The availability of these techniques, in combination with developments in phylogenetic methodology, provides the potential for better analysis of microbial niche specialisation. This study aimed to develop an approach for sequencing of bacterial and archaeal amoA genes amplified from soil using bioinformatics pipelines developed for general analysis of functional genes and employed sequence data to reassess phylogeny and niche specialisation in terrestrial bacterial ammonia oxidisers.Characterisation of microbial communities increasingly involves use of high throughput sequencing methods (e.g. MiSeq Illumina) that amplify relatively short sequences of 16S rRNA or functional genes, the latter including ammonia monooxygenase subunit A . The pipeline was used to provide an up-to-date phylogenetic analysis of terrestrial betaproteobacterial The online version of this article (10.1186/s40793-019-0342-6) contains supplementary material, which is available to authorized users. Nitrosospira phylogeny is required using more advanced phylogenetic methods, as this genus dominates soil AOB communities.Despite advances in our understanding of niche specialisation of ammonia oxidisers during the past 20 years, the successive discoveries of archaeal ammonia oxidisers (AOA)  and compNitrosospira in nitrous oxide emissions [Increased phylogenetic resolution improves the ability to assess microbial niche specialisation, at least in organisms whose phylogenetic structure reflects their ecological niche . Severalmissions , 9 justimissions , 5, by uamoA) gene targets an ecosystem function, ammonia oxidation, which is the rate-limiting step in nitrification, a key process in biogeochemical cycling of nitrogen. The amoA gene has been extensively used to estimate the abundance and diversity of bacterial (AOB) and archaeal ammonia oxidisers (AOA) and has provided evidence for their high phylogenetic diversity in natural environments, including soil [amoA gene was chosen as the optimal functional gene to analyse soil AOB diversity and two sets of primers have been mainly employed in terrestrial environments, amoA-1F/amoA-2R [amoA genes, respectively. Developments in high-throughput sequencing (HTS) technologies have greatly increased our ability to characterise natural microbial communities, through significant increases in the depth and accuracy of sequencing of genes of interest, amplified from environmental DNA  to improve the analysis of niche specialisation of terrestrial Nitrosospira to clarify their ecological adaptation and test the extent to which pH is a key factor in their ecological distribution; and iii) to provide an Illumina MiSeq V3 sequencing approach for environmental analysis of the ammonia monooxygenase subunit A (amoA) gene present in both bacterial and archaeal ammonia oxidisers, applicable to other functional genes.The aims of this study were, therefore, i) to revisit the amoA gene Illumina MiSeq sequencing, respectively, on 47 and 33 UK soils for which environmental data were available. In addition, 16S rRNA and amoA genes retrieved from 56 available Nitrosospira strains were used for phylogenetic reconstructions and assessment of phylogenetic congruency between them.The aims of the study were addressed by performing archaeal and bacterial Nitrosomonas and Nitrosospira [Nitrosolobus [Nitrosovibrio [Nitrosospira genus, which dominates betaproteobacterial ammonia oxidiser communities in soil. The 16S rRNA gene sequences from cultivated Nitrosospira isolates and those amplified from environmental samples have previously been classified within seven lineages, for which phylogenetic node support was not high [Nitrosospira strains , while support for more ancestral phylogenetic branching was supported for most of the nodes in both the 16S rRNA gene and the amoA gene phylogenies, even if some paraphyletic branching could not be resolved , it provides evidence for the existence of clear and distinct lineages using both marker genes of cultivated strains  of the environmental sequences obtained from the 33 soils amplified using bacterial primers affiliated to the ronments . These aamoA gene sequences were made using both OTU richness (at 100% sequence similarity) and microbial community composition for archaea and bacteria independently using the different sequencing approaches, 454 (only available for AOA and previously described in [amoA amplicons (490-bp), due to overlapping of the paired-end sequences. Rarefied richness was similar or higher for the ‘gap’ pipeline than the ‘assembly’ pipeline, due to the higher restrictive size selection in the latter , indicated that deletion of the central archaeal amoA gene region did not impact significantly on estimated archaeal community composition . These findings suggest that Illumina MiSeq sequencing can be successfully used to provide a good characterisation of AOA and AOB amoA amplicon sequences to infer their community structure. The sequencing bioinformatics pipelines presented here are freely available on GitHub (https://github.com/AigleAxel/amoA_MiSeq_sequencing/) allowing their implementation for other functional genes of interest. Specific advantages and associated limitations of each bioinformatics pipeline (e.g. high recovered diversity for the ‘gap’ pipeline and high confidence of sequence phylogenetic affiliation for the ‘assembly’ pipeline) indicate the requirement for thorough comparison of approaches for cleaning of sequencing data for any novel analysed gene.Comparisons of the diversity of environmental https://github.com/AigleAxel/amoA_MiSeq_sequencing/). This tool will be useful to the researchers interested in diverse microbiome communities, especially those working on functional genes of interest larger than 500 bp. The developed pipeline was used to provide a revisited and up-to-date phylogenetic analysis of terrestrial betaproteobacterial ammonia oxidiser amoA genes and further analysis provided evidence for soil pH as a key ecological factor controlling the niche specialisation of those microbes.This study offers an optimal bioinformatics pipeline for high-throughput sequencing of functional genes, adaptable to any amplicon size and freely available on GitHub  and from 7 soil samples from long-term experimental field plots maintained for more than 60 years at a range of soil pH values . Soils were selected to include a wide range of characteristics  and PCR-indexing was performed using the Nextera XT Index Kit according to the manufacturer’s protocol. Following further cleaning, library quantification, normalisation and pooling of 144 samples per sequencing run were performed prior to paired-end V3 MiSeq sequencing, enabling production of 2 × 300-bp reads.amoA genes generated by Illumina MiSeq sequencing necessitated use of two read assembly strategies. The first, with overlapping reads and termed the ‘assembly’ pipeline, was used for bacterial amoA only. The second, with non-overlapping reads and termed the ‘gap’ pipeline, was used for both archaeal and bacterial amoA. For the ‘assembly’ pipeline, reads were demultiplexed using NextGENe software and, for each sample, paired-end raw reads were trimmed (−- paired) with very low quality filtration (−q 15) using Trim Galore  and fur, as a compromise between selection of high-quality reads  and conservation of high-quality information (carried by higher number of nucleotides) to maximise the output number of reads. The reverse reads were reverse-complemented and concatenated with the forward reads (instead of being assembled via PEAR). The following steps  were the same as in the ‘assembly’ pipeline.amoA databases, respectively) and the proportions of sequences affiliated to each phylogenetic cluster within each soil sample were represented in a heatmap. As the two different bioinformatics pipelines produced AOB sequences of different lengths, the community dissimilarity between full-length ‘assembly’ and ‘assembly-gapped’ sequences (i.e. ‘assembly’ sequences for which the corresponding gap region of the ‘gap’ pipeline was deleted) were tested on the AOB Illumina MiSeq dataset. Similarly, the significance of the deleted sequence produced in the ‘gap’ AOA Illumina MiSeq dataset was tested using previously obtained 454 sequences (i.e. 454 full-length sequences vs 454 sequences without the DNA region corresponding to the MiSeq ‘gap’ and termed ‘454-gapped’). Pearson correlation and Bray-Curtis similarity indices were estimated for each soil sample independently using the cor and the vegdist functions from the vegan package on R V3.5.1 [amoA sequences (see details of phylogenetic reconstruction below) by estimating the Euclidian distance between the two amoA trees (built with either full- or truncated-length) using treecompare in the DendroPy library [Comparison of the different sequencing methodologies was performed for the seven Craibstone samples following a blast-assignment of sequences to the different phylogenetic clusters . The amoA and 16S rRNA gene sequences of 56 Nitrosospira strains were recovered from NCBI and JGI databases  and another phylogenetic tree was constructed to assess global diversity in soil. Finally, the dereplicated AOA amoA sequences previously produced on 46 UK soils using 454 were trimmed in the central sequence region (corresponding to the gap region of the MiSeq ‘gap’ pipeline and resulting in 264 bp) and a Bayesian phylogenetic tree was constructed to compare to a previously published phylogenetic tree [Known terrestrial AOB are affiliated to the amoA datasets, any sequence in which a recombination event was detected using at least 3 of 4 methods  implemented in RDP4 software [All sequence datasets were aligned using Mafft  and furtsoftware  was remosoftware ) for eacsoftware  and the amoA and 16S rRNA trees) were constructed using IQ-TREE [amoA trees) by inferring the best-fit substitution model using ModelFinder [http://tree.bio.ed.ac.uk/software/figtree/) and phylogenetic clusters were defined based on strong bootstrap values (> 80% in most cases). Visual assessment of congruence between the two reference trees (amoA vs 16S rRNA gene trees) was performed using phylo.io [All AOB phylogenetic trees  and maximum clade credibility trees from converged MCMC runs were generated using TreeAnnotator version 1.7 [For the AOA ‘454-gapped’ Bayesian phylogenetic tree, the best substitution model per codon position estimated using PartitionFinder  was SYM sion 1.7  after 50sion 1.7  and treesion 1.7 .amoA sequence composition were identified by canonical correspondence analysis followed by permutation tests performed on relative abundance matrices using the vegan package [amoA sample sequence obtained from the 50 soils against the bacterial non-redundant database clustered at different cut-offs , as performed for the archaeal dataset [Environmental factors , moisture content and vegetation type) associated with AOB  package  in R. Re dataset . As pH aAdditional file 1:Figure S1. Full amoA (A) and 16S rRNA (B) gene trees for the bacterial ammonia oxidiser reference sequences. Figure S2. Congruence of amoA and 16S rRNA gene phylogenetic trees for the bacterial ammonia oxidiser reference sequences. Shades of blue indicate similarity between the most common nodes between the two trees. Figure S3. Full bacterial amoA gene tree including the environmental sequences (assembled using the MiSeq ‘Assembly’ pipeline) and the reference sequences. Figure S4. Congruence between two phylogenetic trees of 370 archaeal amoA sequences  with (A) or without (B) the sequence gap corresponding to the MiSeq AOA gap pipeline. Branch colour corresponds to congruence between the two trees. Table S1. Identification of sequences of the 56 terrestrial Nitrosospira AOB strains used in this study. For strains with > 1 copy, only the sequences used in this study presented. n.a. – not applicable. Table S2. Statistical results of the canonical correspondence and permutation analyses performed on the AOB amoA communities clustered at different identity cut-offs. Table S3. Number of sequences and richness of AOA and AOB amoA sequences retrieved in each Craibstone soil sample with different sequencing technologies, with or without rarefaction to the smallest number of sequences obtained in one of the two technologies. Table S4. Characteristics of the 33 UK soils (26 CEH followed by 7 Craibstone soils) used in the multivariate statistics analysis. (DOCX 1560 kb)


Wound infection is a common complication following caesarean section. Factors influencing the risk of infection may include the suture material for skin closure, and closure of the subcutaneous fascia. We assessed the effect of skin closure with absorbable versus non-absorbable suture, and closure versus non-closure of the subcutaneous fascia on risk of wound infection following Caesarean section.Women undergoing caesarean birth at an Adelaide maternity hospital were eligible for recruitment to a randomised trial using a 2 × 2 factorial design. Women were randomised to either closure or non-closure of the subcutaneous fascia and to subcuticular skin closure with an absorbable or non-absorbable suture.Participants were randomised to each of the two interventions into one of 4 possible groups: Group 1 - non-absorbable skin suture and non-closure of the subcutaneous fascia; Group 2 - absorbable skin suture and non-closure of the subcutaneous fascia; Group 3 - non-absorbable skin suture and closure of the subcutaneous fascia; and Group 4 - absorbable skin suture and closure of the subcutaneous fascia.The primary outcomes were reported wound infection and wound haematoma or seroma within the first 30 days after birth.A total of 851 women were recruited and randomised, with 849 women included in the analyses .p = 0.035). There was no significant difference in risk of wound infection for absorbable vs non-absorbable sutures in women who did not undergo fascia closure.In women who underwent fascia closure, there was a statistically significant increase in risk of wound infection within 30 days post-operatively for those who had skin closure with an absorbable suture (Group 4), compared with women who had skin closure with a non-absorbable suture (Group 3) (adjusted RR 2.17; 95% CI 1.05, 4.45; The combination of subcutaneous fascia closure and skin closure with an absorbable suture may be associated with an increased risk of reported wound infection after caesarean section.ACTRN12608000143325, on the 20th March, 2008.Prospectively registered with the Australian and New Zealand Clinical Trials Registry, number  Caesarean section is one of the most commonly performed surgical procedures for women worldwide . Rates oWomen who birth by caesarean section have a 5 to 20 times increased risk of peri-partum infective complications, compared with women who birth vaginally –12. SurgOperative techniques vary widely between surgeons , 17. WhiWe performed a 2 × 2 factorial randomised trial comparing the effects of absorbable versus non-absorbable skin suture, and closure or non-closure of the subcutaneous fascia, on wound infection and complication rates.Single centre 2 × 2 factorial randomised trial, allocated 1:1.The trial was reviewed and approved by the Human Research Ethics Committee of the Women’s and Children’s Hospital, North Adelaide .Eligible women were identified and recruited from a single tertiary metropolitan maternity hospital in Adelaide, South Australia. Women were identified in antenatal clinic, given a trial information sheet and counselled by a researcher, after which written informed consent was obtained. Women who were undergoing a caesarean section via a transverse suprapubic incision were eligible for inclusion. Women with a known lethal fetal anomaly or where the procedure was planned via a midline skin incision were excluded.2 versus > 25.0 kg/m2). Treatment packs were assembled according to the prepared schedule by a researcher not involved with recruitment or clinical care and were opaque and externally identical. Each treatment pack contained a sterile pack of suture material (3/0 absorbable or 3/0 non-absorbable) and a card documenting closure or non-closure of the subcutaneous fascia. The treatment pack was opened by the scrub nurse after delivery of the baby. Instructions were relayed to the operating surgeon, and the suture material opened onto the operating tray. The operating surgeon and participants were unblinded to treatment assignment after opening of the pack. Participant follow up and outcome data collection were performed by investigators not involved in the primary procedure and who were not aware of treatment allocation. Data analysis was performed following a statistical analysis plan finalised prior to analyses being conducted.Randomisation occurred at the time of decision to perform a caesarean section, by taking the next sequentially numbered treatment pack containing the skin suture material and indication of whether or not to perform subcutaneous fascia closure. The computer-generated randomisation schedule used balanced variable blocks, and was prepared by an investigator not involved with recruitment or clinical care of the participants. The randomisation schedule was stratified according to the type of caesarean section (emergency vs elective) and maternal BMI at hospital booking visit (BMI ≤ 25.0 kg/mSkin closure: subcuticular closure of the skin with a 3/0 absorbable (Caprosyn™) or non-absorbable (Prolene™) synthetic monofilament suture materialFascia closure: closure or non-closure of subcutaneous fascia, performed with 2/0 Vicryl® sutureThe two interventions were:Group 1 - non-absorbable skin suture and non-closure of the subcutaneous fascia;Group 2 - absorbable skin suture and non-closure of the subcutaneous fascia;Group 3 - non-absorbable skin suture and closure of the subcutaneous fascia; andGroup 4 - absorbable skin suture and closure of the subcutaneous fascia.Participants were randomised to each of the two interventions into one of 4 possible groups:Wound complications were identified by asking women to complete a questionnaire by telephone. Additionally, hospital case-notes were reviewed at 30 days postpartum, to identify any diagnosis or signs or symptoms of wound complications, readmission to hospital, or presentation to the emergency department.Wound infection reported in first 30 days after operation defined in accordance with CDC definition of superficial incisional surgical site infection , includiWound haematoma or seroma formation, defined as a collection of blood or serous fluid beneath the skin, diagnosed clinically or by ultrasound assessment, in first 30 days after operation .The primary outcomes were:Secondary outcomes included, emergency presentation to health care for wound management, need for surgical intervention including drainage or debridement in theatre or outpatient setting; need for hospital readmission for wound complications in first 30 days after operation (both self-report and case note review), and pain score day three post operative.Allowing for additive treatment effect with the two interventions in a factorial study design, a sample size of 1230 participants was determined on the basis of 80% power to detect a relative reduction of 40% in the incidence of wound infection (from 12%  to 7%), 2 versus > 25 kg/m2), and baseline variables prespecified as potential confounders . All analyses were performed in Stata v16 .Analyses were performed on an intention to treat basis, according to treatment group allocated at randomisation, and followed the Statistical Analysis Plan. Analyses were performed using log binomial regression models for binary outcomes, and linear regression models for continuous outcomes. In the case of outcomes where the number of events was too small for the planned analysis to be performed, a Fisher’s Exact test was performed to test the hypothesis of no association between treatment group and outcome. An interaction term (suture type x fascia method) was included to test for synergistic effects . Separate effect estimates were therefore derived for each treatment effect by level of the other treatment and are presented as RR  and 95% CI for binary outcomes, and differences in means  and 95% CI for continuous outcomes. Both unadjusted and adjusted analyses were performed, with adjusted models including stratification variables  and one who did not undergo a caesarean birth (randomised to Group 2), leaving 849 women for whom primary outcome data was available.2 (SD 7.36 kg/m2). The majority of women underwent an elective caesarean section (701 women [82.57%]), at 38.50 weeks (SD 1.85 weeks) gestation. Most women were in their second or subsequent ongoing pregnancy (656 women [77.27%]), with a previous caesarean birth (562 women [66.20%]).Baseline demographic characteristics of women were comparable between treatment groups Table . The meaWound infection and complication outcomes by group are presented in Table Women who underwent fascia closure and skin closure with a non-absorbable suture (Group 3) had the lowest rate of postoperative wound infection (10 women 4.72%]), while women who underwent fascia closure and skin closure with an absorbable suture had the highest rate of postoperative wound infection (Group 4) (22 women [10.43%]) (Table %), whileThe occurrence of wound haematoma and seroma were very low, with only 4 women (0.47%) in the whole cohort having this complication. Two of these women (0.95%) were in Group 2 (fascia non-closure and absorbable skin suture), with a further woman each in Group 1 (0.47%) (fascia non-closure and non-absorbable skin suture) and Group 4 (0.47%) (fascia closure and absorbable skin suture)  (Table p = 0.984) (Table Among women who underwent fascia closure and had skin closure with an absorbable suture (Group 4), there was a statistically significant increased risk of developing a wound infection in the first 30 days following operation, compared with women who underwent fascia closure and then had skin closure with a non-absorbable suture (Group 3)  (Table p = 0.169) , compared with women who did not have fascia closure (Group 2) , there were too few events to perform modelling on this outcome , is associated with an increased risk of postoperative wound infection, in comparison with subcutaneous fascia closure and skin suturing with non-absorbable monofilament suture (Prolene™). While previous studies have shown no statistically significant difference in wound infection rates when comparing the use of absorbable versus non-absorbable skin sutures –28, the One possible reason for our findings is that the burden of absorbable suture material is related to the increased relative risk of wound infection, among women who underwent subcutaneous fascia closure and skin closure with absorbable suture. Both the suture used for subcutaneous fascia closure (Vicryl®), and the suture used for skin closure (Caprosyn™) are absorbable. Vicryl® is a poly-filament, braided absorbable synthetic suture, which is completely absorbed within 56–70 days . Caprosy2 or more.Potentially at odds with this theory is the observation that women who had skin closure with a non-absorbable suture and did not have closure of the subcutaneous fascia had rates of infection similar to women with fascia closure and absorbable skin sutures. This may be due to an additive effect of two sources of an inflammatory response to foreign material and the possibility that closure of the subcutaneous fascia is somewhat protective from post-operative wound infection, particularly in a population of women where more than 60% have a BMI of 25 kg/mIt is generally acknowledged that routine closure of the subcutaneous fascia is not recommended, having not been shown to reduce the risk of wound infection (NICE guidelines 2015) . ClosureMaternal overweight and obesity is an increasingly common issue in current obstetric practice, with approximately 50% of women across developed countries entering pregnancy overweight or obese , 38–40. A major strength of our study is the large sample size of 851 women randomised. To our knowledge, this represents the largest randomised trial of wound closure techniques published to date. Although we did not attain our pre-specified sample size of 1230 women due to slow recruitment, our results represent a significant addition to the published literature on the effect of different wound and subcutaneous fascia closure techniques on wound infection and complication rates. However, this study remains underpowered for the primary outcome, with a lower than anticipated rate of both wound infection and associated complications rates, and the possibility of synergistic effects between the two interventions, a much larger sample size would be required to properly study these techniques.Our finding of a statistically significant increased relative risk of wound infection among women who had fascia closure and skin closure with an absorbable suture is supported by the direction of the effect estimate towards a lower relative risk of wound infection among women who underwent fascia closure and skin closure with a non-absorbable suture, despite this effect estimate not being statistically significant. Additionally, while there were too few events to allow for effect estimates to be calculated with regards to our secondary outcome of wound reoperation, four out of five women who underwent a wound reoperation had undergone fascia closure and skin closure with an absorbable suture, further supporting our findings.Use of non-absorbable skin suture should be considered at caesarean section, as should consideration of closure of the subcutaneous fascia. These findings may be of significant clinical relevance in light of increasing rates of maternal overweight and obesity. Further research should consider fascia closure or non-closure in women with high BMI and the appropriate skin suture material in this population.


The lung is comprised of more than 40 distinct cell types that support a complex 3-dimensional (3D) architecture that is required for efficient lung function. Loss of this proper architecture can accommodate and promote lung disease, highlighting researchers’ growing need to analyze lung structures in detail. Additionally, in vivo cellular and molecular response to chemical and physical signals, along with the recapitulation of gene-expression patterns, can be lost during the transition from complex 3D tissues to 2D cell culture systems. Therefore, technologies that allow for the investigation of lung function under normal and disease states utilizing the entirety of the lung architecture are required to generate a complete understanding of these processes. Airway cell-derived organoids that can recapitulate lung structure and function ex vivo while being amenable to experimental manipulation, have provided a new and exciting model system to investigate lung biology. In this perspective, we discuss emerging technologies for culturing lung-derived organoids, techniques to visualize organoids using high-resolution microscopy and the resulting information extracted from organoids supporting research focused on lung function and diseases.The online version contains supplementary material available at 10.1007/s00418-020-01955-w. The lung is one of the most complex organs in the human body, comprised of more than 40 cell types that form a unique architecture to enable its primary function of efficient gas exchange  and cytokeratin 5 (KRT5) are stem/progenitor cells that maintain airway homeostasis and the first organoids derived from mouse tracheal basal cells were referred to as tracheospheres  are characterized by the production of pulmonary surfactant proteins, while alveolar epithelial type I cells (AEC1) cover most of the surface area of the alveoli and perform the function of gas exchange. Interestingly, AEC2 cells can form alveolospheres and differentiate into organoid structures that contain both AEC2s and AEC1s , which includes embryonic stem cells and induced pluripotent stem cells (iPSCs). The development of human pluripotent stem cell (hPSC)-derived organoids  make it possible to visualize cellular details as well as overall tissue architecture within a single biological sample. Using organoid cultures derived from tumor bearing mouse lungs, and laser scanning confocal microscopy, a detailed yet heterogenous architecture can be observed in 3D Fig. .Fig. 3LuRecently, a detailed protocol for high-resolution 3D imaging of fixed and cleared organoids was published  Supplementary Video 1: Stepwise transition through 2 µm thick optical sections of tumor bearing mouse derived lung organoids. Note spherical organoids that are an extended protrusion from larger airways.Below is the link to the electronic supplementary material.


Our recent studies, using (SWRxNZB)F1 (SNF1) mice, showed a potential contribution of the gut microbiota and pro-inflammatory immune responses of the gut mucosa to systemic autoimmunity in lupus. Here, using this mouse model, we determined the abundance and the nAg reactivity of IgA antibody produced in the intestine under lupus susceptibility. Intestinal lymphoid tissues from SNF1 mice, females particularly, showed significantly higher frequencies of nAg (dsDNA and nucleohistone) reactive IgA producing B cells compared to B6 females. Most importantly, younger age fecal IgA -abundance and -nAg reactivity of lupus-prone mice showed a positive correlation with eventual systemic autoimmunity and proteinuria onset. Depletion of gut microbiota in SNF1 mice resulted in the diminished production of IgA in the intestine and the nAg reactivity of these antibodies. Overall, these observations show that fecal IgA features, nuclear antigen reactivity particularly, at preclinical stages/in at-risk subjects could be predictive of autoimmune progression. Importantly, women are more predisposed to SLE than men, and the disease prevalence ratio of women is about 9:1 over men2. Autoantibody production and gender bias in SLE is caused by a combination of genetic and environmental factors4. Disproportionate functioning of genes as well as sex hormones, estrogen in particular, contribute to the onset and development of disease activities in SLE8. Recent studies that used human samples and rodent models have shown that gut microbiota composition influences the rate of disease progression and the overall disease outcome15. We have demonstrated that minor dietary deviations alter the composition of gut microbiota and SLE in a mouse model13. We have also found that gut microbiota influences the autoimmune progression differently in lupus-prone male and female mice, leading to a gender bias in disease incidence16.Systemic lupus erythematosus (SLE) is an autoimmune disease which arises when abnormally functioning B lymphocytes, in at risk subjects, produce auto-(self-reactive) antibodies to nuclear antigens such as DNA and proteins. High levels of circulating autoantibodies and immune complex deposition in the kidney, leading to tissue damage and glomerulonephritis are the hallmarks of SLE17. We also showed that pro-inflammatory responses including higher cytokine expression, recruitment of large number of immune cells, and presence of higher number of antibody positive plasma cells in the gut mucosa of lupus-prone females, compared to males, can be detected as early as at juvenile age. These pro-inflammatory immune features of female mouse gut mucosa progressively increase at later ages, prior to systemic autoimmunity and kidney pathology. These observations and reports by others showing the involvement of microbiota in systemic autoimmune progression in lupus19 suggest that autoantibody production and systemic autoimmunity in lupus-prone subjects are initiated in the gut mucosa, microbiota dependently and there is a need for additional studies to assess antibody production in the intestine.Our recent studies that used lupus-prone (SWRxNZB)F1 (SNF1) mice showed a potential contribution of pro-inflammatory immune response initiated in the gut mucosa, and gut microbiota in triggering the disease associated gender bias observed in SLE22. Intriguingly, a recent report showed, in addition to differences in the gut microbiota composition, relatively higher levels of total IgA in stool samples of SLE patients compared to that of healthy controls9. On the other hand, serum IgA levels, but not IgG or IgM levels, were diminished in lupus-prone mice that received oral treatment with Lactobacillus, which suppresses lupus nephrites23. Importantly, anti-DNA antibodies of IgA class are found in the serum of patients with SLE29, suggesting that they may be of gut primed B cell origin. These reports along with our studies17 showing pro-inflammatory immune phenotype and higher plasma cell frequency by lupus-prone female mouse intestine suggests the degree of IgA secretion in the gut lumen could show gender bias and may be indicative of lupus susceptibility and autoimmune progression. Nevertheless, the relationship between fecal IgA levels and gender bias in lupus is unknown. Further, the reactivity of fecal IgA in a lupus-prone background with nuclear antigens and the potential association with disease onset has never been studied.IgA is the most abundant Ig isotype released in to the gut lumen and it plays an important role in the protection against microbial infection as well as in maintaining a healthy gut microbiotaIn the present study, we investigated the degree of IgA production in the intestine, and the abundance and nAg reactivity of fecal IgA in lupus-prone SNF1 mice. We have then assessed the relationship between these features and autoimmune progression in male and female mice, and if the gut microbiota has an influence on fecal IgA abundance and nAg reactivity. Our studies, for the first time, show not only that higher amounts of IgA are produced in the gut mucosa- associated immune tissues of lupus-prone mice, but also these antibodies have significant nAg reactivity, even at juvenile age. Correspondingly, the abundance of IgA is profoundly higher in the feces of lupus-prone mice, females particularly, and these antibodies show significant nAg (dsDNA and nucleohistone) reactivity. These fecal IgA features of pre-clinical stages correlate significantly with the eventual systemic autoantibody levels and the proteinuria onset in lupus-prone mice, suggesting that fecal IgA features could be valuable for predicting systemic autoimmune progression, early on, prior to sero-positivity and lupus nephritis.30 to deplete the gut microbiota. Briefly, female SNF1 mice were given a broad-spectrum antibiotic cocktail  -containing drinking water, starting at week 4 of age. Depletion of gut microbiota was confirmed by the culture of fecal pellet suspension on blood agar and brain heart infusion agar plates, under aerobic and anaerobic conditions, as described before30. Urine and tail vein blood samples were collected at different time-points to detect proteinuria and autoantibodies. All animal experiments were performed according to ethical principles and guidelines were approved by the institutional animal care committee (IACUC) of MUSC. Animal experimental protocol of this study was approved by the IACUC of MUSC.SWR/J (SWR), NZB/BlNJ (NZB), C57BL/6J (B6), and Balb/cJ mice were purchased from the Jackson Laboratory  and housed under specific pathogen free (SPF) conditions at the animal facilities of Medical University of South Carolina (MUSC). (SWRxNZB)F1 (SNF1) hybrids were generated at the SPF facility of MUSC by crossing SWR females with NZB males. Fresh fecal pellets were collected separately from individual mice at different time-points . Fecal pellets from Balb/cJ mice were collected immediately upon receipt from the vendor. In some experiments, SNF1 mice were given a broad spectrum antibiotic cocktail as described in our recent report17. Proteinuria was scored as follows; 0: 0–1 mg/ml, 1: 1–2 mg/ml, 2: 2–5 mg/ml, 3: 5–10 mg/ml and 4: ≥ 10 mg/ml. Mice that showed proteinuria > 5 mg/ml were considered to have severe nephritis.Urine samples were tested weekly for proteinuria. Protein level in the urine was determined by Bradford assay (BioRad) against bovine serum albumin standards as described before17. These cells were subjected to flow cytometric analysis to detect IgA + B cells as described before17, and/or cultured in the presence of bacterial LPS (2 µg/ml) and anti-CD40 antibody (5 µg/ml) for 48 h. Optimally diluted, spent media were tested for relative levels of total IgA and nucleohistone- and dsDNA-reactive IgA by ELISA.Male and female SNF1 mice, and female B6 mice of 4 and 16 weeks of age were euthanized, single cell suspensions of spleen and Peyer’s patches (PP) or enriched immune cells from ileum portion of the small intestine (Si) and large intestine (Li) were prepared as described in our recent reports17. Briefly, 0.5 µg/well of nucleohistone (Sigma-Aldrich) or dsDNA from calf thymus (Sigma-Aldrich) was coated as antigen, overnight, onto ELISA plate wells. Serial dilutions of the sera were made, total IgG against these antigens were detected using HRP-conjugated respective anti-mouse antibody  and the reaction was detected using TMB substrate (BD biosciences). For determining antibody levels in fecal samples, extracts of fresh fecal pellets from individual mice were collected separately and used. Weighed fecal pellets were suspended in proportionate volume of PBS  by breaking the pellet using a pipette tip and high speed vortex, and continuous shaking at 800 rpm overnight at 4 °C. Suspensions were centrifuged at 14,800 rpm for 15 min and top 2/3 of the supernatants were collected. Supernatants were diluted 1:100 for determining total IgA concentration. Total IgA levels were determined by employing in-house quantitative sandwich ELISA. Briefly, purified anti-mouse IgA monoclonal antibody (0.1 µg/well in 50 µl) coated wells were incubated with diluted samples for 2 h, incubated with biotin linked polyclonal anti-mouse IgA antibody for 1 h, and finally with avidin-HRP for 30 min, before developing the reaction using TMB substrate. Purified mouse IgA (Southern biotech) was used in each plate for generating the standard curve. In initial assays, to test anti-dsDNA and anti-nucleohistone reactivity of IgA in different sample types, optimum dilution (1:10) of aforementioned extracted samples were incubated in dsDNA or nucleohistone coated plates and further incubated with anti-mouse IgA-HRP before developing the assay. In ELISA assays where dsDNA or nucleohistone antibody titers were determined, serial dilutions of fecal extracts (starting at 1:10 dilution) were incubated in dsDNA or nucleohistone coated plates, incubated with biotin linked anti-mouse IgA antibody, and followed by avidin-HRP. Known positive and negative control samples identified from the initial screening were used in all plates to validate the results for determining a reliable titer. Highest dilution of the sample that produced an OD value of ≥ 0.05 above background value was considered as the nAg reactive titer. IgA concentrations and nAg reactive titers per gram of feces were then calculated for the data presented here.Antibodies against nAgs (nucleohistone and dsDNA) in mouse sera were evaluated by ELISA as described in our recent reports with minor modificationst test or Mann–Whitney test was employed to calculate p-values when two means were compared. Proteinuria scores were analyzed using the Chi-square test. Pearson's correlation coefficient/bivariate correlation approach was employed for measuring linear correlation between two variables. For correlating concentration and titer of two specific factors, Spearman correlation approach was employed. A P value ≤ 0.05 was considered statistically significant.GraphPad Prism or Microsoft excel was used to calculate the statistical significance. Two-tailed 33, have been widely used for understanding the disease etiology. We have shown that significant amounts of circulating autoantibodies against nucleohistone and dsDNA are detectable in these mice by 16 weeks of age and severe nephritis indicated by high proteinuria is observed after 20 weeks of age17. While the timing of detectable levels of serum autoantibodies as well as proteinuria are highly heterogeneous in these mice, about 80% of the female and 20% of the male SNF1 mice develop severe nephritis within 32 weeks. We have reported that gut mucosa of female SNF1 mice, as compared to their male counterparts, harbor higher frequencies of activated B cells including plasma cells, as well as express high levels of pro-inflammatory cytokines as early as at juvenile age (4 weeks) and have amplified levels of these factors at adult ages17.SNF1 mice that develop lupus symptoms and proteinuria spontaneously and show gender bias in disease incidence, similar to human SLE patients17.In the current study, we assessed if the degree of IgA production in the gut relates to the pro-inflammatory immune phenotype of the gut mucosa. Examination of the B cells revealed that SNF1 mice have relatively higher occurrence of total and IgA + B cells in the intestinal compartment compared to lupus resistant B6 mice  were obtained from a much larger scale experiment. Figure Since Fig. Since fecal IgA levels were higher in lupus-prone SNF1 mice compared to B6 mice and in SNF1 females compared to male counterparts, we determined if the fecal IgA antibodies from SNF1 mice recognize nuclear antigens such as dsDNA and nucleohistones, and this feature correlates with systemic autoimmunity and disease incidence. Both longitudinal and cross-sectional samples described for Fig. As described for total IgA analysis of Fig. Since Fig. 35. Our recent study showed that depletion of gut microbiota suppresses the intestinal pro-inflammatory immune phenotype at juvenile and adult ages, systemic autoimmune progression and proteinuria onset in female SNF1 mice. Therefore, we examined if IgA production in the gut mucosa, and abundance and nAg reactivity of fecal IgA are influenced by gut microbiota, by treating female SNF1 mice with broad spectrum antibiotic cocktail (Abx). As reported in our recent study16, Abx treatment effectively depleted majority of the gut microbiota and caused a profound delay in proteinuria onset  model50.It is well established that gut microbiota including symbionts in general, and specific microbial communities including pathobionts in particular, can influence the magnitude of antibody production in the gut mucosa and the IgA abundance in feces55, suggesting that cross-reactive antibodies against microbial and host molecules are generated in the gut mucosa. This also prompts the notion that antibody production in the gut mucosa against these microbial components could eventually spread to the systemic compartment in lupus-prone background as autoimmunity against host nuclear antigens. It is possible that pro-inflammatory gut mucosa in a lupus-susceptible background, as we observed in lupus-prone mice56, could facilitate this process. In fact, our observation that fecal IgA in lupus-prone female SNF mice are dsDNA and nucleohistone reactive as early as juvenile age, much earlier than the appearance of circulating autoantibodies, supports this notion. Importantly, age-dependent progressive increase in the dsDNA and nucleohistone reactive fecal IgA and strong correlation between the levels of these antibodies at younger ages and the eventual systemic autoimmune progression and proteinuria was observed in this study. This suggests a vicious cycle of pro-inflammatory responses in the gut mucosa contributing to the generation of a large number of host nuclear antigen and microbial antigen cross-reactive B cells in the gut mucosa and their eventual spread to the systemic compartment leading to the pathogenic events of systemic autoimmunity in lupus.Autoantibody response against antigens such as nucleic acids and nuclear proteins including histones, is the key feature of lupus autoimmunity in humans and rodent models. Importantly, nucleic acids and histone-like and other nucleic acid binding proteins can be structurally homologous to their mammalian host counterparts59. Gut microbial communities including symbionts and pathobionts could differently impact the degree of IgA production in the gut mucosa62. Therefore, we postulated that if anti-nuclear antigen reactive IgA response in the gut mucosa of lupus-prone mice is originated against microbial antigens, then depletion of gut microbiota could suppress nAg reactive IgA levels. Of note, recently we showed that the depletion of gut microbiota using broad spectrum antibiotics suppressed autoimmunity and gender bias, by causing slower disease progression in SNF1 females16. Further, another group has reported that selective depletion of gut microbiota using vancomycin suppresses systemic autoimmunity and serum IgA abundance in lupus-prone MRL/lpr mice63. Here we show that depletion of gut microbiota not only caused the suppression of fecal total IgA levels, but also affected the degree of dsDNA and nucleohistone reactivity.IgA-antibody production in the gut mucosa is largely influenced by microbiota and the germ free (GF mice) as well as microbiota-depleted SPF mice show significantly lower amounts of fecal IgA64. Nevertheless, our systematic comparison between lupus-prone SNF1 mice and lupus-resistant B6 mice show a higher abundance and nAg reactivity of fecal IgA in lupus-prone SNF1 mice. Most importantly, we also found that fecal IgA in lupus-prone mice show nAg reactivity at younger ages, long before the detection of systemic autoantibodies and lupus nephritis. Furthermore, fecal IgA abundance and nAg reactivity correlate with the onset of lupus-like disease associated gender bias in this mouse model. These novel observations suggest that fecal IgA levels and nuclear antigen reactivity could serve as an early biomarker to detect the systemic autoimmune activities in at-risk subjects, before the seropositive stage and disease onset. Since the abundance and nAg reactivity of fecal IgA in lupus-prone subjects have not been studied, systematic longitudinal follow-up studies are needed to determine the clinical translation value of IgA features as biomarkers to predict the clinical disease.Overall, our study demonstrates, for the first time, that fecal IgA profiles are different in lupus-prone SNF1 mice and lupus-resistant B6 mice as early as juvenile age and this feature is reflective of late adult age systemic autoimmunity and nephritis. Most importantly, we also found that fecal IgA in lupus-prone mice show nAg reactivity at younger ages, long before the detection of systemic autoantibodies and lupus nephritis. Furthermore, fecal IgA abundance and nAg reactivity correlate with the onset of lupus-like disease associated gender bias in this mouse model. Of note,  fecal pellets from a single age group of Balb/cJ females from Jackson lab also showed lower total and nAg reactive fecal IgA levels compared to that of SNF counterparts. However, additional systematic longitudinal comparative studies are needed using different age groups of Balb/cJ and other lupus-resistant strains of mice that are housed and bred in our colony to realize the strain to strain difference in fecal IgA features including nAg reactivity. In this regard, a recent reports showed that, as compared to B6 mice, Balb/c mice of three different vendors , mice that harbor segmented filamentous bacteria particularly, produce higher amounts of polyreactive IgA in the gut mucosa and fecal samplesSupplementary information


The preservation of genome integrity in the mammalian female germline from primordial follicle arrest to activation of growth to oocyte maturation is fundamental to ensure reproductive success. As oocytes are formed before birth and may remain dormant for many years, it is essential that defence mechanisms are monitored and well maintained. The phosphatase and tensin homolog of chromosome 10 (PTEN)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B  is a major signalling pathway governing primordial follicle recruitment and growth. This pathway also contributes to cell growth, survival and metabolism, and to the maintenance of genomic integrity. Accelerated primordial follicle activation through this pathway may result in a compromised DNA damage response (DDR). Additionally, the distinct DDR mechanisms in oocytes may become less efficient with ageing. This review considers DNA damage surveillance mechanisms and their links to the PTEN/PI3K/Akt signalling pathway, impacting on the DDR during growth activation of primordial follicles, and in ovarian ageing. Targeting DDR mechanisms within oocytes may be of value in developing techniques to protect ovaries against chemotherapy and in advancing clinical approaches to regulate primordial follicle activation. In mammalian females, oocytes are formed before birth and are surrounded by somatic cells (granulosa cells) to form structures known as follicles. Oocytes have entered meiosis and are arrested at the dictyate stage of prophase I with the most immature stage  forming the store of female germ cells that will be utilised throughout reproductive life (reviewed in ). The poThe phosphatase and tensin homolog of chromosome 10 (PTEN)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B  pathway is one of the major non-gonadotropic insulin signalling pathways that coordinates the activation, growth and differentiation of follicles ,11. The A great deal of evidence, discussed below, suggests that the PI3K/PTEN/Akt pathway is essential in regulating cell-cycle checkpoint initiation and DNA repair and that the lack of PTEN in cells may cause genomic instability ,39. The In this review, we will limit the discussion to the DNA damage/DSBs repair pathway, and primarily focus on the mechanisms used by oocytes within primordial follicles to protect themselves against DNA damage throughout their lifespan. The DDR mechanism in granulosa cells will be discussed where relevant. Crosstalk between the PI3K/PTEN/Akt pathway and DDR has been linked to increased DNA damage and impaired DNA repair protein interactions in ovarian follicles activated in vitro . Such fiPublished articles including original research, peer-reviewed and reviews were searched systematically in PubMed (Medline) database using specific terms such as ‘DNA damage’, ‘oocytes’, ‘primordial follicle activation’, ‘PI3K/Akt’, ‘ovarian ageing’ and ‘chemotherapy’. Abstracts and conferences proceeding were not included. The search yielded 757 relevant references of English language literature. Article selection was conducted using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The refThe DNA of a cell is continuously threatened by various types of damage that may cause a reduction in cellular function, cell cycle progression and DNA repair . ExogenoDNA DSBs can be repaired by two main mechanisms: non-homologous end-joining (NHEJ) , and homHR requires the recognition of the DNA DSBs by the meiotic recombination 11 (MRE11)-Rad50-nijmegen breakage syndrome 1 (NBS1) (MRN) complex. The binding of MRN complex to DSB free ends allows the NBS1 protein to interact with ataxia telangiectasia mutated (ATM) dimers leading to autophosphorylation of ATM at a serine residue  . DetectiBRCA1; ATM in turn phosphorylates a specific histone protein, H2AX, at the C-terminal serine 139 to generate γH2AX, which binds specifically to the DNA damage sites and controls the recruitment of DNA repair proteins. The critical role of ATM in DDR is demonstrated by a study using mouse ovaries in culture exposed to phosphoramide mustard (PM), a metabolite of cyclophosphamide (CP). Increased γH2AX in oocytes occurred 24 h after exposure and consequently induced substantial follicle loss. Interestingly, the administration of the ATM inhibitor KU55933, reduced the adverse impact of PM on follicle depletion, emphasising the importance of ATM in the DDR . PhosphoPhosphorylation of ATM is the first step in the initiation of G2 checkpoint activation in the DNA damage repair pathway . ActivatThe role of Rad51 is of paramount importance in the final step of HR and in preventing oocyte death, as is evident from studies in mouse and bovine ,62,76. IIn the presence of DSBs, Chk2 activation delays the cell cycle transiently to provide sufficient time for DNA repair . DNA damIn conditions resulting in severe DNA damage or with ineffective DNA repair, DNA DSBs accumulation is more likely to initiate the activity of p53 family members. This process is critical to abolish oocytes with unrepaired DNA damage and safeguard against germline mutations. The apoptosis process of oocytes within primordial follicles is mediated by a distinct cell surveillance mechanism involving N-terminal transactivation domain p63 (TAp63α) ,80,81,82p63 gene encodes two major isoforms of TAp63, one with the transactivation (TA) domain and the other, ΔN-p63 , lacking the TA domain [The A domain . TAp63α A domain ,81,83. TA domain . ExposurA domain ,86,87 anA domain ,81,84. Mouse oocytes within primordial follicles also express all necessary kinases required to trigger p63 activation. Once DNA damage ensues, it may activate p63 directly, resulting in enhanced oocyte sensitivity to DNA damage compared to granulosa cells . This vuTAp63 activation in oocytes within primordial follicles requires consecutive phosphorylation by Chk2 at serine 582 . TAp63α The regulation of recruitment of primordial follicles to grow is strictly controlled by a delicate balance between inhibitory and stimulatory factors to preserve the primordial follicle pool from premature exhaustion. Evidence from genetically modified mice supports the central role of the PTEN/PI3K/Akt signalling pathway in controlling the initiation of primordial follicle growth . Thus, tUpregulation of the PI3K/Akt signalling pathway within the oocyte triggers a cascade of reactions that ultimately initiates activation of primordial follicles . PI3K isPDK1 is indispensable in maintaining primordial follicle survival and preserving reproductive lifespan. It seems likely that both PTEN and PDK1 loss leads to premature ovarian failure (POF) but through different mechanisms. PTEN loss is associated with excessive primordial follicle activation and subsequent follicular atresia, whereas PDK1 deficiency instigates accelerated clearance of primordial follicles straight from their quiescent state . Both tymTORC1 is a further downstream substrate of Akt. mTORC1 is upregulated by the destabilisation of the heterodimeric complex of tuberous sclerosis complex 1 (TSC1) and 2 (TSC2). mTORC1 phosphorylates S6 protein kinase (S6K1), which promotes cell growth and proliferation and activates ribosomal protein S6 (rpS6), which increases protein translation . ForkheaPTEN is a tumour suppressor gene and is an essential factor in promoting normal cell proliferation and coordinating oocyte growth alongside granulosa cell proliferation [PI3K-related protein kinases (PIKKs) are considered to be the main regulators of DNA damage repair capacity of cells. Akt activation implicates the cell cycle checkpoint kinase 1 (Chk1), which has an important role in the DNA damage repair mechanism as it delays the cell cycle progression in S and G2 phase to correct an error of DNA damage before cell division . PTEN isferation ,30. Oocyferation ,18. Hyperactivation of Akt due to PTEN inhibition may impair HR activity leading to genomic instability. Notably, a high endogenous level of Akt may be of significant importance in the pathology of cancer as Akt inhibits apoptosis and increases cell proliferation . In cancDespite the role of PI3K/Akt in the pathology of cancer, the modulation of this pathway has been adopted as a potential approach for women with premature ovarian insufficiency (POI) and pregnancies have been achieved ,113. HowA recent finding from our lab utilising bovine ovarian cortical fragments exposed to the PTEN inhibitor bpv(HOpic) for 24 h showed increased primordial follicle activation after six days of culture. However, γH2AX expression in oocytes was upregulated and not associated with increased expression of the DNA repair enzymes ATM and Rad51. A low dose of bpv(HOpic) did not affect BRCA1 and 2 expression and more follicles in this group survived after six days of culture compared to high doses of bpv(HOpic). Nevertheless, a marked decrease in BRCA1 and 2 expression was observed after exposure to high doses suggesting a compromised DDR. Interestingly, despite high γH2AX expression being observed in granulosa cells of secondary stage follicles, DNA repair capacity of these cells was not significantly affected, as indicated by increased MRE11, ATM and Rad51 expression and a non-significant decline of BRCA1 and 2  Figure . AlthougSeveral publications utilising mouse models have provided evidence that oocytes within resting follicles may be directly targeted by chemotherapy treatments, including CP, cisplatin and doxorubicin ,117,118.In addition to chemotherapy, another clinical problem that is linked to DNA damage, PI3K/Akt signalling pathway and ovarian ageing is endometriosis. Increased PI3K/Akt activity has been suggested in endometriosis ,126,127,The effects of PI3K/Akt/mTOR on primordial follicle activation following chemotherapy treatment have led to research utilising this mechanism to reduce the adverse impact of chemotherapy on the ovary. In human, as mTOR hyperactivation is a common feature of cancers, mTOR inhibitors are becoming a therapeutic target in certain type of cancers. In a study utilising mouse embryonic fibroblast cell lines, constitutive mTOR activation enhanced apoptosis triggered by chemotherapy through persistent DNA damage as was shown by the upregulation of γH2AX. In parallel, the absence of both PTEN and TSC2 upregulates γH2AX expression. Intriguingly, mTOR inhibition prior to treatment is able to protect cells from etoposide-induced apoptotic cell death . SubstraOvarian ageing as a physiological process varies substantially among women depending on the number of primordial follicles and the rate of follicle loss ,142. It BRCA1 but not BRCA2 perturb ovarian stimulation leading to smaller litter size. Interestingly, DNA damage was not evident in pre-granulosa cells within primordial follicles [BRCA1, Rad51 and H2AX were reduced in aged female rat and buffalo oocytes within primordial follicles [There is increasing evidence of an association between DNA damage and repair capacity of oocytes and maternal age, with DNA repair becoming less efficient with ageing ,20,21,58ollicles ,58.BRCA2 mutations do not show a reduced response to ovarian stimulation [BRCA1 but not BRCA2 mutations [BRCA1 mutations are more susceptible to DNA damage accumulation, as shown by high γH2AX expression in primordial follicles [Women with mulation . Howevermulation , indicatmulation . A genommulation , particuutations  supportsutations . In addiollicles .As ageing is thus associated with a reduction in DNA repair capacity, oocytes from older women may be more susceptible to genotoxic insults with increased primordial follicle loss due to apoptosis . It is eROS accumulation in mitochondria can be an underlying factor in ageing, by increasing oxidative damage leading to a gradual decrease in follicle quality . IncreasIt is clear that mammalian oocytes have distinct DNA damage surveillance mechanisms. There is evidence linking the regulation of primordial follicle growth activation through the PI3K pathway with increased DNA damage/reduced repair, and this provides a model for the development of new approaches to the investigation and potentially therapeutic intervention in both these key aspects of oocyte biology. Evidence from both genetic mouse models and the culture of mammalian ovarian cortical fragments supports the contention that imbalance in signalling events between oocytes and granulosa cells may contribute to impaired follicle function after aberrant primordial follicle growth activation.Data reviewed here explore the links between the regulation of primordial follicle growth activation and DNA damage repair pathways. The primordial follicle and particularly the oocyte within it has unique physiological challenges, being required to maintain genomic integrity and quality from birth over several decades without cell growth or replication. Thus, the opportunity for repeated DNA surveillance during cell division is absent, and oocyte-specific pathways from DNA damage to apoptosis exist. There is increasing research activity linking follicle growth regulation with oocyte DNA damage and repair capacity in the context of potential prevention of ovarian damage against chemotherapy, radiation or environmental toxicants. The elucidation of the possibility to confer resistance against chemotherapy through identification of key factors in the oocyte apoptotic pathway may lead to clinical trials building on the differences in these pathways between the oocyte and somatic cells.Rad51, a critical protein involved in oocyte resilience to apoptosis, is a feasible candidate to promote DNA repair capacity in oocytes and ultimately conserve fertility in women undergoing cancer treatment. Administration of recombinant Rad51 into mouse oocytes has been demonstrated to increase DDR, prevent apoptosis, improve the defective DNA repair capacity in oocyte and restore embryo development ,76. FutuTargeting the PI3K/PTEN/Akt/mTOR pathway, mTOR inhibition with rapamycin  and everIt has been shown that either complete loss of PUMA or partial loss of TAp63 in mice oocytes could retain the primordial follicle pool following CP and cisplatin exposure. This is a promising approach to reduce the negative effects of chemotherapy on the ovaries ,117 as tAn intriguing novel approach to the protection of ovaries against chemotherapy has been suggested by a recent study introducing microRNAs (miRNAs) . It is rIn vitro activation (IVA) methods have generated controversy regarding efficacy and safety with in vitro studies indicating that manipulating activation by pharmacological methods has an impact on subsequent quality of oocytes ,36,37. P


P4HA1, MPZL3, TMC4, PLEKHA6, CA8, AREG) were inversely associated with monoethyl phthalate  concentration (p < 0.05) among postmenopausal women; all six genes loaded on to one of seven factors identified by factor analysis. Transcription factor enrichment analysis revealed that genes in this factor were enriched for androgen receptor binding sites. These six genes were also significantly down-regulated in pre-malignant adjacent tissues compared to the corresponding tumor tissues in pair-wise analyses (p < 0.05). Results from our translational study indicate that low level exposure to diethyl phthalate results in measurable genomic changes in breast tissue with implications in breast carcinogenesis.Phthalates are commonly included as ingredients in personal care products such as cosmetics, shampoos and perfumes. Diethyl phthalate (DEP) has been found to be anti-androgenic and linked with adverse reproductive effects on males, but effects on females are poorly understood. We designed an integrative and translational study to experimentally examine the effects of DEP exposure at a human-equivalent dose on the mammary transcriptome in rats and to subsequently examine the DEP gene signature in breast tissues  from a population study. In Sprague-Dawley rats treated orally with DEP from birth to adulthood, we identified a signature panel of 107 genes predominantly down-regulated by DEP exposure. Univariate analysis of this 107 DEP gene signature in pre-malignant breast tissues revealed that six genes ( One of the congeners, diethyl phthalate (DEP), is commonly used as a plasticizer and solvent in personal care products such as cosmetics, shampoos and perfumes and in pharmaceutical products1. The United States (U.S.) Food and Drug Administration found DEP to be the most common phthalate in personal care products2, a major source of exposure to DEP3. The urinary metabolite of DEP, monoethyl phthalate (MEP), is detectable in U.S. population samples at levels that are often an order of magnitude higher than other phthalates such as di(2-ethylhexyl) phthalate and dibutyl phthalate4. Due to non-covalent bonding to its parent materials, DEP is leached easily into the environment resulting in widespread human exposure1.Phthalates are a family of chemicals with diverse properties that are added to a wide variety of consumer products and used in industrial and medical applications5 and increased sperm DNA damage7. MEP has been detected in breast milk, suggesting lactational exposure, and was associated with lowered testosterone in infant males8. Links between phthalate exposures and female developmental and reproductive and cancer outcomes, however, have been less well studied, despite their potential as endocrine disruptors. Reduced fecundity with MEP exposure was reported in a Danish cohort9. An epidemiologic study in Mexican women showed an association between MEP exposure and increased breast cancer risk10. High serum level of MEP in postmenopausal women was associated with elevated breast density11, a marker for breast cancer risk12. We recently reported results from the Long Island Breast Cancer Study Project (LIBCSP) in which we did not observe any significant associations between urinary level of MEP and breast cancer risk, while inverse associations were observed for mono(3-carboxypropyl) phthalate (MCPP) and monocarboxyoctyl phthalate (MCOP), metabolites of anti-androgenic phthalates13.DEP has been shown to have anti-androgenic effects in males, including shorter anogenital distance14. Given the hormone-disrupting properties of DEP and its widespread exposure through the use of personal care and consumer products, there is an urgent need to systematically study whether DEP exposures may impact breast cancer development. In rats, fetal and prepubertal exposures to high doses of butyl benzyl phthalate led to changes in gene expression profiles of mammary glands16. Less is known about the effects of DEP exposure particularly at doses that are relevant to human exposure.Breast cancer is considered a hormone-driven disease17. We identified a DEP gene signature in normal developing mammary glands in SD rats, and subsequently examined this signature in pre-malignant and breast cancer tissues from a subsample of women who participated in a population-based study. The overall goal of the study is to examine whether human level exposure to DEP induces measurable transcriptomic changes in target tissues, thus shedding light on the possible causal relationship between phthalate exposure and breast cancer.The aim of our present study is to employ an animal model to experimentally identify a gene signature of DEP exposure at a human-equivalent dose and to subsequently translate these findings to humans. We exposed female Sprague-Dawley (SD) rats to DEP orally from birth to adulthood at a dose that was previously shown to produce a urinary metabolite (MEP) level similar to that of the U.S. population20. DEP was stored in the dark at room temperature (20 °C). The solutions were prepared weekly on the basis of mean body weight of each group and were continuously stirred before and during the treatment. To minimize external contamination, olive oil and DEP were stored in glass containers and administered using 5 mL glass syringes. Biological samples were collected in polypropylene vials. Chemical analyses and stability testing have been described previously21.Diethyl phthalate (DEP)  was supplied in plastic containers . Olive oil, supplied in glass bottles , was used as the vehicle to prepare the dosing solution. The experimental oral dose was 0.1735 mg/Kg/day, which represented 1/10,000 the no observed adverse effect levels (NOAEL) of DEPad libitum and were both periodically analyzed to exclude biological and chemical contamination .Animal studies were carried out at Cesare Maltoni Cancer Research Centre/Ramazzini Institute (CMCRC/RI)  in accordance with the rules of Italian law for Animal Welfare , following the principles of Good Laboratory Practices and Standard Operating Procedures of the CMCRC/RI facility, which include authorization by the ethical committee. At Mount Sinai, the study was approved by the Institutional Animal Care and Use Committee (IACUC). The experiment used female Sprague-Dawley (SD) rats which belong to the colony that has been used for over 40 years in the laboratory of the CMCRC/RI. There were no siblings in each treatment group and they were randomized so as to have minimal differences in body weight among them (standard deviation <10% of the average). Animals were housed in makrolon cages (41 × 25 × 15 cm) at two or three per cage, with a stainless steel wire top and a shallow layer of white wood shavings as bedding . All animals were kept in a single room at 23 ± 3 °C and 40–60% relative humidity. Lighting was artificial and the light/dark cycles were approximately 12 hours each. All animals were given the same standard “Corticella” pellet diet . Feed and tap water were available There were four experimental groups: parous and age-matched nulliparous, treated with DEP or vehicle, olive oil. There were five animals per experimental group. Animals were weighed weekly to determine treatment dose. F0 generation corresponds to breeders of the experimental animals (F1). F1 animals were treated daily from postnatal day (PND) one to PND 28 through milk of their dams (F0), which were gavaged with DEP or vehicle. After weaning at PND 28, F1 animals were treated with DEP or vehicle by gavage three times a week until PND 180 and sacrificed at PND 181. Parous rats were mated (outbred) at PND 97 and treatment was continued through pregnancy, delivery of pups (F2) and lactation. At the time of sacrifice of parous rats on PND 181, F2 pups had completed weaning. Animals were euthanized via carbon dioxide inhalation and necropsy was immediately performed for collection of mammary tissues. Animals were sacrificed on the same day and at random during the estrous cycle.22. Quality control of .CEL files and pre-processing by robust multiarray average (RMA) method were done using expression console software . Batch effects due to RNA extraction method were removed using ComBat package23 in RStudio (R 3.0.2). We applied a signal intensity filter to retain only those probesets with high and stable expression . A variance-based filter was used to retain the top 50% of probesets with high interquartile range resulting in a final dataset containing 7,532 genes. Microarray data have been deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE95554 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE95554)The fifth left and right caudal mammary glands were collected, pulverized in liquid nitrogen and total RNA was extracted using Maxwell 16 LEV simplyRNA Blood kit  or by Direct-zol RNA MiniPrep kit . RNA concentration was determined using NanoDrop  and RNA quality was assessed using a 2100 Bioanalyzer ; samples with RNA Integrity Number ≥ 7 were used for microarrays. Transcriptomes were profiled by GeneChip Rat Gene 2.0 ST arrays  at the Yale Center for Genome Analysis  as described previously24. Approval of the Institutional Review Board (Program for the Protection of Human Subjects) was obtained by all participating institutions and informed consent was obtained from all study participants. Study participants included 1,508 women diagnosed with first primary in situ or invasive breast cancer between 1996 and 1997 who resided in Nassau and Suffolk counties on Long Island, NY. At the time of the in-person baseline interview, 93% of participants donated 25 mL spot urine samples, which were shipped overnight on ice, processed, and banked at −20 °C. Archived tumor tissue of the first primary breast cancer was obtained from the diagnosing hospitals25. Demographic, reproductive and lifestyle characteristics of women with available pre-malignant adjacent tissue and urinary MEP concentrations (n = 294), are provided in Table We utilized resources from the Long Island Breast Cancer Study Project (LIBCSP), which includes a population-based sample of women newly diagnosed with first primary breast cancer who participated in the main study interview within two to three months of diagnosisTumor tissue was excised prior to treatment initiation. Formalin-fixed, paraffin-embedded (FFPE) tumor sections were histopathologically reviewed by a trained pathologist and the cancer tissue was separated using manual microdissection for 745 women, and histologically normal pre-malignant adjacent tissue was retrieved for 586 of them. Following RNA extraction and quality control, samples were cross-referenced with DEP exposure information to obtain the final number of samples used in this study: 294 pre-malignant adjacent tissues, of which 243 had a paired tumor tissue.26.Monoethyl phthalate (MEP) was measured in spot urine samples donated by 892 women with breast cancer using standard laboratory techniques by staff at the Centers for Disease Control and Prevention. Briefly, metabolites were deconjugated enzymatically, matrix removal and analyte enrichment were accomplished by solid phase extraction, and instrumental analysis was done with high performance liquid chromatography–tandem mass spectrometry using isotope dilution quantification as described before27, resulting in a panel of 107 genes. These genes were included as part of a custom-designed code-set  to determine expression of the DEP gene signature in breast tumor and pre-malignant adjacent tissues of the LIBCSP. Total RNA was extracted from FFPE breast tumor and adjacent tissues using the Qiagen miRNeasy FFPE kits . RNA concentration and quality were determined using NanoDrop . Only those samples exceeding concentration of 10 ng/µL were used. 100 ng RNA was incubated with reporter and capture probes overnight at 65 °C. Following hybridization, unbound probes were removed, and the purified complexes were aligned and immobilized on imaging cartridges using an nCounter Prep station. Code count detection was carried out by scanning cartridges in an nCounter Digital Analyzer to determine gene expression levels. Raw nCounter code counts were first normalized against the geometric mean of spike-in control probes to minimize the impact of sample preparation and detection. For background determination, counts that fell below two standard deviations of the mean of negative control probes were deemed unexpressed and assigned a value equivalent to the background threshold divided by the square root of two. Finally, data were normalized against the geometric mean of 6 housekeeping genes with high and stable expression: CLTC, GAPDH, GUSB, HPRT1, PGK1 and TUBB. The final dataset was log2 transformed.Genes found to be differentially expressed by DEP exposure in rat mammary tissues were converted to orthologous human IDs using the Gene Annotator tool in the rat genome database28. To obtain a robust DEP gene signature, we bootstrapped samples by choosing four out of five samples in treatment groups and in control groups resulting in 25 rounds of differential gene expression analysis each for parous and for nulliparous groups. A lenient false discovery rate (FDR) of 25% using Benjamini-Hochberg (BH) correction29 and a fold change of ≥1.5 fold was used. For functional enrichment analysis of differentially expressed genes we used gene ontology (GO)30 via EnrichR31. Fisher’s exact test32 was used to assess significance of enrichment at a FDR of 5% by BH method.Differential gene expression analysis between DEP and control in rats was performed using linear models for microarray data (limma) package33 before and after stratifying by menopausal status. Natural log transformed urinary MEP concentration was quartiled, and the median of each quartile was considered as the predictor. To reduce the dimensionality of the dataset, we performed factor analysis to group highly correlated genes into latent factors using the psych package34 with a correlation cutoff of 0.3 to load a gene into a factor. We subsequently carried out an examination of the association between quartiles of urinary MEP concentration with the latent factors, such as oral contraceptive use, hormone replacement therapy, lactation, smoking status, religion etc. Differential gene expression analysis between pre-malignant adjacent and corresponding tumors in this study (n = 243) as well as in the breast cancer TCGA microarray data (n = 61 tumors and adjacent samples) filtered to include only the DEP gene signature, was carried out using Wilcoxon signed rank test35. To gain insight about upstream molecular regulators of genes contained within factors, we performed enrichment for transcription factors using ‘ChEA 2016’ via Enrichr31 which uses Fisher’s exact test32. Multiple comparisons were adjusted using BH-based FDR of 5%.Gene expression data of pre-malignant adjacent tissues as well as urinary MEP concentration were available for 294 breast cancer cases. Examination of the association between urinary MEP concentration and expression of each gene in the DEP gene signature was carried out using generalized linear models13.A full analysis of the association between phthalate exposure and breast cancer incidence in the LIBCSP cases and population-based controls has been reported previouslyAnimal studies were performed following the principles of Good Laboratory Practices and Standard Operating Procedures of the CMCRC/RI facility, which include authorization by the ethical committee; the study was approved by the Institutional Animal Care and Use Committee (IACUC) of Mount Sinai. For the Long Island Breast Cancer Study Project (LIBCSP), Institutional Review Board approval was obtained by all participating institutions and informed consent was obtained from all study participants; study was approved by the Institutional Review Board of Mount Sinai (Program for the Protection of Human Subjects). All experiments were performed in accordance with relevant guidelines and regulations.Rps16 which was up-regulated in the nulliparous rat group. Eighteen genes overlapped between the parous and nulliparous signature, resulting in a final gene panel of 107 genes (‘DEP gene signature’) , ‘response to hypoxia’ , ‘response to steroid hormone’  and ‘cellular amine metabolic process’  (FDR < 0.05).Our final dataset of ~7,500 genes was obtained after filtering the mammary microarray data to retain only those genes with both high expression and variation. We then performed differential gene expression analysis between DEP-treated rats and controls to identify a phthalate gene signature. To identify a robust signature, we bootstrapped samples in parous and nulliparous rats separately given the potential difference in mammary transcriptome. This resulted in 339 and 43 differentially expressed genes by DEP treatment in the parous and nulliparous rat groups, respectively. For the parous rat group, we ranked genes by their difference in median expression between treatment and control and selected the top 100 genes. Finally, we identified the human orthologs of the rat genes resulting in 91 orthologous human genes in the parous group (‘DEP parous gene signature’) and 34 orthologous human genes in the nulliparous group (‘DEP nulliparous gene signature’) (Supplementary Table e’) Fig. . PathwayAmong the LIBCSP case cohort, we have 294 subjects with both gene profile and urinary MEP measurement. We compared the full group of cases with urinary MEP information (n = 892) with the subset (n = 294) used in our investigation and found no major differences in clinical, demographic and reproductive factors Table , nor didAREG, TMEM50B, CA8, C4ORF19) regardless of menopausal status. Given the different risk factor profiles associated with pre- and postmenopausal breast cancer38 we stratified our analysis by menopausal status and found that nine and three genes were significantly associated with MEP in the pre-malignant adjacent breast tissue among postmenopausal (n = 189) and premenopausal (n = 99) women, respectively  had decreasing expression with increasing levels of MEP, which was consistent with results from rats where we observed reduced gene expression levels with DEP exposure. Given the limited sample size in premenopausal women, we carried out subsequent analysis in postmenopausal women only. To reduce multiple comparisons and to take advantage of the strong correlation structure among genes, a dimensionality reduction technique, i.e. factor analysis, was carried out  included SLC44A3, P4HA1, CXADR, AADAT, GALNT3 and ABCG2. We then restricted our analysis to AR + cases, which we defined as AR expression above the lower whisker of Tukey’s boxplot39 (n = 167) and found six more genes to be inversely associated with urinary MEP  were also validated in the TCGA dataset (FDR < 0.05); however, AREG and CA8 showed the opposite trend.We compared expression levels of 107 DEP signature genes in adjacent and their tumor counterparts (n = 243 pairs), and found that 33 genes were down-regulated and 34 genes were up-regulated in adjacent compared to corresponding tumor tissues at FDR < 0.05 , we were able to show that even at a low, human level exposure, measurable changes in the transcriptome were detectable. The 107 modified genes in adult rats were predominantly down-regulated by DEP exposure and were enriched for biological processes such as ‘mammary gland alveolus development’. Interestingly, overexpression of at least eight genes among the DEP gene signature has been previously reported in breast cancer tissues; these include the genes FOXA141, PRLR42, TPH143, AREG44, AR45, XBP146, TGFB347 and HMGCS248. Coordinated down-regulated expression of these genes by DEP exposure in our study seemed to suggest an inverse relationship between DEP and breast cancer in animal models. While we could not obtain such information in our animal study , the suggestive inverse association in our population study13 seems to corroborate this postulation.We first identified a panel of 107 genes modified by DEP exposure in rats treated chronically from birth to adulthood using a human-level exposure that has previously been shown to result in urinary MEP concentrations within the range reported for the U.S. populationP4HA1 encodes a component of prolyl 4-hydroxylase, a key enzyme in collagen synthesis; it was shown to be an activator of the HIF-1 pathway in breast cancer and biomarker of poor prognosis in various types of cancers including melanomas49, pancreatic cancer50 and head and neck squamous cell carcinomas51. MPZL3 (Myelin protein zero like 3) is involved in mediating cell adhesion but little is known about its possible role in cancer pathways. TMC4 encodes a transmembrane channel protein52; while its expression was upregulated in tumor-versus-adjacent tissue in our dataset as well as TCGA, other studies reported its downregulation in high-metastatic breast cancer cell lines compared to their low metastatic counterparts53 as well as breast-cancer cells compared to normal breast epithelium54, and its increased expression in tumors was associated with better survival of breast cancer patients55. PLEKHA6 (Pleckstrin Homology Domain Containing 6) has been shown to be differentially methylated in head and neck cancer56; its high expression was shown to be a positive prognostic factor in lung57 and breast58 cancers. CA8 (Carbonic Anhydrase 8) encodes an inhibitor of inositol trisphosphate inhibitors which regulate intracellular calcium release fundamental to many cellular processes including mitochondrial energy production and cell fate59. AREG (Amphiregulin) is a member of the epidermal growth factor family that is expressed in the mammary gland during development and has been shown to promote the growth of normal epithelial cells while inhibiting the growth of aggressive carcinoma cell lines52.We next examined the DEP gene signature in pathologically non-malignant tissues because we hypothesized that gross genomic abnormalities in tumor tissues would mask the likely subtle effects of low-dose environmental chemical exposures. In particular we identified by univariate analysis, a subset of six out of 107 genes to be negatively associated with urinary MEP in pre-malignant adjacent tissues of postmenopausal women. The down-regulated expression of these six genes with urinary MEP in humans was consistent with direction of expression change observed in rats by DEP exposure. These six genes have diverse functions. et al.10 (higher urine levels of MEP associated with increased BC risk) and Sprague et al.11 (elevated serum levels of MEP associated with increased breast density) for which we currently don’t have an explanation.The strong correlation structure underlying genes in adjacent tissues enabled us to use factor analysis to derive seven factors, clusters of highly correlated genes, of which one factor, factor 2, showed independent negative association with urinary MEP. Interestingly, all six genes identified by univariate analysis loaded into factor #2, indicating similar expression patterns of these genes. Furthermore, we showed that the six genes that were negatively associated with urinary MEP were also coordinately down-regulated in adjacent compared to paired tumor tissue, and four out of six of these genes were also down-regulated in adjacent compared to paired tumors of the TCGA dataset. Collectively, our results from humans and animals seem to suggest an inverse relationship between DEP exposure and breast cancer development. However, we acknowledge the cross-sectional nature of our study, where urinary MEP concentrations were obtained only at one time-point after breast cancer diagnosis, and may not necessarily reflect lifetime exposure to DEP or whether the exposure occurred prior to diagnosis of breast cancer. Additionally, there is no control (cancer-free) population in the study. Hence, whether phthalate-induced transcriptome changes modify breast cancer susceptibility needs to be further investigated to clarify the etiologic mechanism of phthalates in breast carcinogenesis. Design of the current study does not allow us to claim the direct link between DEP exposure and breast cancer risk. We also have to acknowledge a seeming discrepancy of our results with the studies of López-Carrillo 62. While the mechanism of anti-androgenic activity of phthalates has been shown to be mediated by inhibition of testosterone synthesis leading to Leydig cell dysfunction63, several phthalates have been shown to be able to bind to AR weakly suggesting that phthalates may elicit their effects through both receptor- and non-receptor-mediated pathways64. Even though in our study AR was among the signature panel of genes disrupted by DEP in rats, its expression was not significantly altered with urinary MEP exposure in human tissues. However, we noted that among postmenopausal women with high AR expression, six more genes were significantly associated with urinary MEP, suggesting that AR could play a role in the association between phthalate exposure and gene expression.To gain insight about upstream molecular regulators of genes associated with urinary MEP in postmenopausal tumor-adjacent breast tissues, we performed transcription factor enrichment analysis of the 20 genes belonging to factor 2 and observed enrichment for AR binding sites among these genes. The anti-androgenic activity of phthalates has been extensively reported, which include endpoints such as shorter anogenital distance and reduced testosterone levels36, be associated with poorer survival37 and have distinct molecular profiles compared to postmenopausal breast cancers38. There was no major difference in distribution of MEP concentrations between premenopausal and postmenopausal women in our dataset. Whether there are different environmental etiologies of premenopausal compared to postmenopausal breast cancers remains to be elucidated.Our results indicated stronger associations of DEP signature genes among adjacent tissues of postmenopausal women, and not premenopausal women; part of this could be attributed to the smaller sample size among premenopausal (n = 99) compared to postmenopausal (n = 189) women. Premenopausal breast cancers tend to be more aggressive65. That we did not observe significant gene expression changes between parous and nulliparous rats suggests that the parous rats in our study had finished involution. Parity is known to reduce the risk of developing breast cancer66; however how environmental factors interact with parity to influence cancer susceptibility is unknown. Our results suggesting that phthalate exposure had a more profound effect on the transcriptome of parous compared to nulliparous rats warrants further investigation, for example, by examining rates of spontaneous mammary tumor formation among exposed and control groups later in life.We found a larger number of genes modified among parous rats (339 genes) compared to nulliparous rats (43 genes) suggesting that parity may play a role in modifying the effects of phthalate exposure on the mammary gland transcriptome. The parous rats in our study completed pregnancy and lactation and were sacrificed 35 days after the end of lactation. Post-lactational involution occurs within a few days of end of lactation whereby apoptosis of secretory structures remodels the gland to its pre-pregnant ductal architecture resembling its virgin counterpart67 as well as species-specific differences in response to environmental chemicals68. Nevertheless, Sprague-Dawley rats have been shown to be one of the most physiologically relevant and genetically defined animal models for studying human sporadic breast cancer70. For example, they share similar age-equivalent distribution of mammary tumors as in human population; mammary carcinomas from this animal model also share many morphological and molecular features, including estrogen-dependence, chromosomal instability, aneuploidy, and deregulation of cell cycle with breast cancer in human populations71. Additionally, we did not have a technical possibility to run additional assays such as in situ hybridization experiments or immunohistochemical staining to confirm the expression changes in the caudal mammary glands. Second is the translation of findings from normal mammary glands in rats to cancer-adjacent breast tissues in humans. Extratumoral benign-appearing tissues lying adjacent to breast tumors have been shown to harbor genomic alterations such as somatic loss of heterozygosity, methylation changes and shortened telomeres, and in some cases may have been infiltrated with tumor cells72 – the so-called “field effect” – making it harder to untangle the subtle effects of low-dose environmental chemical exposures on transcriptome pattern. Finally, the route of exposure used in animal studies involved oral gavage, while exposures to many environmental chemicals, including phthalates have been shown to occur via multiple routes other than oral, such as dermal or by inhalation62. However, we attempted to resolve this issue by calibrating the exposure dose to achieve similar urinary biomarker concentration in rats and humans.We acknowledge some limitations of our study. First is the translation of findings from rats to humans. While rats have been a useful model system to study breast cancer, important species-specific differences exist, including an accelerated lifespan leading to condensed mammary developmental timing in rats, six pairs of mammary glands in rats compared to one in humansUsing an integrative and translational approach, we identified a DEP gene signature in an animal model which was partially validated in a human population. Functional relevance of the gene signature indicated by in silico pathway analysis further support the potential causal role of phthalate in breast carcinogenesis.Table S1.