Although receptive to follow-up, the situational factors of being a new mother need to be taken into account to engage successfully with this patient group. Further research is needed to help clarify the extent to which a history of preeclampsia is an independent factor for future cardiovascular disease to provide a solid foundation for effective risk communication.

In MCMA twins surviving beyond 24 weeks of gestation, there was a higher survival rate compared to previous decades presumably due to early diagnosis, close surveillance and elective birth around 32-34 weeks of gestation. High perinatal mortality at early gestations was mainly attributed to extreme prematurity due to preterm spontaneous labour. This article is protected by copyright. All rights reserved.

It is widely debated whether fetal membranes possess a genuine microbiome, and if bacterial presence and load is linked to inflammation. Chorioamnionitis is an inflammation of the fetal membranes. This research focussed on inflammatory diagnosed histological chorioamnionitis (HCA) and aims to determine whether the bacterial load in fetal membranes correlates to inflammatory response, including histological staging and inflammatory markers in HCA. Methods Fetal membrane samples were collected from patients with preterm spontaneous labour and histologically phenotyped chorioamnionitis (HCA; n=12), or preterm (n=6) and term labour without HCA (n=6). The bacterial profile of fetal membranes was analysed by sequencing the V4 region of the 16S rRNA gene. Bacterial load was determined using qPCR copy number/mg of tissue. The association between bacterial load and bacterial profile composition was assessed using correlation analysis. Results Bacterial load was significantly greater within HCA amnion (p=0.002) and chorion (p=0.042), compared to preterm birth without HCA. Increased bacterial load was positively correlated with increased histological staging (p=0.001) and the expression of five inflammatory markers; IL8, TLR1, TLR2, LY96 and IRAK2 (p=<0.050). Bacterial profiles were significantly different between membranes with and without HCA in amnion (p=0.012) and chorion (p=0.001), but no differences between specific genera were detected. Discussion 4 Inflammatory HCA is associated with infection and increased bacterial load in a dose response relationship. Bacterial load is positively correlated with HCA severity and the TLR signalling pathway. Further research should investigate the bacterial load threshold required to generate an inflammatory response in HCA. Short title: Fetal membrane bacterial load is increased in HCA Highlights:-Increased bacterial load was significantly associated with inflammation-Bacterial load is correlated with HCA severity in a dose dependent manner-Bacterial load is correlated to the TLR signalling pathway-Non-HCA samples and negative controls are not distinct in bacterial load.

Histological chorioamnionitis (HCA) is an established marker of ascending infection, a major cause of preterm birth. No studies have characterised the global change in expression of genes involved in the toll-like receptor (TLR) signalling pathways in the presence of HCA in the setting of preterm birth (pHCA). Fetal membranes were collected immediately after delivery and underwent histological staging for inflammation to derive 3 groups; term spontaneous labour without HCA (n = 9), preterm birth <34 weeks gestation without HCA (n = 8) and pHCA <34 weeks (n = 12). Profiling arrays ran in triplicate for each group were used to determine the expression of 84 genes associated with TLR signalling and screen for genes of interest (fold change >2; p<0.1). Expression of identified genes was validated individually for all samples, relative to GAPDH, using RT-PCR. Expression of TLR 1, TLR 2, lymphocyte antigen 96, interleukin 8 and Interleukin-1 receptor-associated kinase-like 2 was increased in pHCA (p<0.05). Degree of expression was positively associated with histological staging of both maternal and fetal inflammation (p<0.05). The inflammatory expression profile at the maternal/fetal interface associated with pHCA, a reflection of ascending infection, is extremely heterogeneous suggesting polymicrobial involvement with activation of a common pathway. Antagonism of TLR 1 and TLR 2 signalling in this setting warrants further assessment.