanmar. The total number of people infected with COVID- 19 in the six countries from January to 31 October 2020 were 936,866 cases and the mortality rate was 2.42%. Indonesia had 410,088 cases with a mortality rate of 3.38%, Philippines had 380,729 cases with a mortality rate of 1.90%, Myanmar had 52,706 cases with a mortality rate of 2.34%, Thailand had 3,780 cases with a mortality rate of 1.56%, Malaysia had 31,548 cases with a mortality rate of 0.79%, and Singapore had 58,015 cases with a mortality rate of 0.05% over the 10- month period. Each country response varied depending on its real-time situations based on the number of active cases and economic situation of the country., Conclusion: The number of COVID-19 cases in these countries waxed and waned over the 10-month period, the number of cases may be coming down in one country, and vice versa in another. Each country, if acting alone, will not be able to control this pandemic. Sharing of information and resources across nations is the key to successful control of the pandemic. There is a need to reflect on how the pandemic affects individuals, families and the community as a whole. There are many people who cannot afford to be isolated from their families and daily wage workers who cannot afford to miss work. Are we as a medical community, only empathising with our patients or are we doing our utmost to uphold them during this time of crisis? Are there any other avenues which can curb the epidemic while reducing its impact on the health and socio-economic condition of the individual, community and the nation?

Background and Objective: Optimal sample storage conditions are essential for non-invasive prenatal testing of cell-free fetal and total DNA. We investigated the effect of long-term storage of plasma samples and extracted cfDNA using qPCR., Materials and Methods: Fetal and total cfDNA yield and fetal fraction were calculated before and after storage of plasma for 0-6 years at -25°C. Dilution experiments were performed to investigate PCR inhibition. Extraction with or without proteinase K was used to examine protein dissociation. Storage of extracted cfDNA was investigated by testing aliquots immediately, and after 18 months and 3 years of storage at -25°C., Results: We observed a marked increase in the levels of amplifiable fetal and total DNA in plasma stored for 2-3 years, and fetal fraction was slightly decreased after 3 years of storage. cfDNA detection was independent of proteinase K during DNA extraction in plasma samples stored >2 years, indicating a loss of proteins from DNA over time, which was likely to account for the observed increase in DNA yields. Measured fetal and total DNA quantities, as well as fetal fraction, increased in stored, extracted cfDNA., Conclusion: Fetal and total cell-free DNA is readily detectable in plasma after long-term storage at -25°C. However, substantial variation in measured DNA quantities and fetal fraction means caution may be required when using stored plasma and extracted cfDNA for test development or validation purposes., (© 2020 International Society of Blood Transfusion.)

Among the transcription factors that are conserved across phylogeny, the grainyhead family holds vital roles in driving the epithelial cell fate. In Drosophila , the function of grainyhead ( grh ) gene is essential during developmental processes such as epithelial differentiation, tracheal tube formation, maintenance of wing and hair polarity, and epidermal barrier wound repair. Three main mammalian orthologs of grh : Grainyhead-like 1-3 (GRHL1, GRHL2, and GRHL3) are highly conserved in terms of their gene structures and functions. GRHL proteins are essentially associated with the development and maintenance of the epithelial phenotype across diverse physiological conditions such as epidermal differentiation and craniofacial development as well as pathological functions including hearing impairment and neural tube defects. More importantly, through direct chromatin binding and induction of epigenetic alterations, GRHL factors function as potent suppressors of oncogenic cellular dedifferentiation program - epithelial-mesenchymal transition and its associated tumor-promoting phenotypes such as tumor cell migration and invasion. On the contrary, GRHL factors also induce pro-tumorigenic effects such as increased migration and anchorage-independent growth in certain tumor types. Furthermore, investigations focusing on the epithelial-specific activation of grh and GRHL factors have revealed that these factors potentially act as a pioneer factor in establishing a cell-type/cell-state specific accessible chromatin landscape that is exclusive for epithelial gene transcription. In this review, we highlight the essential roles of grh and GRHL factors during embryogenesis and pathogenesis, with a special focus on its emerging pioneering function., (Copyright © 2020 Sundararajan, Pang, Choolani and Huang.)

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2, has been declared a pandemic by the World Health Organization. As the pandemic evolves rapidly, there are data emerging to suggest that pregnant women diagnosed as having coronavirus disease 2019 can have severe morbidities (up to 9%). This is in contrast to earlier data that showed good maternal and neonatal outcomes. Clinical manifestations of coronavirus disease 2019 include features of acute respiratory illnesses. Typical radiologic findings consists of patchy infiltrates on chest radiograph and ground glass opacities on computed tomography scan of the chest. Patients who are pregnant may present with atypical features such as the absence of fever as well as leukocytosis. Confirmation of coronavirus disease 2019 is by reverse transcriptase-polymerized chain reaction from upper airway swabs. When the reverse transcriptase-polymerized chain reaction test result is negative in suspect cases, chest imaging should be considered. A pregnant woman with coronavirus disease 2019 is at the greatest risk when she is in labor, especially if she is acutely ill. We present an algorithm of care for the acutely ill parturient and guidelines for the protection of the healthcare team who is caring for the patient. Key decisions are made based on the presence of maternal and/or fetal compromise, adequacy of maternal oxygenation (SpO 2 >93%) and stability of maternal blood pressure. Although vertical transmission is unlikely, there must be measures in place to prevent neonatal infections. Routine birth processes such as delayed cord clamping and skin-to-skin bonding between mother and newborn need to be revised. Considerations can be made to allow the use of screened donated breast milk from mothers who are free of coronavirus disease 2019. We present management strategies derived from best available evidence to provide guidance in caring for the high-risk and acutely ill parturient. These include protection of the healthcare workers caring for the coronavirus disease 2019 gravida, establishing a diagnosis in symptomatic cases, deciding between reverse transcriptase-polymerized chain reaction and chest imaging, and management of the unwell parturient., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The current coronavirus disease 2019 (COVID-19) pneumonia pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading globally at an accelerated rate, with a basic reproduction number (R0) of 2-2.5, indicating that 2-3 persons will be infected from an index patient. A serious public health emergency, it is particularly deadly in vulnerable populations and communities in which healthcare providers are insufficiently prepared to manage the infection. As of March 16, 2020, there are more than 180,000 confirmed cases of COVID-19 worldwide, with more than 7000 related deaths. The SARS-CoV-2 virus has been isolated from asymptomatic individuals, and affected patients continue to be infectious 2 weeks after cessation of symptoms. The substantial morbidity and socioeconomic impact have necessitated drastic measures across all continents, including nationwide lockdowns and border closures. Pregnant women and their fetuses represent a high-risk population during infectious disease outbreaks. To date, the outcomes of 55 pregnant women infected with COVID-19 and 46 neonates have been reported in the literature, with no definite evidence of vertical transmission. Physiological and mechanical changes in pregnancy increase susceptibility to infections in general, particularly when the cardiorespiratory system is affected, and encourage rapid progression to respiratory failure in the gravida. Furthermore, the pregnancy bias toward T-helper 2 (Th2) system dominance, which protects the fetus, leaves the mother vulnerable to viral infections, which are more effectively contained by the Th1 system. These unique challenges mandate an integrated approach to pregnancies affected by SARS-CoV-2. Here we present a review of COVID-19 in pregnancy, bringing together the various factors integral to the understanding of pathophysiology and susceptibility, diagnostic challenges with real-time reverse transcription polymerase chain reaction (RT-PCR) assays, therapeutic controversies, intrauterine transmission, and maternal-fetal complications. We discuss the latest options in antiviral therapy and vaccine development, including the novel use of chloroquine in the management of COVID-19. Fetal surveillance, in view of the predisposition to growth restriction and special considerations during labor and delivery, is addressed. In addition, we focus on keeping frontline obstetric care providers safe while continuing to provide essential services. Our clinical service model is built around the principles of workplace segregation, responsible social distancing, containment of cross-infection to healthcare providers, judicious use of personal protective equipment, and telemedicine. Our aim is to share a framework that can be adopted by tertiary maternity units managing pregnant women in the flux of a pandemic while maintaining the safety of the patient and healthcare provider at its core., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Conventionally Surface-enhanced Raman spectroscopy (SERS) is realized by adsorbing analytes onto nano-roughened planar substrate coated with noble metals (silver or gold) or their colloidal nanoparticles (NPs). Nanoscale irregularities in such substrates/NPs could lead to SERS sensors with poor reproducibility and repeatability. Herein, we demonstrate a suspended core photonic crystal fiber (PCF) based SERS sensor with extremely high reproducibility and repeatability in measurement with a relative SD of only 1.5% and 4.6%, respectively, which makes it more reliable than any existing SERS sensor platforms. In addition, our platform could improve the detection sensitivity owing to the increased interaction area between the guided light and the analyte, which is incorporated into the holes that runs along the length of the PCF. Numerical calculation established the significance of the interplay between light coupling efficiency and evanescent field distribution, which could eventually determine the sensitivity and reliability of the developed SERS active-PCF sensor. As a proof of concept, using this sensor, we demonstrated the detection of haptoglobin, a biomarker for ovarian cancer, contained within the ovarian cyst fluid, which facilitated in differentiating the stages of cancer. We envision that with necessary refinements, this platform could potentially be translated as a next-generation highly sensitive SERS-active opto-fluidic biopsy needle for the detection of biomarkers in body fluids., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Keloid formation occurs in Caucasian, African, and Asian populations and is a severe psychosocial burden on patients. There is no permanent treatment for this problem as its pathogenesis is not properly understood. Furthermore, differences in keloid behavior between ethnic groups are not known. It has been hypothesized that keloids behave like benign tumors because of their uncontrolled growth. The present study evaluated the tumoricidal properties of human Wharton's jelly stem cell-conditioned medium (hWJSC-CM) on fresh Asian keloid cells (AKCs)., Methods: Human Wharton's jelly stem cells (hWJSCs) and AKCs were isolated based on our previous methods. hWJSCs and human skin fibroblasts (HSF) (controls) were used to collect hWJSC-CM and HSF-conditioned medium (HSF-CM). AKCs were treated with hWJSC-CM and HSF-CM in vitro and in vivo in a human keloid xenograft SCID mouse model. The inhibitory effect of hWJSC-CM on AKCs was tested in vitro using various assays and in vivo for attenuation/abrogation of AKC tumors created in a xenograft mouse model., Results: qRT-PCR analysis showed that the genes FN1, MMP1, and VCAN were significantly upregulated in AKCs and ANXA1, ASPN, IGFBP7, LGALS1, and PTN downregulated. AKCs exposed to hWJSC-CM in vitro showed significant decreases in cell viability and proliferation, increases in Annexin V-FITC+ cell numbers, interruptions of the cell cycle at Sub-G1 and G2/M phases, altered CD marker expression, downregulated anti-apoptotic-related genes, and upregulated pro-apoptotic and autophagy-related genes compared to controls. When AKCs were administered together with hWJSC-CM into immunodeficient mice there were no keloid tumors formed in 7 mice (n = 10) compared to the untreated control mice. When hWJSC-CM was injected directly into keloid tumors created in mice there were significant reductions in keloid tumor volumes and weights in 30 days., Conclusions: hWJSC-CM inhibited the growth of AKCs in vitro and in xenograft mice, and it may be a potential novel treatment for keloids in the human. The specific molecule(s) in hWJSC-CM that induce the anti-keloid effect need to be identified, characterized, and tested separately in larger preclinical and clinical studies.

The COVID-19 pandemic has stressed patients and healthcare givers alike and challenged our practice of antenatal care, including fetal diagnosis and therapy. This document aims to review relevant recent information to allow us to optimize prenatal care delivery. We discuss potential modifications to obstetric management and fetal procedures in SARS-CoV2-negative and SARS-CoV2-positive patients with fetal anomalies or disorders. Most fetal therapies are time sensitive and cannot be delayed. If personnel and resources are available, we should continue to offer procedures of proven benefit, acknowledging any fetal and maternal risks, including those to health care workers. There is, to date, minimal, unconfirmed evidence of spontaneous vertical transmission, though it may theoretically be increased with some procedures. Knowing a mother's preoperative SARS-CoV-2 status would enable us to avoid or defer certain procedures while she is contagious and to protect health care workers appropriately. Some fetal conditions may alternatively be managed neonatally. Counseling regarding fetal interventions which have a possibility of additional intra- or postoperative morbidity must be performed in the context of local resource availability. Procedures of unproven benefit should not be offered. We encourage participation in registries and trials that may help us to understand the impact of COVID-19 on pregnant women, their fetuses, and neonates., (© 2020 S. Karger AG, Basel.)

Background: The administration of aspirin <16 weeks gestation to women who are at high risk for preeclampsia has been shown to reduce the rate of preterm preeclampsia by 65%. The traditional approach to identify such women who are at risk is based on risk factors from maternal characteristics, obstetrics, and medical history as recommended by the American College of Obstetricians and Gynecologists and the National Institute for Health and Care Excellence. An alternative approach to screening for preeclampsia has been developed by the Fetal Medicine Foundation. This approach allows the estimation of patient-specific risks of preeclampsia that requires delivery before a specified gestational age with the use of Bayes theorem-based model., Objective: The purpose of this study was to examine the diagnostic accuracy of the Fetal Medicine Foundation Bayes theorem-based model, the American College of Obstetricians and Gynecologists, and the National Institute for Health and Care Excellence recommendations for the prediction of preterm preeclampsia at 11-13 +6 weeks gestation in a large Asian population STUDY DESIGN: This was a prospective, nonintervention, multicenter study in 10,935 singleton pregnancies at 11-13 +6 weeks gestation in 11 recruiting centers across 7 regions in Asia between December 2016 and June 2018. Maternal characteristics and medical, obstetric, and drug history were recorded. Mean arterial pressure and uterine artery pulsatility indices were measured according to standardized protocols. Maternal serum placental growth factor concentrations were measured by automated analyzers. The measured values of mean arterial pressure, uterine artery pulsatility index, and placental growth factor were converted into multiples of the median. The Fetal Medicine Foundation Bayes theorem-based model was used for the calculation of patient-specific risk of preeclampsia at <37 weeks gestation (preterm preeclampsia) and at any gestation (all preeclampsia) in each participant. The performance of screening for preterm preeclampsia and all preeclampsia by a combination of maternal factors, mean arterial pressure, uterine artery pulsatility index, and placental growth factor (triple test) was evaluated with the adjustment of aspirin use. We examined the predictive performance of the model by the use of receiver operating characteristic curve and calibration by measurements of calibration slope and calibration in the large. The detection rate of screening by the Fetal Medicine Foundation Bayes theorem-based model was compared with the model that was derived from the application of American College of Obstetricians and Gynecologists and National Institute for Health and Care Excellence recommendations., Results: There were 224 women (2.05%) who experienced preeclampsia, which included 73 cases (0.67%) of preterm preeclampsia. In pregnancies with preterm preeclampsia, the mean multiples of the median values of mean arterial pressure and uterine artery pulsatility index were significantly higher (mean arterial pressure, 1.099 vs 1.008 [P<.001]; uterine artery pulsatility index, 1.188 vs 1.063[P=.006]), and the mean placental growth factor multiples of the median was significantly lower (0.760 vs 1.100 [P<.001]) than in women without preeclampsia. The Fetal Medicine Foundation triple test achieved detection rates of 48.2%, 64.0%, 71.8%, and 75.8% at 5%, 10%, 15%, and 20% fixed false-positive rates, respectively, for the prediction of preterm preeclampsia. These were comparable with those of previously published data from the Fetal Medicine Foundation study. Screening that used the American College of Obstetricians and Gynecologists recommendations achieved detection rate of 54.6% at 20.4% false-positive rate. The detection rate with the use of National Institute for Health and Care Excellence guideline was 26.3% at 5.5% false-positive rate., Conclusion: Based on a large number of women, this study has demonstrated that the Fetal Medicine Foundation Bayes theorem-based model is effective in the prediction of preterm preeclampsia in an Asian population and that this method of screening is superior to the approach recommended by American College of Obstetricians and Gynecologists and the National Institute for Health and Care Excellence. We have also shown that the Fetal Medicine Foundation prediction model can be implemented as part of routine prenatal care through the use of the existing infrastructure of routine prenatal care., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Development of cell transplantation for treating liver cirrhosis hinges critically on the availability of animal models for studying human stem cell transplantation. We report an immune-permissive murine model of liver cirrhosis with full clinical correlates of decompensated liver disease, and allows testing efficacy of stem cell transplantation. Liver cirrhosis was induced in Nod-scid gamma(NSG) mice with oral thioacetamide(TA) and compared to controls over 12 months. 4 month TA treated cirrhotic mice were then transplanted intrasplenically with 2million human fetal liver progenitor cells(HFH) and compared with cirrhotic controls 2 months after transplantation. NSG-TA mice developed shrunken and nodular livers with histological evidence of fibrosis as compared to controls. This was associated with evidence of worsening decompensated liver disease, with jaundice, hypoalbuminemia, coagulopathy, and encephalopathy in NSG-TA mice. Transplantation of HFH resulted in improvement in both fibrosis and markers of decompensated liver disease. We have demonstrated that NSG-TA mice can recapitulate the full clinical picture of structural and functional cirrhosis, both of which can be improved by transplantation of human fetal liver cells. This model serves as a valuable tool for validation of in vivo liver stem cell transplantation and opens up opportunities for studying the mechanism how stem cells reverse fibrosis.

Purpose: Multiple chromosomal aneuploidies may be associated with maternal malignancies and can cause failure of noninvasive prenatal screening (NIPS) tests. However, multiple chromosomal aneuploidies show poor specificity and selectivity for diagnosing maternal malignancies., Methods: This multicenter retrospective analysis evaluated 639 pregnant women who tested positive for multiple chromosomal aneuploidies on initial NIPS test between January 2016 and December 2017. Women were assessed using genome profiling of copy-number variations, which was translated to cancer risk using a novel bioinformatics algorithm called the cancer detection pipeline (CDP). Sensitivity, specificity, and positive predictive value (PPV) of diagnosing maternal malignancies were compared for multiple chromosomal aneuploidies, the CDP model, and the combination of CDP and plasma tumor markers., Results: Of the 639 subjects, 41 maternal malignant cancer cases were diagnosed. Multiple chromosomal aneuploidies predicted maternal malignancies with a PPV of 7.6%. Application of the CDP algorithm to women with multiple chromosomal aneuploidies allowed 34 of the 41 (83%) cancer cases to be identified, while excluding 422 of 501 (84.2%) of the false positive cases. Combining the CDP with plasma tumor marker testing gave PPV of 75.0%., Conclusion: The CDP algorithm can diagnose occult maternal malignancies with a reasonable PPV in multiple chromosomal aneuploidies-positive pregnant women in NIPS tests. This performance can be further improved by incorporating findings for plasma tumor markers.

A woman's chances of having a child with Down syndrome increases with age. By age 40, the risk of conceiving a child with Down syndrome is about 1 in 100. We report a rare case of dizygotic dichorionic diamniotic twin pregnancy conceived via in vitro fertilisation, with both twins having trisomy 21. Both fetuses were independently detected to be at high risk of autosomal trisomy, initially via first-trimester screening and subsequently via invasive definitive diagnostic tests (ie, chorionic villus sampling and amniocentesis).Diagnosis of trisomy 21 has to be made via initial non-invasive prenatal screening, followed by further rigorous and accurate invasive pregnancy testing for confirmation. The gravity of the results necessitates high detection rates and high specificity of prenatal screening tests. Management of the patient must be multidisciplinary and supportive in nature, involving extensive and non-directive pregnancy counselling and management, genetic counselling and management of psychological distress., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

The evaluation of intra-tumour heterogeneity (ITH) from a transcriptomic point of view is limited. Single-cell cancer studies reveal significant genomic and transcriptomic ITH within a tumour and it is no longer adequate to employ single-subtype assignment as this does not acknowledge the ITH that exists. Molecular assessment of subtype heterogeneity (MASH) was developed to comprehensively report on the composition of all transcriptomic subtypes within a tumour lesion. Using MASH on 3431 ovarian cancer samples, correlation and association analyses with survival, metastasis and clinical outcomes were performed to assess the impact of subtype composition as a surrogate for ITH. The association was validated on two independent cohorts. We identified that 30% of ovarian tumours consist of two or more subtypes. When biological features of the subtype constituents were examined, we identified significant impact on clinical outcomes with the presence of poor prognostic subtypes (Mes or Stem-A). Poorer outcomes correlated with having higher degrees of poor prognostic subtype populations within the tumour. Subtype prediction in several independent datasets reflected a similar prognostic trend. In addition, paired analysis of primary and recurrent/metastatic tumours demonstrated Mes and/or Stem-A subtypes predominated in recurrent and metastatic tumours regardless of the original primary subtype. Given the biological and prognostic value in delineating individual subtypes within a tumour, a clinically applicable MASH assay using NanoString ® technology was developed as a classification tool to comprehensively describe constituents of molecular subtypes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at ∼0.4 gestation. The initial cohort received 1 × 10 12 vector genomes (vgs) of AAV5-hFIX ( n = 5; 0.45 × 10 13 vg/kg birth weight), resulting in ∼3.0% hFIX at birth and 0.6-6.8% over 19-51 mo. The next cohort received 0.2-1 × 10 13 vg boluses. AAV5-hFX animals ( n = 3; 3.57 × 10 13 vg/kg) expressed <1% at birth and 9.4-27.9% up to 42 mo. AAV8-hFIX recipients ( n = 3; 2.56 × 10 13 vg/kg) established 4.2-41.3% expression perinatally and 9.8-25.3% over 46 mo. Expression with AAV8-hFX ( n = 6, 3.12 × 10 13 vg/kg) increased from <1% perinatally to 9.8-13.4% >35 mo. Low expressers (<1%, n = 3) were postnatally challenged with 2 × 10 11 vg/kg AAV5 resulting in 2.4-13.2% expression and demonstrating acquired tolerance. Linear amplification-mediated-PCR analysis demonstrated random integration of 57-88% of AAV sequences retrieved from hepatocytes with no events occurring in or near oncogenesis-associated genes. Thus, early-IUGT in macaques produces sustained curative expression related significantly to integrated AAV in the absence of clinical toxicity, supporting its therapeutic potential for early-onset monogenic disorders.-Chan, J. K. Y., Gil-Farina I., Johana, N., Rosales, C., Tan, Y. W., Ceiler, J., Mcintosh, J., Ogden, B., Waddington, S. N., Schmidt, M., Biswas, A., Choolani, M., Nathwani, A. C., Mattar, C. N. Z. Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Human Wharton's jelly stem cells (hWJSCs) isolated from the human umbilical cord are a unique population of mesenchymal stem cells (MSCs) with significant clinical utility. Their broad differentiation potential, high rate of proliferation, ready availability from discarded cords, and prolonged maintenance of stemness properties in culture make them an attractive alternative source of MSCs with therapeutic value compared with human bone marrow MSCs (hBMMSCs). We aimed to characterize the differences in gene expression profiles between these two stem cell types using single-cell RNA sequencing (scRNA-Seq) to determine which pathways are involved in conferring hWJSCs with their unique properties. We identified 436 significantly differentially expressed genes between the two cell types, playing roles in processes, including immunomodulation, angiogenesis, wound healing, apoptosis, antitumor activity, and chemotaxis. Expression of immune molecules is particularly high in hWJSCs compared with hBMMSCs. These differences in gene expression may help to explain many of the advantages that hWJSCs have over hBMMSCs for clinical application. Although cell surface protein marker expression indicates that isolated hWJSCs and hBMMSCs are both homogenous populations, using scRNA-Seq we can clearly identify extreme variability in expression levels between individual cells within a certain cell type. If the cells are examined as bulk populations, it is not possible to appreciate that a single cell may be making a major unique contribution to the apparent overall expression level. We demonstrated how the fine tuning of expression within hWJSCs and hBMMSCs may be achieved by expression of molecules with opposing function between two cells. We hypothesize that a greater understanding of these differences in gene expression between the two cell types may aid in the development of new therapies using hWJSCs.

Introduction: Significant limitations with existing treatments for major haemoglobinopathies motivate the development of effective intrauterine therapy. We assessed the feasibility of fetoscopic and ultrasound-guided intrauterine haemopoietic cell transplantation (IUHCT) in macaque fetuses in early gestation when haemopoietic and immunological ontogeny is anticipated to enable long-term donor cell engraftment., Material and Methods: Fluorescent-labelled bone marrow-derived mononuclear cells from 10 pregnant Macaca fascicularis were injected into their fetuses at E71-114 (18.9-170.0E+6 cells/fetus) by fetoscopic intravenous (n = 7) or ultrasound (US)-guided intracardiac injections, with sacrifice at 24 h to examine donor-cell distribution., Results: Operating times ranged from 35 to 118 min. Chorionic membrane tenting and intrachorionic haemorrhage were observed only with fetoscopy (n = 2). Labelled cells were stereoscopically visualised in lung, spleen, liver, and placenta. Donor-cell chimerism was highest in liver, spleen, and heart by flow cytometry, placenta by unique polymorphism qPCR, and was undetected in blood. Chimerism was 2-3 log-fold lower in individual organs by qPCR than by flow cytometry., Discussion: Both fetoscopic and US-guided IUHCT were technically feasible, but fetoscopy caused more intraoperative complications in our pilot series. The discrepancy in chimerism detection predicts the challenges in long-term surveillance of donor-cell chimerism. Further studies of long-term outcomes in the non-human primate are valuable for the development of clinical protocols for IUHCT., (© 2019 S. Karger AG, Basel.)

Objective: Human primitive erythroblasts produced during early embryogenesis have been found in maternal circulation at early gestation and are considered good target cells for noninvasive prenatal diagnosis. We aimed to gain a better understanding of the biology of primitive erythroblasts and maximize their potential utility for noninvasive prenatal diagnosis., Methods: Cells were obtained from first trimester human placental tissues. Biological properties including surface antigen composition, differentiation, proliferation, enucleation, and degeneration were studied as gestation progressed. A microdroplet culture system was developed to observe the behavior of these cells in vitro., Results: Histology showed that primitive erythroblasts undergo maturation from polychromatic to orthochromatic erythroblasts and can differentiate spontaneously in vitro. Cell surface markers and nuclear gene expression suggest that the cells do not possess stemness properties, despite being primitive in nature. They have limited proliferative activity and highly deacetylated chromatin, but a microdroplet culture system can prolong their viability under normoxic conditions. No apoptosis was seen by 11 weeks' gestation, and there was no enucleation in vitro., Conclusion: These properties confirm that viable cells with intact nuclei can be obtained at very early gestation for genetic analysis., (© 2018 John Wiley & Sons, Ltd.)

Polyethylene glycol-linked multiwalled carbon nanotube-coated poly-acrylamide hydrogel (CNT-PA) was customized to mimic human liver stiffness and nanostructured surface in liver cells for modulating differentiation of human amniotic epithelial cells (hAECs) into functional hepatocyte-like cells (HLCs) in vitro. This composite of CNT-PA matrix enhanced the hepatic differentiation of hAECs into HLCs with suppression of pluripotent markers and up-regulation of hepatic markers at both transcript and protein levels. Furthermore, the HLCs on CNT-PA demonstrated hepatocytic functions in terms of albumin secretion, higher uptake of indocyanine green, and comparable CYP3A4 enzymatic function and inducibility when matched against HepG2 cells. Taken together, CNT-PA provides an efficient and scalable platform for the expansion of HLCs from hAECs and could be explored further for downstream development., (Copyright © 2018 John Wiley & Sons, Ltd.)

Major hemoglobinopathies place tremendous strain on global resources. Intrauterine hemopoietic cell transplantation (IUHCT) and gene transfer (IUGT) can potentially reduce perinatal morbidities with greater efficacy than postnatal therapy alone. We performed both procedures in the thalassemic HbbTh3/+ mouse. Intraperitoneal delivery of co-isogenic cells at embryonic days13-14 produced dose-dependent chimerism. High-dose adult bone marrow (BM) cells maintained 0.2-3.1% chimerism over ~24 weeks and treated heterozygotes (HET) demonstrated higher chimerism than wild-type (WT) pups (1.6% vs. 0.7%). Fetalliver (FL) cells produced higher chimerism than BM when transplanted at thesame doses, maintaining 1.8-2.4% chimerism over ~32 weeks. We boosted transplanted mice postnatally with BM cells after busulfan conditioning. Engraftment was maintained at >1% only in chimeras. IUHCT-treated nonchimeras and non-IUHCT mice showed microchimerism or no chimerism. Improved engraftment was observed with a higher initial chimerism, in HET mice and with the addition of fludarabine. Chimeric HET mice expressed 2.2-15.1% engraftment with eventual decline at 24 weeks (vs. <1% in nonchimeras) and demonstrated improved hematological indices and smaller spleens compared with untreated HETmice. Intravenous delivery of GLOBE lentiviral-vector expressing human β-globin (HBB) resulted in a vector concentration of 0.001-0.6 copies/cell. Most hematological indices were higher in treated than untreated HET mice, including hemoglobin and mean corpuscular volume, but were still lower than in WT. Therefore, direct IUGT and IUHCT strategies can be used to achieve hematological improvement but require further dose optimization. IUHCT will be useful combined with postnatal transplantation to further enhance engraftment., (Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome (TTTS), but is technically challenging and can lead to significant complications. Acquiring and maintaining the necessary surgical skills require consistent practice, a critical caseload, and time. Training on realistic surgical simulators can potentially shorten this steep learning curve and enables several proceduralists to acquire procedure-specific skills simultaneously. Here we describe realistic simulators designed to allow the user familiarity with the equipment and specific steps required in the surgical treatment of TTTS, including fetoscopic handling, approaches to anterior and posterior placenta, recognition of anastomoses, and efficient coagulation of vessels. We describe the skills that are especially important in conducting placental laser coagulation that the surgeon can practice on the model and apply in a clinical case. These models can be adapted easily depending on the availability of materials and require standard fetoscopy equipment. Such training systems are complementary to traditional surgical apprenticeships and can be useful aids for fetal medicine units that provide this clinical service.

Objective: To evaluate whether targeted sequencing and relative mutation dosage can be used to diagnose correctly inheritance of maternal β-thalassaemia mutations in cell-free DNA., Design: Feasibility study using samples collected in a prenatal clinic., Setting: South East Asia., Population: Couples where both partners were known to be carriers of one of four common β-thalassaemia mutations or an HbE mutation, and therefore at risk of carrying a fetus affected with β-thalassaemia., Methods: 49 samples previously identified as having inherited a paternal β-thalassaemia mutation were amplified using nested polymerase chain reaction (PCR), and then sequencing. Relative mutation dosage was used to classify the fetus as having inherited the wild-type or mutant maternal allele., Main Outcome Measures: Classification of the fetus as 'unaffected' (if the maternal wild-type allele was inherited) or 'affected' with β-thalassaemia (if the maternal mutant allele was inherited)., Results: A classification for inheritance of maternal allele was obtained in 48/49 samples (98.0%). A concordant call was made in 44/48 cases (91.7%): one false-positive and three false-negatives were obtained. Thus, we had an overall sensitivity of 87.5% [95% confidence interval (CI) 67.6-97.3%] and a specificity of 95.8% (95% CI 78.9-99.9%) for inheritance of maternal genotype., Conclusions: RMD for detection of inheritance of maternal β-thalassaemia mutations has potential for clinical use. Our sequential approach could be applied to other single-gene disorders., Tweetable Abstract: NIPT for β-thalassaemia achieved using nested-PCR followed by relative mutation dosage., (© 2017 Royal College of Obstetricians and Gynaecologists.)

Objective: To investigate haptoglobin within ovarian cyst fluid (OCF) as a diagnostic biomarker for epithelial ovarian cancer (EOC) and develop an in vitro diagnostic point-of-care device test (IVDPCT) for use in the operating theatre., Design: Retrospective and prospective cohort study., Setting: South-East Asia., Population: Women with suspicious ovarian cysts., Methods: Proteomic, immunohistochemical and ELISA methods measured haptoglobin in OCF to differentiate benign and EOCs. Diagnostic performance of haptoglobin was compared with CA125, risk malignancy indices (RMI) and frozen section. Blinded validation of the IVDPCT was performed., Main Outcome Measures: Prediction of malignancy., Results: Haptoglobin concentration measured by ELISA was 0.70 ± 0.09 mg/ml in patients with benign cysts (n = 87), 6.22 ± 0.53 mg/ml in early stage-EOC (n = 17), and 6.57 ± 0.65 mg/ml in late stage-EOC (n = 20). Haptoglobin in EOCs was significantly higher than in benign cysts (P < 0.0001). Haptoglobin using rapid colorimetric assay (RCA) on a training set had a sensitivity of 97.3% and a specificity 92.0%, comparable to ELISA and frozen sections. The haptoglobin AUROC curve was 0.999 (95% CI 0.997-1.000) compared with 0.895 (95% CI 0.814-0.977, P < 0.05) for CA125. Haptoglobin performed significantly better than all the RMIs (P < 0.01). Blinded validation studies showed a minor drop in average diagnostic performance (sensitivity 85.2% and specificity 90.5%) compared with the training set. However, when compared with frozen section, haptoglobin was no worse in diagnostic accuracy for malignancy., Conclusion: Haptoglobin was identified as a biomarker for the detection of EOC with potential as a point-of-care diagnostic tool., Tweetable Abstract: Haptoglobin within ovarian cyst fluid: a biomarker for epithelial ovarian cancer and point-of-care diagnostics., (© 2017 Royal College of Obstetricians and Gynaecologists.)

Liver progenitor cells have the potential to repair and regenerate a diseased liver. The success of any translational efforts, however, hinges on thorough understanding of the fate of these cells after transplant, especially in terms of long-term safety and efficacy. Here, we report transplantation of a liver progenitor population isolated from human fetal livers into immune-permissive mice with follow-up up to 36 weeks after transplant. We found that human progenitor cells engraft and differentiate into functional human hepatocytes in the mouse, producing albumin, alpha-1-antitrypsin, and glycogen. They create tight junctions with mouse hepatocytes, with no evidence of cell fusion. Interestingly, they also differentiate into functional endothelial cell and bile duct cells. Transplantation of progenitor cells abrogated carbon tetrachloride-induced fibrosis in recipient mice, with downregulation of procollagen and anti-smooth muscle actin. Paradoxically, the degree of engraftment of human hepatocytes correlated negatively with the anti-fibrotic effect. Progenitor cell expansion was most prominent in cirrhotic animals, and correlated with transcript levels of pro-fibrotic genes. Animals that had resolution of fibrosis had quiescent native progenitor cells in their livers. No evidence of neoplasia was observed, even up to 9 months after transplantation. Human fetal liver progenitor cells successfully attenuate liver fibrosis in mice. They are activated in the setting of liver injury, but become quiescent when injury resolves, mimicking the behavior of de novo progenitor cells. Our data suggest that liver progenitor cells transplanted into injured livers maintain a functional role in the repair and regeneration of the liver. Stem Cells 2018;36:103-113., (© 2017 AlphaMed Press.)

Strategies that induce immunogenic cell death (ICD) or downregulate CD47 or PD-L1 expression have resulted in successful therapeutic options for tumor eradication. Several groups have reported the tumoricidal effects of human umbilical cord Wharton's jelly stem cells (hWJSCs) or its conditioned medium (hWJSC-CM) on certain cancers but the mechanisms have not been elucidated. Since hWJSCs possess immunomodulatory properties, we investigated whether one of the tumoricidal mechanisms was via ICD. We first concentrated hWJSC-CM into a 3 kDa concentrate and then exposed various concentrations of this concentrate to human lymphoma cells to find out which concentration had the greatest tumoricidal effect. We observed that a 500 µg/ml concentration of the concentrate had the greatest inhibitory effect. Thereafter, lymphoma cells were exposed to 500 µg/ml of the hWJSC-CM-3 kDa concentrate and then subjected to analysis for morphology, viability, apoptosis, mitochondrial and endoplasmic reticulum stress, danger associated molecular patterns (DAMP), extracellular HMGB1, CD47 and PD-L1 markers and dendritic cell phenotype. Extensive nuclear chromatin and mitochondrial changes with significantly decreased cell viability and increased apoptosis were observed in the treated lymphoma cells compared to controls. There were also significant increases in the release of DAMPs, extracellular HMGB1 and dendritic cell activation and maturation, with concomitant decreases in CD47 and PD-L1 expression in the treated cells compared to controls. In other ongoing studies we observed increased expression of specific tumor-suppressor molecules (miRNA-146a and miRNA-126, MCP-1, IL-6, IL-8 and IL-12) in hWJSC-CM suggesting that one or more of these molecules may be the modulators of the ICD.