Within sample (alpha) diversity

Within sample, alpha diversity can be quantified using a number of indices. These tend to calculate a score based on the total number of species/OTUs present within a sample (richness), and, sometimes, evenness of read distributions between these groups. Table 6.15 summarises estimates of Richness and Shannon indices across a subset of the whole cohort, rarefied to 1000 reads and averaged over 100 iterations. A total of 269/321 samples were retained after rarefaction. The mean number of OTUs in any one sample was 48.5. As discussed above, this is likely to be an inflation of the true species richness within samples. This potential inflation is a result of errors in the experimental and computational processes that create these sequencing cohorts, which serve to introduce false variation into the nucleotide sequences.

Table 6.15 – Summary of Shannon diversity and total richness estimates for whole cohort.

Index Mean (SD) Median (IQR) Range

Shannon index 2.3 (0.95) 2.5 (1.7-3.0) 0.05-4.5

Total richness 48.5 (25.4) 43.6 (31.7-48.5) 5.1-169.2

As demonstrated in Figure 6.7, placental infection within certain sPTB samples appeared to be driven by growth of a single organism. It follows that placenta with more significant infections, as reflected by higher bacterial loads, may then have lower alpha diversity measures. To explore this, total bacterial load (see Chapter 5), was plotted against the Shannon index for each individual sample. The Shannon index increases with the increasing evenness and richness of a community.

Figure 6.9 shows that as 16S load increases, mean Shannon index decreases. This implies that a larger bacterial presence in placental tissue is associated with a reduction in alpha diversity. This reduction in diversity is both in terms of evenness and richness. This scenario could be interpreted as an expansion of one organism at the expense of many others. When a linear regression model is fitted to this relationship, for every 1 increase in 16S copy number, there is a -0.24 reduction in Shannon index (P=0.00017). A similar, significant observation was noted when Simpsons index was used instead of Shannon in this model (data not shown). No relationship was observed when mean richness was used instead of the Shannon value.

Figure 6.9 – The mean Shannon index reduces with increasing bacterial load in placental tissue from a rarefied cohort.

6.3.6.1 Alpha diversity and pregnancy outcome

To investigate associations between alpha diversity and pregnancy outcome, multi- level regression models were run, with participant as a random intercept. If a significant proportion of infectious mediated preterm births within the cohort were driven by overgrowth of a single, or a small number of species, it would be reasonable to hypothesise that sPTB may be associated with a reduction in alpha diversity. However, no significant difference was observed in the mean Shannon diversity index between sPTB and nsPTB (P=0.53, adjusted model), or between sPTB and term (P=0.14, adjusted model) placenta, in univariate analyses, as well as those adjusted for the potential effects of delivery method, ethnicity, BMI, smoking, and tissue type. There was also no association observed when mean richness was the outcome (P=0.29, P=0.39, for adjusted comparisons between sPTB and nsPTB or term placenta, respectively). Delivery method was not associated with any difference in Shannon diversity (P=0.96, adjusted model) or total richness (P=0.1, adjusted model).

6.4 Discussion

This study has shown that bacterial DNA from a wide variety of organisms was present in the placenta of both normal and complicated pregnancies. In order to understand the clinical significance of such observations, identifying where the sequenced DNA originates from is critical. This will facilitate the development of clinical interventions to mitigate the adverse impacts of infection during pregnancy. From the analyses conducted in this chapter, it is clear that delivery method contributed significantly to the taxonomic composition of the sequenced placental samples. Nevertheless, a number of organisms were clearly enriched within sPTB placental samples, independently of mode of delivery.

Analyses focusing on diversity metrics also identified some associations of interest. Shannon diversity was negatively associated with bacterial load, implying that those placental samples that were most ‘infected’, were also less diverse and less evenly colonised. This perhaps represents overgrowth of a single or small number of species in a small number of extreme cases. Despite this, alpha diversity did not associate with pregnancy outcome specifically, and beta diversity measures differed minimally between groups. These observations together, imply that it is largely in the presence of single or small numbers of organisms of interest that risk of sPTB is increased. Evidence for the existence of a shared ‘preterm placental microbiome’ was less conclusive. A summary of the key findings from this chapter are as follows:

1. The majority of organisms recovered from the placenta were assigned to five phyla:

Firmicutes, Proteobacteria, Tenericutes, Bacteroidetes and Actinobacteria.

2. Relative abundances of some of the most prevalent genera varied by delivery method. Vaginally delivered placenta were dominated by Lactobacilli spp. and CS deliveries by common skin commensals.

3. A number of OTUs and genera were consistently enriched in sPTB placenta, some of which are well known organisms hypothesised to be involved in sPTB, although few reached statistical significance when adjusted for multiple testing.

4. For some enriched organisms, the associations appear to be largely driven by highly infected, ‘outlying’ placental infections of single pregnancies.

5. Using three measures of beta diversity, the placental microbiome did not cluster by outcome group. Although statistical tests of difference were significant, the proportion of variance accounted for by the model was very low (<2%) and thus assumed to be of limited clinical significance.

6. With increasing bacterial load, evenness and richness was reduced within samples, as quantified by the Shannon index.

7. Alpha diversity did not associate with the pregnancy outcomes of interest.

Aagaard et al (2014) described a somewhat similar high-level taxonomic community in their microbiome analysis of placental parenchyma samples as those found in this study. Three of the top four most abundant phyla in their study were in the top five most abundant in this cohort (Bacteroidetes, Firmicutes, and Tenericutes). The five most abundant phyla found in our cohort contain many well-known human commensals, from the gut, to the skin, oral, and vaginal microbiomes (Huttenhower et al., 2012).

Many of the organisms recovered from our placental samples are also known vaginal and skin commensals, such as Lactobacilli and Corynebacterium spp. respectively. It was shown that some of these most abundant organisms did associate strongly with delivery method. These patterns were also observed in parenchyma tissue, which was collected from beneath the protective fetal membrane. Similar observations were drawn from villous samples that were analysed separately. Given the hypothesis that at least some intra-uterine colonisation will be the result of ascending vaginal infection, it would be overly conservative to label all vaginal species, or indeed all skin-associated organisms, as delivery contaminants. Indeed, none of the delivery-associated organisms were exclusive to one or other delivery type. However, these associations do make it difficult to describe conclusively a placental microbiome, which is distinct from the ‘delivery microbiome’. Further, the observation of a low-level, relatively diverse placental microbial signature is supported by other recent molecular studies working in the same tissues (Doyle et al., 2014, Jones et al., 2009, Aagaard et al., 2014).

The most powerful observations from this cohort surround comparisons of normalised abundances of OTU and genus level counts, between sPTB and other pregnancy

these associations survived conservative cut-offs following multiple correction. Some, such as Mycoplasma, Ureaplasma, and Fusobacteria spp., have been known as opportunistic intra-uterine pathogens for decades (Combs et al., 2014, DiGiulio, 2012, Jones et al., 2009, Hitti et al., 1997). Others, such as Capnocytophaga, are less well studied with respect to sPTB pathogenesis. Interestingly, the organisms that associated with sPTB in the Aagaard et al. (2014) study, such as Burkholderia spp., did not overlap with those found here. A smaller study describing the placental membrane microbiome from preterm and term pregnancies reported results that were much more similar to our own (Doyle et al., 2014). For example Fusobacteria, Mycoplasma, and Ureaplasma spp. were also enriched in their vaginally delivered sPTB samples, when compared to term placenta.

There was a notable prevalence of oral bacteria in the organisms enriched in sPTB placenta within this cohort. These associations contribute to a growing body of research supporting a link between organisms isolated from the placental and the oral cavities. These associations are supported by both human and animal data. Han et al. (2004) injected F. nucleatum into the tail of pregnant mice, ultimately inducing preterm delivery and fetal loss. These live F. nucleatum cells were found to have proliferated within uterine tissues examined from these animals, but they did not persist in other organs such as the spleen or liver. As previously discussed, Aagaard et al. (2014) also reported a significant overlap of their data with the oral rather than vaginal HMP dataset. Indeed, from our data it was clear that at least one sPTB placenta was significantly infected with Fusobacterium spp. This was a relatively early, 29th _week delivery by CS, in which the infant was admitted to intensive care. This type of single organism overgrowth, in which a very large proportion of reads mapped to a single genus, was also observed in at least two other instances for Mycoplasma hominis and, curiously, OTUs mapping to the Erwinia genus. Erwinia spp. were present in four placental samples from the same pregnancy (two technical replicates of parenchyma, one more parenchyma biological replicate, and one villous sample) at between 86 and 89% abundance. The abundances were very similar between all four samples from the same pregnancy, demonstrating the robustness of this observation. Furthermore, the placenta was taken from a very early vaginal birth at 24 weeks: this infant also went to the NICU. Erwinia species have not been previously associated with sPTB. Indeed it is a genus of bacteria containing mostly plant pathogens, which makes this observation somewhat unexpected. However, it has previously been isolated and cultured from

meconium samples from a number of very preterm neonates (Moles et al., 2013). This unexpected but interesting association therefore requires further exploration.

Capnocytophaga, was present across a number of samples at a relatively low total

abundance, when compared to genera such as Mycoplasma. However, it was one of the two genera that remained significantly associated with sPTB placenta following adjustment for multiple testing. This anaerobic organism is usually isolated from the oral cavity, and rarely the genital tract. Capnocytophaga infections tend to be most commonly reported in immunocompromised children and are known to be involved in periodontitis (Campbell and Edwards, 1991). The genus has been previously associated with intra-uterine infection in a handful of publications (Douvier et al., 1999, Lopez et al., 2010b, Hill, 1998). In a recent review of the literature, Lopez et al. (2010b) identified a total of 19 reported cases of Capnocytophaga infection of the amniotic cavity and/or a preterm infant and concluded that:

‘Infection with Capnocytophaga sp. is therefore probably an underestimated

aetiology of occult chorioamnionitis and preterm delivery leading to neonatal

infection.’

Capnocytophaga’s occasional, but independently reported, associations with adverse

pregnancy outcomes, as well as its status as an oral opportunistic pathogen, provide support for its association with sPTB in our cohort. Furthermore, it is often in immunocompromised patients that infections of this organism take hold. This is interesting, considering the immuno-suppressed state of pregnancy. Taken together, this may well be a promising and underappreciated candidate for placental mediated sPTB pathogenesis. Capnocytophaga was also isolated from one nsPTB placenta in our cohort. The infant associated with this placenta was delivered by CS at 29 weeks. It was significantly growth restricted, with a birthweight of only 634g, and was admitted to intensive care. This mother had previously delivered another preterm infant. Whether this Capnocytophaga infection contributed in some way to the fetal growth of this infant is worthy of further exploration.

The significant enrichment of Tepidimonas within sPTB versus term placenta, and its particularly high log2 fold change value, is perhaps the most enigmatic result from these analyses. Over 12,000 reads mapped to 5 Tepidimonas labelled OTUs. This was

case, where there was a bone marrow infection in a leukemia patient (Ko et al., 2005). This lack of previous human associated evidence questions the validity of the widespread presence of this genus in our cohort. This genus was first described as an isolate from a hot spring in central Portugal (Moreira et al., 2000). Thus this observation may be a false positive association between outcome and a contaminating genus that is specific to our cohort.

Bacteroides, Peptoniphilus, Streptococcus, and Suterella species were some of the most significantly enriched genera and OTUs within our sPTB cohort. These genera have also previously been associated with preterm birth (Combs et al., 2014, Wang et al., 2013, Hitti et al., 1997). In one sample with a particularly high Peptoniphilus abundance, reads mapping to Sneathia, another well-known sPTB associated organism, made up over 25% of total reads. Some genera, such as Mogibacterium and Finegoldia contain oral and vaginal species respectively, with the former notably associated with periodontitis and the latter with BV (Nakazawa et al., 2000, MacIntyre et al., 2015). These initial associations require further exploration for validation.

These data support the notion that certain bacteria, when present in the placenta, may contribute to an inflammatory response and a subsequent preterm delivery. However, the data do not support the existence of a ‘unique preterm microbiome’ in terms of a structured community, which is shared amongst this particular obstetric group, and distinguishable from other outcome groups. This interpretation is supported both by the graphical ordination of the beta diversity data, and the quantitative statistical tests that were conducted. Although the P-value of all adonis tests could lead us to reject the null hypothesis of no difference in community structure between groups, only around 2% of variation in the beta diversity matrices was ever accounted for by these groupings. This is a very small proportion and unlikely to be of clinical relevance. Given the data at hand, it is reasonable to assert that the high-level community structure of the placental microbiome in this cohort is not significantly associated with pregnancy outcome.

There were a number of limitations to this study. The first was that we were unable to sequence the whole cohort for which qPCR data were available, necessarily limiting power to identify all associations of interest. This will hopefully be addressed in the future when more resources become available. Despite this, our study provides one of the largest cohorts of 16S sequenced placental tissue from sPTBs in the literature.

Furthermore, some of these cases were delivered at extremely low GAs, making this a particularly valuable and rare sample set.

A further limitation of this study was that some clinical categories of interest, such as delivery method or maternal smoking status, were incomplete. This limited the size of some of the final adjusted models. It would also have been of interest to examine the association of microbiome profiles with the well-known clinical phenotype of chorioamnionitis, but this data was unavailable in the BBB clinical records. In the future it would also be of great interest to collect parallel samples from other maternal body sites. This could help to identify the origins of organisms of interest.

Sequencing a tissue of low biomass is a challenge for reasons discussed throughout this PhD. Those microbiome studies focusing on tissues of high biomass, such as the vagina, oral cavity, or intestine, produce 16S sequencing outputs of even and high per-sample read depth. This was not the case for our placental cohort. Although for some samples very large numbers of reads were procured, the likely underlying variation in starting material within this tissue meant that normalisation of template amount was a particular challenge for this cohort. As a result, very few reads were produced for certain samples and there was a very wide range of sequencing depths across the cohort. This presented challenges for statistical interpretation of the data, which were met via normalisation methods originally designed for RNA-seq and micro-array data. Additionally, 73 samples (19%) were discarded during the filtering process, as their read depth, following filtering, was not considered sufficient to be representative of the entire diversity of the population sampled. The development of techniques, some of which are already starting to be used in which microbial DNA enrichment precedes sequencing, will be a powerful improvement in future studies.

The dissection and extraction procedures for this study were carried out using rigorously controlled, sterile procedures. However, the samples were not originally collected with the intention to be used in microbial analyses. Placenta were not collected under strictly sterile conditions and it is acknowledged that this may limit the reliability of observations noted here. Similarly, the storage and cleaning reagents,

RNAlater and PBS, which were used by the collection team were not available to use as

However, adjustment in final models for hospital collection site, will have accounted for at least some site-specific contamination patterns.

The specific study of the placental microbiome remains in its infancy. This research and other similar data suggest that there may be a low-level non-pathogenic placental microbiome present in many, if not all, placenta. However, differentiating this from organisms picked up at delivery, or during experimental handling is an on-going challenge. A recent publication examining placental tissue from healthy pregnancy concluded that these signals could not be differentiated in their cohort when rigorous contamination controls were used (Lauder et al., 2016). Furthermore, it remains impossible to assert from our data whether those organisms identified from placental samples functioned as mutualists that directly promoted the health of the developing fetus, or were commensal, and essentially neutral in their role. Metagenomic analyses, in which gene level information is retrieved, will serve to enlighten the functional role of this community in the future, in a way that 16S analyses cannot.

To summarise, these data show that sPTB is associated with the placental presence of some well-known and some novel genera of interest. Some of these sPTB cases appear to have involved infectious triggers originating from the overgrowth of a single, or small number of, organism(s), originating from the vaginal or oral cavities. This study gathered novel data from a tissue that remains largely unexplored from an unbiased microbiological perspective. The cohort consisted of a large number of spontaneous, early preterm births, for which a large number of biological replicates were also available. This improved the power of the study to detect colonisation patterns of interest. Furthermore, the use of a specifically defined ‘non-spontaneous’ preterm birth group was a novel addition to previous studies. These nsPTB placenta provided a comparison group that was essentially matched for GA with the sPTB cases, but very