Objective: Pre-eclampsia is a severe placenta related complication of pregnancy and aetiological knowledge, with limited early diagnostic and therapeutic options. Phenotyping of native placental three-dimensional (3D) morphology offers a novel approach to improve our understanding of the functional and structural placental abnormalities underlying this clinical syndrome. The aim of this project was to develop a 3D placental imaging protocol using multiphoton microscopy (MPM) and demonstrate quantifiable imaging targets for phenotyping 3D features of pre-eclampsia. Design: Exploratory pilot study. Setting: Single centre, MUMC. Sample: Formalin fixed placental biopsies from: term control (n=3), pre-eclampsia (n=3), preterm birth (n=2), 2nd trimester placenta (n=1), and intra-uterine growth restriction cases without pre-eclampsia (n=2). Methods: Placental slabs were visualised with MPM. Collagen and cytoplasm (based on inherent signal), and fluorescently stained nuclei and blood vessels, enabled the visualization of villous tissue with subcellular resolution. Segmentation based on pixel classification, deep learning, and clustering algorithms were used to generate quantifiable features. Main outcome measures: Trophoblast arrangement, 3D-villous tree structure, syncytial knots, fibrosis, and 3D-vascular networks were identified as imaging targets. Villous morphology, vascular fraction, vascular network (i.e., branchpoint density and diameter), nuclear density, and knot fraction were quantified to describe placental phenotypes. Results: Pre-eclamptic placentas had disorganized trophoblast arrangement, decreased vascular fraction, and altered vessel diameters, compared to control placentas. The developed 3D-methodology indicated that placental vasculature, syncytial knotting, and villous growth are altered in pre-eclampsia. Conclusion: Our preliminary data demonstrate the potential of the developed quantification method for phenotyping pre-eclampsia, to improve future disease stratification.

Dear Editor, We would like to thank Dr. Lamont and colleagues for their interesting commentary.1 The authors point out very pressing topics that deserve more attention than we could provide in the original manuscript.They expressed concerns in their letter towards the heterogeneity of the data that were used in our meta-analysis. If incidences differ between regions and populations, absolute numbers on colonisation and neonatal infections might not be comparable. However, new policies were implemented in various settings, where differences in outcomes were measured under the same local circumstances. So as rightly mentioned by Lamont et al, data from the US cannot and should not be used as a blueprint for other parts of the world.1 Yet the general trend in developed countries that we observed, might shed some new light on the respective effectiveness of the strategies currently available.

Dear Editor,We read the letter from colleagues Dr. Seedat and Dr. Marshall, commenting on our article, with great interest (1, 2). Their clarifications on the UK National Screening Committee (UK NSC) position are very clear. The UK NSC decided against a general screening since they cannot assess the benefits and harms in the patient populations of women (3) but they could indeed in newborns. The on-going clinical trial (GBS3 Trial; ISRCTN49639731) in the UK will compare the current risk-based strategy to two different screening tests. A lab based culture test at 3 to 5 weeks before anticipated delivery date will use an established microbiological technique [Enriched Culture Medium Testing] to reduce false-negative results and a molecular point of care test at the onset of labour. The latter test reduces the time period between screening and the start of labour. The predictive value of antenatal GBS cultures decreases if the interval between culture and delivery is longer than 5 weeks. The results of the trial will help to determine the appropriate screening technique and the rational use of antibiotics for the prevention of early onset GBS sepsis in newborn babies.Perinatal empirical therapy of newborns at risk for or with suspected EOS represents the main contributor to the use of antibiotics in early life (4). There is growing concern about the effects that unnecessary exposure to antibiotics in the perinatal period may have on the future health of these children (5, 6). Antibiotic-related alterations in the microbiome may have downstream effects on the developing immune system and may increase the risk of allergic, autoimmune, and metabolic diseases (5, 6).Seedat and Marshall state that according to another study, the use of IAP would indeed increase if screening were implemented, and that the portion of women receiving IAP would be ‘low risk women’ who… ‘would not have a neonate with EOGBS in the absence of IAP’ (1). In this statement is embedded the assumption that the currently established risk factors are indeed a good prediction of EOGBS transmission. However, 50% of neonates with early onset sepsis with GBS did not have risk factors. To the contrary, we confirm in our meta-analysis and systematic review that universal screening lowered the incidence of early onset GBS sepsis in newborn whereas risk-based approaches did not (2). This might indicate that although screening is imperfect, risk factors might be worse in predicting EOGBS outcomes.Besides, we found no evidence that the rate of intrapartum antibiotic treatment was different in risk-based screening than in universal screening. Administration of antibiotics in risk-based policies was in our study neither lower nor associated with a reduction in the burden of disease in early onset GBS sepsis (2). We are looking forward to the results of the GBS3 trial since there is a need for unbiased evidence on the appropriate policy. A trial comparing screening with risk-factor based intrapartum antibiotic prophylaxis is hard to conduct in areas that currently have a screening policy. Recruitment of participants is very challenging and a premature stop for futility is very likely. A lot of women might not want a risk-based protocol if screening is already the standard of care or easily available. Therefore, the UK data will be very helpful in guiding the future way.References1. Seedat F, Marshall J. Re: Universal screening versus risk-based protocols for antibiotic prophylaxis during childbirth to prevent early-onset Group B streptococcal disease: A systematic review and meta-analysis. (First comment letter. Reference to be added). BJOG. 2020.2. Hasperhoven GF, Al-Nasiry S, Bekker V, Villamor E, Kramer B. Universal screening versus risk-based protocols for antibiotic prophylaxis during childbirth to prevent early-onset Group B streptococcal disease: a systematic review and meta-analysis. BJOG. 2020. https://doi.org/10.1111/1471-0528.160853. Seedat F, Geppert J, Stinton C, Patterson J, Freeman K, Johnson SA, et al. Universal antenatal screening for group B streptococcus may cause more harm than good. BMJ. 2019;364:l463.4. Achten NB, Klingenberg C, Benitz WE, Stocker M, Schlapbach LJ, Giannoni E, et al. Association of Use of the Neonatal Early-Onset Sepsis Calculator With Reduction in Antibiotic Therapy and Safety: A Systematic Review and Meta-analysis. JAMA Pediatr. 2019.5. Cotten CM. Adverse consequences of neonatal antibiotic exposure. Curr Opin Pediatr. 2016;28(2):141-9.6. Esaiassen E, Fjalstad JW, Juvet LK, van den Anker JN, Klingenberg C. Antibiotic exposure in neonates and early adverse outcomes: a systematic review and meta-analysis. J Antimicrob Chemother. 2017;72(7):1858-70.